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  • 101.
    Klingström, Tomas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Plewczynski, Dariusz
    Protein-protein interaction and pathway databases, a graphical review2011In: Briefings in Bioinformatics, ISSN 1467-5463, E-ISSN 1477-4054, Vol. 12, no 6, p. 702-713Article, review/survey (Refereed)
    Abstract [en]

    The amount of information regarding protein-protein interactions (PPI) at a proteomic scale is constantly increasing. This is paralleled with an increase of databases making information available. Consequently there are diverse ways of delivering information about not only PPIs but also regarding the databases themselves. This creates a time consuming obstacle for many researchers working in the field. Our survey provides a valuable tool for researchers to reduce the time necessary to gain a broad overview of PPI-databases and is supported by a graphical representation of data exchange. The graphical representation is made available in cooperation with the team maintaining www.pathguide.org and can be accessed at http://www.pathguide.org/interactions.php in a new Cytoscape web implementation. The local copy of Cytoscape cys file can be downloaded from http://bio.icm.edu.pl/similar to darman/ppi web page.

  • 102. Klingström, Tomas
    et al.
    Soldatova, Larissa
    Stevens, Robert
    Roos, Erik
    Swertz, Morris
    Müller, Kristian
    Kalas, Matus
    Lambrix, Patrick
    Taussig, Michael
    Litton, Jan-Eric
    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.
    Bongcam-Rudloff, Erik
    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.
    Workshop on laboratory protocol standards for the molecular methods database2013In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 30, no 2, p. 109-113Article in journal (Refereed)
    Abstract [en]

    Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point.

  • 103.
    Koechling, Michaela
    et al.
    Univ Hosp Munster, Dept Neurosurg, D-48149 Munster, Germany..
    Ewelt, Christian
    Univ Hosp Munster, Dept Neurosurg, D-48149 Munster, Germany..
    Fuertjes, Gina
    Univ Hosp Munster, Dept Neurosurg, D-48149 Munster, Germany..
    Peetz-Dienhart, Susanne
    Univ Hosp Munster, Inst Neuropathol, D-48149 Munster, Germany..
    Koos, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Hasselblatt, Martin
    Univ Hosp Munster, Inst Neuropathol, D-48149 Munster, Germany..
    Paulus, Werner
    Univ Hosp Munster, Inst Neuropathol, D-48149 Munster, Germany..
    Stummer, Walter
    Univ Hosp Munster, Dept Neurosurg, D-48149 Munster, Germany..
    Brokinkel, Benjamin
    Univ Hosp Munster, Dept Neurosurg, D-48149 Munster, Germany..
    hTERT promoter methylation in pituitary adenomas2016In: BRAIN TUMOR PATHOLOGY, ISSN 1433-7398, Vol. 33, no 1, p. 27-34Article in journal (Refereed)
    Abstract [en]

    Telomerase reverse transcriptase (TERT) expression is a hallmark in tumorigenesis and upregulated due to mutations and methylation of the human (h)TERT promoter. As mutations are rare but methylation is common in pituitary adenomas (PA), we determined promoter methylation and its clinical impact in 85 primary and 15 recurrent PA by methylation-specific PCR. 40 females (47 %) and 45 males (53 %) with a median age of 53 years harboring micro-, macro-, and giant adenomas in 12, 82, and 6 % were included (prolactinomas, corticotroph, somatotroph, gonadotroph, thyreotroph, plurihormonal, and null cell adenomas in 11, 18, 10, 29, 1, 10, and 21 %, respectively). In primary diagnosed tumors, methylation rate was 27 % and higher in males than in females (40 vs. 13 %, p = 0.001) after uni- and multivariate analyses. Methylation differed among PA subtypes (0-42 %, p = n.s.) and was not significantly correlated with tumor size, cavernous sinus invasion, or serum hormone levels. Ki67 labeling index and recurrence (N = 16, 19 %) were independent of methylation. In recurrent tumors, methylation was similar to primary PA (N = 5/15, 33 %) and remained unchanged along follow-up. Thus, while being commonly observed in PA, hTERT promoter methylation is stable along follow-up and independent of most clinical variables, PA subtype, proliferation, and without prognostic value.

  • 104.
    Koos, Björn
    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.
    Andersson, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    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.
    Grannas, Karin
    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.
    Klaesson, Axel
    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.
    Cane, Gaëlle
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Analysis of protein interactions in situ by proximity ligation assays2014In: High-Dimensional Single Cell Analysis: Mass Cytometry, Multi-parametric Flow Cytometry and Bioinformatic Techniques / [ed] Fienberg, Harris G., Nolan, Garry P., Springer, 2014, Vol. 377, p. 111-26Chapter in book (Refereed)
    Abstract [en]

    The fate of the cell is governed by interactions among proteins, nucleic acids, and other biomolecules. It is vital to look at these interactions in a cellular environment if we want to increase our understanding of cellular processes. Herein we will describe how the in situ proximity ligation assay (in situ PLA) can be used to visualize protein interactions in fixed cells and tissues. In situ PLA is a novel technique that uses DNA, together with DNA modifying processes such as ligation, cleavage, and polymerization, as tools to create surrogate markers for protein interactions of interest. Different in situ PLA designs make it possible not only to detect protein-protein interactions but also post-translational modifications and interactions of proteins with nucleic acids. Flexibility in DNA probe design and the multitude of different DNA modifying enzymes provide the basis for modifications of the method to make it suitable to use in many applications. Furthermore, examples of how in situ PLA can be combined with other methods for a comprehensive view of the cellular activity status are discussed.

  • 105.
    Koos, Björn
    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.
    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.
    David, Leonor
    Sobrinho-Simoes, Manuel
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Next-Generation Pathology: Surveillance of Tumor Microecology2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 11, p. 2013-2022Article, review/survey (Refereed)
    Abstract [en]

    A tumor is a heterogeneous population of cells that provides an environment in which every cell resides in a microenvironmental niche. Microscopic evaluation of tissue sections, based on histology and immunohistochemistry, has been a cornerstone in pathology for decades. However, the dawn of novel technologies to investigate genetic aberrations is currently adopted in routine molecular pathology. We herein describe our view on how recent developments in molecular technologies, focusing on proximity ligation assay and padlock probes, can be applied to merge the two branches of pathology, allowing molecular profiling under histologic observation. We also discuss how the use of image analysis will be pivotal to obtain information at a cellular level and to interpret holistic images of tissue sections. By understanding the cellular communications in the microecology of tumors, we will be at a better position to predict disease progression and response to therapy.

  • 106.
    Koos, Björn
    et al.
    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.
    Closing in on life - proximity dependent methods for life sciences2015In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 6, no 20, p. 17867-17868Article in journal (Other academic)
  • 107. Kourti, Maria
    et al.
    Ikonomou, Georgia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Giakoumakis, Nikolaos-Nikiforos
    Rapsomaniki, Maria Anna
    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.
    Siniossoglou, Symeon
    Lygerou, Zoi
    Simos, George
    Mylonis, Ilias
    CK1 delta restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1 alpha/ARNT complex formation2015In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 27, no 6, p. 1129-1140Article in journal (Refereed)
    Abstract [en]

    Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1 alpha, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1 delta phosphorylates HIP-la in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIP-1 alpha interactions within the cell nucleus using the in situ proximity ligation assay (PIA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1 delta-dependent modification of HIF-1 alpha impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1 delta increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1 alpha- and lipin-1-dependent manner. These data reveal a novel role for CK1 delta in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.

  • 108.
    Kuhnemund, Malte
    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.
    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.
    Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore2015In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 67, no SI, p. 11-17Article in journal (Refereed)
    Abstract [en]

    Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.

  • 109.
    Kuhnemund, Malte
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Witters, Daan
    Nilsson, Mats
    Lammertyn, Jeroen
    Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection2014In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 16, p. 2983-2992Article in journal (Refereed)
    Abstract [en]

    We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rotting circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products mu L-1 as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.

  • 110.
    Kühnemund, Malte
    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. Stockholm Univ, Dept Biophys & Biochem, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Wei, Qingshan
    Univ Calif Los Angeles, Dept Elect Engn, Los Angeles, CA 90095 USA.;Univ Calif Los Angeles, Dept Bioengn, Los Angeles, CA 90095 USA..
    Darai, Evangelia
    Stockholm Univ, Dept Biophys & Biochem, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Wang, Yingjie
    Univ Calif Los Angeles, Dept Elect Engn, Los Angeles, CA 90095 USA..
    Hernandez-Neuta, Ivan
    Stockholm Univ, Dept Biophys & Biochem, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Yang, Zhao
    Univ Calif Los Angeles, Dept Elect Engn, Los Angeles, CA 90095 USA..
    Tseng, Derek
    Univ Calif Los Angeles, Dept Elect Engn, Los Angeles, CA 90095 USA..
    Ahlford, Annika
    Stockholm Univ, Dept Biophys & Biochem, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden.;Devyser AB, Instrumentvagen 19, S-12653 Stockholm, Sweden..
    Mathot, Lucy
    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.
    Sjöblom, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ozcan, Aydogan
    Univ Calif Los Angeles, Dept Elect Engn, Los Angeles, CA 90095 USA.;Univ Calif Los Angeles, Dept Bioengn, Los Angeles, CA 90095 USA.;Univ Calif Los Angeles, Calif NanoSyst Inst CNSI, Los Angeles, CA 90095 USA..
    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. Stockholm Univ, Dept Biophys & Biochem, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 13913Article in journal (Refereed)
    Abstract [en]

    Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

  • 111.
    La Fleur, Linnea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Falk-Sörqvist, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Smeds, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Sundström, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Mattsson, Johanna Sofia Margareta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Brandén, Eva
    Gavle Cent Hosp, Dept Resp Med, Gavle, Sweden.
    Koyi, Hirsh
    Gavle Cent Hosp, Dept Resp Med, Gavle, Sweden.
    Isaksson, Johan
    Gavle Cent Hosp, Dept Resp Med, Gavle, Sweden.
    Brunnström, Hans
    Reg Labs Reg Skane, Pathol, Lund, Sweden.
    Sandelin, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Lung- allergy- and sleep research.
    Lamberg, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Lung- allergy- and sleep research.
    Landelius, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery.
    Nilsson, Mats
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Moens, Lotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Mutation Profiling by Targeted Next Generation Sequencing of an Unselected NSCLC Cohort2017In: Journal of Thoracic Oncology, ISSN 1556-0864, E-ISSN 1556-1380, Vol. 12, no 1, p. S526-S527Article in journal (Other academic)
  • 112.
    La Fleur, Linnea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Moens, Lotte
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Falk-Sörqvist, Elin
    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.
    Mattsson, Johanna S. M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Koyi, Hirsh
    Gavle Cent Hosp, Dept Resp Med, S-80187 Gavle, Sweden..
    Branden, Eva
    Gavle Cent Hosp, Dept Resp Med, S-80187 Gavle, Sweden..
    Brunnström, Hans
    Lund Univ, Div Oncol & Pathol, Dept Clin Sci Lund, Lund, Sweden..
    Ekman, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Sandelin, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Respiratory Medicine and Allergology.
    Isaksson, Johan
    Gavle Cent Hosp, Dept Resp Med, S-80187 Gavle, Sweden..
    Jirström, Karin
    Lund Univ, Div Oncol & Pathol, Dept Clin Sci Lund, Lund, Sweden..
    Micke, Patrick
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Nilsson, Mats
    Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden..
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mutation Profiling by Targeted Next-Generation Sequencing for Diagnostics and Patient Cohort Screening in FFPE NSCLC Samples2015In: Journal of Thoracic Oncology, ISSN 1556-0864, E-ISSN 1556-1380, Vol. 10, no 9, p. S697-S697Article in journal (Other academic)
  • 113.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    AFFINOMICS and the prospects for large-scale protein analyses2016In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, no 5, p. 491-493Article in journal (Other academic)
  • 114.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Offshoots of the ESF functional genomics programme2013In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 30, no 3, p. 296-298Article in journal (Refereed)
    Abstract [en]

    After the conclusion of the second five-year period of the European Science Foundation (ESF) programme on functional genomics, it is time to take stock and evaluate its accomplishments. The programme networked leading scientists from a large number of European countries for strategy discussions about the promotion of functional genomics research, and to arrange scientific meetings and exchange programmes. In brief, I believe this programme has punched above its weight, and that it has successfully contributed to the overall organisation of molecular biosciences in Europe. With a modest annual budget the programme has created several interesting new opportunities, some of which may have yet to show their full impact. However, these mini-reviews are intended to provide a personal perspective on this functional genomics effort, and accordingly I focus on my personal experiences from the ESF programme.

  • 115.
    Landegren, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Al-Amin, Rasel A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Björkesten, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    A myopic perspective on the future of protein diagnostics2018In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 45, p. 14-18Article, review/survey (Refereed)
    Abstract [en]

    Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.

  • 116.
    Landegren, Ulf
    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.
    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.
    Hammond, Maria
    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.
    Wu, Di
    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.
    Ullerås, Erik
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Opportunities for sensitive plasma proteome analysis2012In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 4, p. 1824-1830Article in journal (Refereed)
    Abstract [en]

    Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. While discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal, and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed.

  • 117. Larssen, Pia
    et al.
    Wik, Lotta
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Czarnewski, Paulo
    Eldh, Maria
    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.
    Ronquist, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Dubois, Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Freyhult, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gallant, Caroline
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Oelrich, Johan
    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.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Ronquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Villablanca, Eduardo
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gabrielsson, Susanne
    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.
    Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay2017In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 3, p. 502-511Article in journal (Refereed)
    Abstract [en]

    Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

  • 118.
    Le Jan, Sébastien
    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.
    Hayashi, Makoto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kasza, Zsolt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bishop, Joseph R
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jakobsson, Lars
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Esko, Jeffrey D
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis2012In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 32, no 5, p. 1255-1263Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis.

    METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, TGFβ, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of TGFβ and platelet-derived growth factor B signal transduction.

    CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

  • 119.
    Lekholm, Emilia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Eriksson, Mikaela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Klaesson, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. 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.
    Perland, Emelie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Schweizer, Nadine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Glucose deprived mouse embryonal cortex cultures respond by altering MFS transporter expression and localizationManuscript (preprint) (Other academic)
  • 120. Lessard, Christopher J.
    et al.
    Adrianto, Indra
    Ice, John A.
    Wiley, Graham B.
    Kelly, Jennifer A.
    Glenn, Stuart B.
    Adler, Adam J.
    Li, He
    Rasmussen, Astrid
    Williams, Adrienne H.
    Ziegler, Julie
    Comeau, Mary E.
    Marion, Miranda
    Wakeland, Benjamin E.
    Liang, Chaoying
    Ramos, Paula S.
    Grundahl, Kiely M.
    Gallant, Caroline J.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Alarcon-Riquelme, Marta E.
    Alarcon, Graciela S.
    Anaya, Juan-Manuel
    Bae, Sang-Cheol
    Boackle, Susan A.
    Brown, Elizabeth E.
    Chang, Deh-Ming
    Cho, Soo-Kyung
    Criswell, Lindsey A.
    Edberg, Jeffrey C.
    Freedman, Barry I.
    Gilkeson, Gary S.
    Jacob, Chaim O.
    James, Judith A.
    Kamen, Diane L.
    Kimberly, Robert P.
    Kim, Jae-Hoon
    Martin, Javier
    Merrill, Joan T.
    Niewold, Timothy B.
    Park, So-Yeon
    Petri, Michelle A.
    Pons-Estel, Bernardo A.
    Ramsey-Goldman, Rosalind
    Reveille, John D.
    Scofield, R. Hal
    Song, Yeong Wook
    Stevens, Anne M.
    Tsao, Betty P.
    Vila, Luis M.
    Vyse, Timothy J.
    Yu, Chack-Yung
    Guthridge, Joel M.
    Kaufman, Kenneth M.
    Harley, John B.
    Wakeland, Edward K.
    Langefeld, Carl D.
    Gaffney, Patrick M.
    Montgomery, Courtney G.
    Moser, Kathy L.
    Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as Susceptibility Loci for Systemic Lupus Erythematosus in a Large-Scale Multiracial Replication Study2012In: American Journal of Human Genetics, ISSN 0002-9297, E-ISSN 1537-6605, Vol. 90, no 4, p. 648-660Article in journal (Refereed)
    Abstract [en]

    Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p(meta-Euro) = 2.08 x 10(-10)), transmembrane protein 39A (TMEM39A; rs1132200; p(meta-all) 8.62 x 10(-9)), and 17q21 (rs1453560; p(meta-all) = 3.48 x 10(-10)) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 x 10(-8) < p(meta-Euro) < 9.99 x 10(-5)) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4 FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.

  • 121.
    Leuchowius, Karl Johan
    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.
    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, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    In situ proximity ligation assay for microscopy and flow cytometry2011In: Current Protocols in Cytometry, ISSN 1934-9297, Vol. Suppl 56, p. Unit 9.36-Article in journal (Refereed)
    Abstract [en]

    The ability to study proteins and protein interactions inside cells and tissues is important for elucidating how cells function in health and disease. The in situ proximity ligation assay (in situ PLA) presented here can be used to visualize proteins, protein-protein interactions, and post-translational modifications in cells and tissues. The method is based upon the use of antibodies that target the proteins involved in an interaction; hence, the method has the advantage that it can be used in clinical specimens, providing localized, quantifiable single molecule detection in single cells. This unit describes how in situ PLA can be used with fluorescence microscopy and flow cytometry to study proteins (obtaining high sensitivity and specificity of detection) and protein interactions. It also includes information on expected results and information on how to troubleshoot the assay.

  • 122.
    Leuchowius, Karl-Johan
    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.
    Clausson, Carl-Magnus
    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.
    Grannas, Karin
    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.
    Erbilgin, Yücel
    Botling, 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.
    Zieba, Agata
    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.
    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.
    Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue2013In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 12, no 6, p. 1563-1571Article in journal (Refereed)
    Abstract [en]

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

  • 123.
    Lind, Anne-Li
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    Wu, Di
    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.
    Freyhult, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bodolea, Constantin
    University of Cluj Napoca, Cluj Napoca, Romania.
    Ekegren, Titti
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Gustafsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Gordh, Torsten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Analyses of CSF reveal decreased levels of four proteins in ALS patients, but no changes upon analgesia in patients with neuropathic painManuscript (preprint) (Other (popular science, discussion, etc.))
  • 124.
    Lindblom, Rickard P F
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery.
    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.
    Axén, Sofie
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Thelin, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Thoracic Surgery.
    Protein Profiling in Serum and Cerebrospinal Fluid Following Complex Surgery on the Thoracic Aorta Identifies Biological Markers of Neurologic Injury.2018In: Journal of Cardiovascular Translational Research, ISSN 1937-5387, E-ISSN 1937-5395, Vol. 11, no 6, p. 503-516Article in journal (Refereed)
    Abstract [en]

    Surgery on the arch or descending aorta is associated with significant risk of neurological complications. As a consequence of intubation and sedation, early neurologic injury may remain unnoticed. Biomarkers to aid in the initial diagnostics could prove of great value as immediate intervention is critical. Twenty-three patients operated in the thoracic aorta with significant risk of perioperative neurological injury were included. Cerebrospinal fluid (CSF) and serum were obtained preoperatively and in the first and second postoperative days and assessed with a panel of 92 neurological-related proteins. Three patients suffered spinal cord injury (SCI), eight delirium, and nine hallucinations. There were markers in both serum and CSF that differed between the affected and non-affected patients (SCI; IL6, GFAP, CSPG4, delirium; TR4, EZH2, hallucinations; NF1). The study identifies markers in serum and CSF that reflect the occurrence of neurologic insults following aortic surgery, which may aid in the care of these patients.

  • 125.
    Liu, Yanling
    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.
    Gu, Jijuan
    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.
    Hagner-McWhirter, Asa
    Sathiyanarayanan, Poojahrau
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gullberg, Mats
    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.
    Johansson, Johan
    Hammond, Maria
    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.
    Ivansson, Daniel
    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.
    Western Blotting via Proximity Ligation for High Performance Protein Analysis2011In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 11, p. O111.011031-Article in journal (Refereed)
    Abstract [en]

    Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor beta by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

  • 126.
    Lovmar, Lovisa
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Ahlford, Annika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Jonsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Silhouette scores for assessment of SNP genotype clusters2005In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 6, article id 35Article in journal (Refereed)
    Abstract [en]

    Background: High-throughput genotyping of single nucleotide polymorphisms ( SNPs) generates large amounts of data. In many SNP genotyping assays, the genotype assignment is based on scatter plots of signals corresponding to the two SNP alleles. In a robust assay the three clusters that define the genotypes are well separated and the distances between the data points within a cluster are short. "Silhouettes" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a cluster. Thus "Silhouettes" can potentially be used as a quality measure for SNP genotyping results and for objective comparison of the performance of SNP assays at different circumstances. Results: We created a program (ClusterA) for calculating "Silhouette scores", and applied it to assess the quality of SNP genotype clusters obtained by single nucleotide primer extension ("minisequencing") in the Tag-microarray format. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment has been arbitrary. In the present study we applied Silhouette scores to compare the performance of four DNA polymerases in our minisequencing system by analyzing 26 SNPs in both DNA polarities in 16 DNA samples. We found Silhouettes to provide a relevant measure for the quality of SNP assays at different reaction conditions, illustrated by the four DNA polymerases here. According to our result, the genotypes can be unequivocally assigned without manual inspection when the Silhouette score for a SNP assay is > 0.65. All four DNA polymerases performed satisfactorily in our Tag-array minisequencing system. Conclusion: "Silhouette scores" for assessing the quality of SNP genotyping clusters is convenient for evaluating the quality of SNP genotype assignment, and provides an objective, numeric measure for comparing the performance of SNP assays. The program we created for calculating Silhouette scores is freely available, and can be used for quality assessment of the results from all genotyping systems, where the genotypes are assigned by cluster analysis using scatter plots.

  • 127.
    Lundin, Karin E.
    et al.
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, Karolinska Univ Hosp, S-14186 Huddinge, Sweden..
    Hamasy, Abdulrahman
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, Karolinska Univ Hosp, S-14186 Huddinge, Sweden..
    Backe, Paul Hoff
    Natl Hosp Norway, Oslo Univ Hosp, Clin Diagnost & Intervent, Dept Microbiol, N-0424 Oslo, Norway.;Univ Oslo, Inst Clin Med, Dept Med Biochem, N-0424 Oslo, Norway..
    Moens, Lotte N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Falk-Sörqvist, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Elgstoen, Katja B.
    Univ Oslo, Inst Clin Med, Dept Med Biochem, N-0424 Oslo, Norway..
    Morkrid, Lars
    Univ Oslo, Inst Clin Med, Dept Med Biochem, N-0424 Oslo, Norway..
    Bjoras, Magnar
    Natl Hosp Norway, Oslo Univ Hosp, Clin Diagnost & Intervent, Dept Microbiol, N-0424 Oslo, Norway.;NTNU, Inst Canc Res & Mol Med, N-7491 Trondheim, Norway..
    Granert, Carl
    Karolinska Univ Hosp, Clin Immunol Sect, Immunodeficiency Unit, S-14186 Stockholm, Sweden..
    Norlin, Anna-Carin
    Karolinska Univ Hosp, Clin Immunol Sect, Immunodeficiency Unit, S-14186 Stockholm, Sweden.;Karolinska Univ Hosp, Karolinska Univ Lab, Clin Immunol & Transfus Med, S-14186 Huddinge, Sweden..
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17121 Stockholm, Sweden..
    Christensson, Birger
    Karolinska Inst, Div Pathol, Dept Lab Med, S-14186 Stockholm, Sweden..
    Stenmark, Stephan
    Umea Univ, Dept Clin Microbiol, S-90187 Umea, Sweden..
    Smith, C. I. Edvard
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, Karolinska Univ Hosp, S-14186 Huddinge, Sweden.;Karolinska Univ Hosp, Clin Immunol Sect, Immunodeficiency Unit, S-14186 Stockholm, Sweden..
    Susceptibility to infections, without concomitant hyper-IgE, reported in 1976, is caused by hypomorphic mutation in the phosphoglucomutase 3 (PGM3) gene2015In: Clinical Immunology, ISSN 1521-6616, E-ISSN 1521-7035, Vol. 161, no 2, p. 366-372Article in journal (Refereed)
    Abstract [en]

    Phosphoglucomutase 3 (PGM3) is an enzyme converting N-acetyl-glucosamine-6-phosphate to N-acetylglucosamine-l-phosphate, a precursor important for glycosylation. Mutations in the PGM3 gene have recently been identified as the cause of novel primary immunodeficiency with a hyper-IgE like syndrome. Here we report the occurrence of a homozygous mutation in the PGM3 gene in a family with immunodeficient children, described already in 1976. DNA from two of the immunodeficient siblings was sequenced and shown to encode the same homozygous missense mutation, causing a destabilized protein with reduced enzymatic capacity. Affected individuals were highly prone to infections, but lack the developmental defects in the nervous and skeletal systems, reported in other families. Moreover, normal IgE levels were found. Thus, belonging to the expanding group of congenital glycosylation defects, PGM3 deficiency is characterized by immunodeficiency, with or without increased IgE levels, and with variable forms of developmental defects affecting other organ systems.

  • 128.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Applications of in situ proximity ligation assays for cancer research and diagnostics2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.

    In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.

    Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.

    In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.     

    Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.    

    A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry. 

     

    List of papers
    1. 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 protein
    Open this publication in new window or tab >>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 protein
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-300107 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-08-26Bibliographically approved
    2. Detergent resistant membranes from erythrocytes can form vesicles and be loaded for delivery of pharmaceutics
    Open this publication in new window or tab >>Detergent resistant membranes from erythrocytes can form vesicles and be loaded for delivery of pharmaceutics
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-300090 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-08-26
    3. Proximity-dependent initiation of hybridization chain reaction
    Open this publication in new window or tab >>Proximity-dependent initiation of hybridization chain reaction
    Show others...
    2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed) Published
    Abstract [en]

    Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

    National Category
    Medical and Health Sciences Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
    Funder
    EU, FP7, Seventh Framework Programme, 278568, 316929, 259796Swedish Foundation for Strategic Research Swedish Research Council
    Available from: 2015-06-17 Created: 2015-06-17 Last updated: 2018-01-25Bibliographically approved
    4. Flow Cytometric Measurement of BCR-ABL Fusion Protein Positive Cells in Blood from Patients with Chronic Myeloid Leukemia
    Open this publication in new window or tab >>Flow Cytometric Measurement of BCR-ABL Fusion Protein Positive Cells in Blood from Patients with Chronic Myeloid Leukemia
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences Clinical Laboratory Medicine
    Research subject
    Molecular Medicine; Pathology
    Identifiers
    urn:nbn:se:uu:diva-300087 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2019-04-02
    5. Detecting extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
    Open this publication in new window or tab >>Detecting extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-300089 (URN)
    Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2016-08-26
  • 129.
    Löf, Liza
    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.
    Ebai, Tonge
    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.
    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.
    Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout – ExoPLA: In Print2018In: Current Protocol in Cytometry, ISSN 9780471142959Article, review/survey (Other academic)
  • 130.
    Löf, Liza
    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.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Siart, Benjamin
    Jansson, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dahlin, Joakim S
    Thörn, Ingrid
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Christiansson, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hermansson, Monica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Ahlstrand, Erik
    Wålinder, Göran
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Rosenquist, Richard
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    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, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-9, article id 623Article in journal (Refereed)
    Abstract [en]

    Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

  • 131.
    Löf, Liza
    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.
    Ebai, Tonge
    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.
    Dubois, Louise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    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.
    Ronquist, K. Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Biochemial structure and function.
    Nolander, Olivia
    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.
    Lundin, Emma
    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.
    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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 34358Article in journal (Refereed)
    Abstract [en]

    Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.

  • 132.
    Löf, Liza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    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.
    FLOW CYTOMETRY-BASED ASSAY FOR DETECTION OF BCR-ABL FUSION PROTEIN IN BLOOD CELLS FROM CML PATIENTS2015In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 100, p. 694-695Article in journal (Other academic)
  • 133.
    Löf, Liza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Olsson-Strömberg, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Pla Flow; A Flow Cytometry-Based Assay For Detection Of Bcr-Abl Fusion Protein In Blood Cells From Cml Patients2016In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 457-458Article in journal (Other academic)
  • 134.
    Lönn, Peter
    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.
    Al-Amin, Rasel A.
    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.
    Heldin, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Gallini, Radiosa
    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.
    Björkesten, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Oelrich, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Science for Life Laboratory, SciLifeLab, 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. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    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.
    High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugsManuscript (preprint) (Other academic)
    Abstract [en]

    Interactions and posttranslational modifications (PTMs) of proteins orchestrate cellular responses to cytokines, drugs or other agents, but it has been difficult to monitor and characterize these dynamic events at high-throughput. Here, we have established a semi-automated system for large-scale in situ proximity ligation assays (isPLA). The protocol combines isPLA in microtiter wells with automated microscopy and computer-based image analysis whereby specific protein phosphorylations and interactions are digitally recorded in cells, along with measurements of morphological features. We demonstrate how this platform can improve analysis of cellular signaling by investigating TGF-b responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells. We depict single cell responses in relation to e.g. local cell crowding and cell cycle progression via measurements of DNA content and nuclear size. Finally, we illustrate the application of the protocol for demonstrating drug effects by screening a library of phosphatase inhibitors. In summary, our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.