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  • 1.
    Acharya, Shikha
    et al.
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Microbiol & Immunol, PO 450, S-40530 Gothenburg, Sweden.
    Jin, Chunsheng
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Med Biochem & Cell Biol, Gothenburg, Sweden.
    Bylund, Johan
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Microbiol & Immunol, PO 450, S-40530 Gothenburg, Sweden.
    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.
    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.
    Jontell, Mats
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Med & Pathol, Gothenburg, Sweden.
    Carlen, Anette
    Univ Gothenburg, Sahlgrenska Acad, Inst Odontol, Dept Oral Microbiol & Immunol, PO 450, S-40530 Gothenburg, Sweden.
    Karlsson, Niclas G.
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Med Biochem & Cell Biol, Gothenburg, Sweden.
    Reduced sialyl-Lewis(x) on salivary MUC7 from patients with burning mouth syndrome2019In: MOLECULAR OMICS, ISSN 2515-4184, Vol. 15, no 5, p. 331-339Article in journal (Refereed)
    Abstract [en]

    We analysed and compared MUC7 O-glycosylation and inflammatory biomarkers in saliva from female patients with burning mouth syndrome (BMS) and gender/age-matched controls. Oligosaccharides from salivary MUC7 from BMS and controls were released. Inflammatory mediators were measured by multiplex proximity extension assay. Presence of sialyl-Lewis(x) (Si-Le(x)) epitope on MUC7 was confirmed using Western blot. MUC7 O-glycans and measured inflammatory biomarkers were found to be similar between BMS and controls. However, oligosaccharides sialyl-Lewis(x) (Si-Le(x)) was found to be reduced in samples from BMS patients. Positive correlation (combined patients and controls) was found between levels of C-C motif chemokine 19 (CCL-19) and the amount of core-2 oligosaccharides on MUC7 as well as fractalkine (CX3CL1) and level of sialylation. Patients with BMS were shown to represent a heterogeneous group in terms of inflammatory biomarkers. This indicates that BMS patients could be further stratified on the basis of low-level inflammation. The results furthermore indicate that reduced sialylation of MUC7, particularly Si-Le(x), may be an important feature in patients with BMS. However, the functional aspects and potential involvement in immune regulation of Si-Le(x) remains unclear. Our data suggests a chemokine driven alteration of MUC7 glycosylation.

  • 2.
    Al-Amin, Abdullah
    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.
    Molecular Approaches to Explore Drug-Target Interactions2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Improved means to assess the clinical potential of drug candidates can critically influence development of new therapeutic entities, a central aim in medical life science. Drug discovery and development relies on construction and selection of small organic compounds or biological agents that bind targets of interest. This thesis includes new methodology to investigate target engagement - that is the tendency for these drugs and drug candidates to bind their intended target molecules versus any off-targets. This is a matter of great importance and current strong interest in the pharmaceutical industry as well as academically and an important aim for precision medicine. Paper I describes the target engagement-mediated amplification (TEMA) technique, an accurate, selective and physiological relevant techniques to monitor target binding by DNA-conjugated low molecular weight drug molecules. The DNA conjugated forms of the drugs are uniquely suited to accurately and sensitively reveal the binding characteristics of drugs directly in relevant tissues. Paper II describes the evaluation of cellular thermal shift assays (CETSA) by multiplex proximity extension assays (PEA), to sensitively measure binding of drugs to their proper targets and off-targets in minimal samples of cells and tissues, and for many targets and samples in parallel. The technique provides valuable advantages during drug development, and potentially also in clinical care. Paper III describes a high-throughput approach to use in situ proximity ligation assays to investigate protein interactions or modifications along with phenotypic responses to drugs or cytokines. The technique allows responses by large numbers of cells to be evaluated by automated microscopy and computer-based analysis. Our approach expands the scope for combined molecular and morphological profiling, offering an information-rich means to profile cellular responses to drugs and other agents at the single cell level.

    List of papers
    1. Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
    Open this publication in new window or tab >>Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
    Show others...
    (English)Manuscript (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. 

    National Category
    Natural Sciences
    Research subject
    Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-374262 (URN)
    Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
    2. Sensitive Measurement of Drug-Target Engagement Using Cellular Thermal Shift Assays with Multiplex Proximity Extension Assay Readout
    Open this publication in new window or tab >>Sensitive Measurement of Drug-Target Engagement Using Cellular Thermal Shift Assays with Multiplex Proximity Extension Assay Readout
    Show others...
    (English)Manuscript (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. 

    National Category
    Natural Sciences
    Research subject
    Medical Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-374264 (URN)
    Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
    3. High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs
    Open this publication in new window or tab >>High-throughput in situ mapping of phosphorylated protein complexes across the cell cycle and in response to drugs
    Show others...
    (English)Manuscript (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.

    National Category
    Natural Sciences
    Research subject
    Biochemistry; Molecular Cellbiology; Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-374248 (URN)
    Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
  • 3.
    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)
  • 4.
    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. 

  • 5.
    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. 

  • 6.
    Arngården, Linda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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

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

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

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

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

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

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

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

    National Category
    Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-279237 (URN)10.1084/jem.20142009 (DOI)000355569300001 ()25987724 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), b2011080Swedish Cancer SocietySwedish Research CouncilNIH (National Institute of Health), CA81554; CA081554EU, European Research Council, 259796EU, FP7, Seventh Framework Programme, 306242
    Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2018-01-10Bibliographically approved
    4. Crosstalk between WNT and Hippo signaling pathways is affected by cell density and loss of or mutated E-cadherin protein, associated to HDGC.
    Open this publication in new window or tab >>Crosstalk between WNT and Hippo signaling pathways is affected by cell density and loss of or mutated E-cadherin protein, associated to HDGC.
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Medical and Health Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-281714 (URN)
    Available from: 2016-03-29 Created: 2016-03-29 Last updated: 2016-05-12
  • 7.
    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)
  • 8. 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)
  • 9.
    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)
  • 10. 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.

  • 11.
    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)
  • 12. 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.

  • 13. 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.

  • 14.
    Bhandage, Amol K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Birnir: Molecular Physiology and Neuroscience.
    Cunningham, Janet L.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ekselius: Psychiatry.
    Jin, Zhe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Birnir: Molecular Physiology and Neuroscience.
    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.
    Bongiovanni, Santiago
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ekselius: Psychiatry.
    Korol, Sergiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Birnir: Molecular Physiology and Neuroscience.
    Syk, Mikaela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ekselius: Psychiatry.
    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.
    Ekselius, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ekselius: Psychiatry.
    Birnir, Bryndis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Birnir: Molecular Physiology and Neuroscience.
    Depression, GABA, and Age Correlate with Plasma Levels of Inflammatory Markers2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 24, article id 6172Article in journal (Refereed)
    Abstract [en]

    Immunomodulation is increasingly being recognised as a part of mental diseases. Here, we examined whether levels of immunological protein markers changed with depression, age, or the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). An analysis of plasma samples from patients with a major depressive episode and control blood donors (CBD) revealed the expression of 67 inflammatory markers. Thirteen of these markers displayed augmented levels in patients compared to CBD. Twenty-one markers correlated with the age of the patients, whereas 10 markers correlated with the age of CBD. Interestingly, CST5 and CDCP1 showed the strongest correlation with age in the patients and CBD, respectively. IL-18 was the only marker that correlated with the MADRS-S scores of the patients. Neuronal growth factors (NGFs) were significantly enhanced in plasma from the patients, as was the average plasma GABA concentration. GABA modulated the release of seven cytokines in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) from the patients. The study reveals significant changes in the plasma composition of small molecules during depression and identifies potential peripheral biomarkers of the disease.

  • 15.
    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.

  • 16.
    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.

  • 17.
    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, Molecular tools. Uppsala universitet.
    Dried blood sampling and digital readout to advance molecular diagnostics2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A drastically increased capacity to measure large sets of molecular features in numerous patient samples in great detail will be required to fulfill the vision of precision medicine and wellness, which may characterize molecular diagnostics in the 21st century. Also sampling procedures need a renaissance to permit continuous sampling at population levels at reasonable cost.

    Blood sampling is typically performed via venipuncture to draw several milliliters of blood for plasma isolation. This is inconvenient, time-consuming and costly, as well as hard to standardize. The effect on plasma protein profiles by pre-centrifugation delay was investigated in Paper II, demonstrating time- and temperature-dependent release of proteins from blood cells upon delayed plasma isolation, but almost no protein degradation as analyzed by two 92-plex protein panels (Olink® Proteomics). An alternative sampling method, where blood drops from a finger stick are collected dried on paper, is relatively non-invasive, potentially home-based and cheap. Dried blood spots can also be shipped via regular mail and compactly stored. The effect of drying and long term storage stability of a large set of proteins from dried blood spots was investigated in Paper I using Olink® technology. The main findings were that drying slightly but consistently influenced the recorded levels of blood proteins, and that long-term storage decreased the detected levels of some of the proteins with half-lives of decades.

    Some molecular diagnostic investigations require great accuracy to be useful, arguing for digital enumeration of individual molecules. Digital PCR is the gold standard but Paper III presents an alternative approach based on rolling circle amplification of single molecules. Another instance where extreme assay performance is required is for rare mutation detection from liquid biopsies. Paper V presents a new method offering essentially error-free genotyping of individual molecules by majority-vote decisions for counting rare mutant DNA in blood. Yet other diagnostic investigations require very simple assays. Paper IV presents a novel one-step method to detect nucleic acid sequences by combining the power of rolling circle amplification and the specificity of DNA strand displacement in a format simple enough to be used at the point of care.   

    Altogether, the thesis spans technologies for advanced molecular diagnostics, from sample collection over assay techniques to an improved readout.

    List of papers
    1. Stability of Proteins in Dried Blood Spot Biobanks.
    Open this publication in new window or tab >>Stability of Proteins in Dried Blood Spot Biobanks.
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    2017 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 7, p. 1286-1296Article in journal (Refereed) Published
    Abstract [en]

    An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. 92 proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4&deg;C or -24&deg;C.</p> <p>Our main findings were that 1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), 2) detection of some proteins was not significantly affected by storage over the full range of three decades (34% and 76% of the analyzed proteins at +4&deg;C and -24&deg;C, respectively), while levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and 3) detectability of proteins was less affected in dried samples stored at -24&deg;C compared to at +4&deg;C, as the median protein abundance had decreased to 80% and 93% of starting levels after 10 years of storage at +4&deg;C or -24&deg;C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.

    Keywords
    Absolute quantification, Affinity proteomics, Biobanking, Bioinformatics splicing, Biomarkers, Blood*, DBS, Diagnostic, Dried Blood Spot, Multiplex protein detection, PCR, Plasma or serum analysis, Predictive markers*, Protein Stability, Proximity Extension Assay
    National Category
    Clinical Laboratory Medicine
    Identifiers
    urn:nbn:se:uu:diva-322568 (URN)10.1074/mcp.RA117.000015 (DOI)000404597500009 ()28501802 (PubMedID)
    Funder
    Swedish Research CouncilEU, FP7, Seventh Framework Programme, 294409Novo Nordisk
    Available from: 2017-05-25 Created: 2017-05-25 Last updated: 2019-11-03Bibliographically approved
    2. Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
    Open this publication in new window or tab >>Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
    Show others...
    2018 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 56, no 4, p. 582-594Article in journal (Refereed) Published
    Abstract [en]

    Background: A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles.

    Methods: Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 degrees C or 22 degrees C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 degrees C and thawing at 22 degrees C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins.

    Results: Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 degrees C or 22 degrees C, respectively. Some increases became noticeable after 8 h delay at 4 degrees C but already after 1 h at 22 degrees C. For samples stored at 4 degrees C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 degrees C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 degrees C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles.

    Conclusions: Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

    Place, publisher, year, edition, pages
    WALTER DE GRUYTER GMBH, 2018
    Keywords
    biobank, protein detection, proteome, proximity extension assay (PEA), sample collection and handling
    National Category
    Clinical Laboratory Medicine
    Identifiers
    urn:nbn:se:uu:diva-350276 (URN)10.1515/cclm-2017-0648 (DOI)000426657400016 ()29040064 (PubMedID)
    Funder
    Swedish Research Council, 829-2009-6285EU, European Research Council, 313010, 294409
    Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2019-11-03Bibliographically approved
    3. Multiplex digital enumeration of circular DNA molecules on solid supports
    Open this publication in new window or tab >>Multiplex digital enumeration of circular DNA molecules on solid supports
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    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Digital PCR is a detection method with unprecedented accuracy for DNA quantification, but with some limitations in the form of complexity of instrumentation and limited multiplexing. Here we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy, to overcome limitations of digital PCR. In this method, sets of small DNA circles, resulting from detection reactions, are captured on a streptavidin-coated surface and subjected to rolling circle amplification (RCA). We found that the addition of 15% polyethylene glycol 4000 to RCA on planar surfaces ensured uniform, easily counted signals, each of which represents an individual reaction product. The DNA circles were immobilized and detected with efficiencies of 50 and 100%, respectively, as determined by droplet digital PCR. We confirmed previous reports about the effect on RCA efficiency by sequence composition and size of the RCA templates at the level of individual amplified molecules, and we developed an efficient one-step de- and re-staining procedure for sequential multiplexing via toehold-triggered DNA strand displacement.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-396324 (URN)
    Available from: 2019-11-03 Created: 2019-11-03 Last updated: 2019-11-03
    4. Rolling circle amplification reporters – a general tool to simplify molecular detections
    Open this publication in new window or tab >>Rolling circle amplification reporters – a general tool to simplify molecular detections
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Methods to detect biomolecules via rolling circle amplification (RCA), for example padlock probes and proximity ligation assay (PLA) tend to be complex and include several reaction steps. Herein, we evaluated a new tool for the toolbox of RCA-based detection methods - RCA Reporters. Briefly, RCA Reporters represent inert DNA structures that, upon contact with a specific nucleic acid sequence, unravel via a highly specific strand displacement process to initiate local enzyme-assisted RCA reactions. The RCA Reporters can be used to directly detect ssDNA or RNA in a sample, or proteins via oligonucleotide-conjugated antibodies. The reagents can also enable faster RCA reactions or extremely selective genotyping of RCA products with repeated copies of a target sequence through a majority-vote mechanism. Further amplification of ongoing RCA reactions via RCA Reporters can allow efficient digital enumeration of single molecules via flow cytometry, with potential for simple and highly accurate molecular counting assays. The intrinsic simplicity of RCA Reporter also renders them attractive for applications at the point of care.

    National Category
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-331743 (URN)
    Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-11-03
    5. Rare Mutation Detection in Blood Plasma Using sRCA Molecule Counting Probes
    Open this publication in new window or tab >>Rare Mutation Detection in Blood Plasma Using sRCA Molecule Counting Probes
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Problems in biology and medicine frequently require the ability to observe, evaluate, andcount even extremely rare macromolecules in biological samples. In particular, rare tumorspecific mutations in plasma provide valuable insights in the course of malignant disease andresponses to therapy, but simpler assay techniques are needed. We describe herein a rapidand exquisitely specific means to recognize and magnify detection signals from individualmolecules to easily recorded levels via a process we call super rolling circle amplification(sRCA). We demonstrate the ability of this technique to enumerate tumor-specific sequencevariants in plasma from cancer patients via flow cytometry at very high efficiency, with specificityadequate to detect single nucleotide mutant sequences among 100,000 copies of thenormal sequence in a 3 hr protocol. And the mutation analysis data generated from patientctDNA samples with our sRCA method are in high accordance with the patients’ primary tumorsequencing data.

    Keywords
    Rolling circle amplification, cfDNA, single molecule, digital counting, rare mutation detection, PoC application
    National Category
    Genetics
    Identifiers
    urn:nbn:se:uu:diva-331737 (URN)
    Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2019-11-03
  • 18.
    Björkesten, Johan
    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. Uppsala universitet.
    Patil, Sourabh
    Fredolini, Claudia
    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.
    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.
    Multiplex digital enumeration of circular DNA molecules on solid supportsManuscript (preprint) (Other academic)
    Abstract [en]

    Digital PCR is a detection method with unprecedented accuracy for DNA quantification, but with some limitations in the form of complexity of instrumentation and limited multiplexing. Here we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy, to overcome limitations of digital PCR. In this method, sets of small DNA circles, resulting from detection reactions, are captured on a streptavidin-coated surface and subjected to rolling circle amplification (RCA). We found that the addition of 15% polyethylene glycol 4000 to RCA on planar surfaces ensured uniform, easily counted signals, each of which represents an individual reaction product. The DNA circles were immobilized and detected with efficiencies of 50 and 100%, respectively, as determined by droplet digital PCR. We confirmed previous reports about the effect on RCA efficiency by sequence composition and size of the RCA templates at the level of individual amplified molecules, and we developed an efficient one-step de- and re-staining procedure for sequential multiplexing via toehold-triggered DNA strand displacement.

  • 19.
    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 se