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  • 201. Clevers, Hans
    et al.
    Rafelski, Susanne
    Elowitz, Michael
    Klein, Allon
    Shendure, Jay
    Trapnell, Cole
    Lein, Ed
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Matthias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Martinez-Arias, Alfonso
    Sanes, Joshua R.
    Blainey, Paul
    Eberwine, James
    Kim, Junhyong
    Love, J. Christopher
    What Is Your Conceptual Definition of "Cell Type'' in the Context of a Mature Organism?2017In: CELL SYSTEMS, ISSN 2405-4712, Vol. 4, no 3, p. 255-259Article in journal (Refereed)
  • 202. Cossu, Rosa Maria
    et al.
    Casola, Claudio
    Giacomello, Stefania
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vidalis, Amaryllis
    Scofield, Douglas G.
    Zuccolo, Andrea
    LTR Retrotransposons Show Low Levels of Unequal Recombination and High Rates of Intraelement Gene Conversion in Large Plant Genomes2017In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 9, no 12, p. 3449-3462Article in journal (Refereed)
    Abstract [en]

    The accumulat on and removal of transposable elements (TEs) is a major driver of genome size evolution in eukaryotes. In plants, long terminal repeat (LTR) retrotransposons (LTR-RTs) represent the majority of TEs and form most of the nuclear DNA in large genomes. Unequal recombination (UR) between LTRs leads to removal of intervening sequence and formation of solo-LTRs. UR is a major mechanism of LTR-RT removal in many angiosperms, but our understanding of LTR-RT-associated recombination within the large, LTR-RT-rich genomes of conifers is quite limited. We employ a novel read based methodology to estimate the relative rates of LTR-RT-associated UR within the genomes of four conifer and seven angiosperm species. We found the lowest rates of UR in the largest genomes studied, conifers and the angiosperm maize. Recombination may also resolve as gene conversion, which does not remove sequence, so we analyzed LTR-RT-associated gene conversion events (GCEs) in Norway spruce and six angiosperms. Opposite the trend for UR, we found the highest rates of GCEs in Norway spruce and maize. Unlike previous work in angiosperms, we found no evidence that rates of UR correlate with retroelement structural features in the conifers, suggesting that another process is suppressing UR in these species. Recent results from diverse eukaryotes indicate that heterochromatin affects the resolution of recombination, by favoring gene conversion over crossing-over, similar to our observation of opposed rates of UR and GCEs. Control of LTR-RT proliferation via formation of heterochromatin would be a likely step toward large genomes in eukaryotes carrying high LTR-RT content.

  • 203. Costa, H.
    et al.
    Xu, X.
    Overbeek, G.
    Vasaikar, S.
    Pawan K. Patro, C.
    Kostopoulou, O. N.
    Jung, M.
    Shafi, G.
    Ananthaseshan, S.
    Tsipras, G.
    Davoudi, B.
    Mohammad, A. -A
    Lam, H.
    Strååt, Klas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Wilhelmi, V.
    Shang, M.
    Tegner, J.
    Tong, J. C.
    Wong, K. T.
    Söderberg-Naucler, C.
    Yaiw, K. -C
    Human cytomegalovirus may promote tumour progression by upregulating arginase-22016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 30, p. 47221-47231Article in journal (Refereed)
    Abstract [en]

    Background: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. Results: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. Methods: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2- overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). Conclusions: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.

  • 204.
    Costea, Paul Igor
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    TagGD: Fast and Accurate Software for DNA Tag Generation and Demultiplexing2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, p. e57521-Article in journal (Refereed)
    Abstract [en]

    Multiplexing is of vital importance for utilizing the full potential of next generation sequencing technologies. We here report TagGD (DNA-based Tag Generator and Demultiplexor), a fully-customisable, fast and accurate software package that can generate thousands of barcodes satisfying user-defined constraints and can guarantee full demultiplexing accuracy. The barcodes are designed to minimise their interference with the experiment. Insertion, deletion and substitution events are considered when designing and demultiplexing barcodes. 20,000 barcodes of length 18 were designed in 5 minutes and 2 million barcoded Illumina HiSeq-like reads generated with an error rate of 2% were demultiplexed with full accuracy in 5 minutes. We believe that our software meets a central demand in the current high-throughput biology and can be utilised in any field with ample sample abundance. The software is available on GitHub (https://github.com/pelinakan/UBD.git).

  • 205.
    Costeira-Paulo, Joana
    et al.
    Uppsala Univ, Dept Chem BMC, Box 576, S-75123 Uppsala, Sweden..
    Gault, Joseph
    Univ Oxford, Dept Chem, Phys & Theoret Chem Lab, South Parks Rd, Oxford OX1 3QZ, England..
    Popova, Gergana
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    Ladds, Marcus J. G. W.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    van Leeuwen, Ingeborg M. M.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    Sarr, Medoune
    Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc NVS, Div Neurogeriatr,Dept Neurobiol, S-14157 Huddinge, Sweden..
    Olsson, Anders
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lane, David P.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Lain, Sonia
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Marklund, Erik G.
    Uppsala Univ, Dept Chem BMC, Box 576, S-75123 Uppsala, Sweden..
    Landreh, Michael
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase2018In: Cell Chemical Biology, ISSN 2451-9456, E-ISSN 2451-9448, Vol. 25, no 3, p. 309-+Article in journal (Refereed)
    Abstract [en]

    The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among cofactor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

  • 206. Daniel, C.
    et al.
    Lagergren, Jens
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Öhman, M.
    RNA editing of non-coding RNA and its role in gene regulation2015In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 117, p. 22-27Article in journal (Refereed)
    Abstract [en]

    It has for a long time been known that repetitive elements, particularly Alu sequences in human, are edited by the adenosine deaminases acting on RNA, ADAR, family. The functional interpretation of these events has been even more difficult than that of editing events in coding sequences, but today there is an emerging understanding of their downstream effects. A surprisingly large fraction of the human transcriptome contains inverted Alu repeats, often forming long double stranded structures in RNA transcripts, typically occurring in introns and UTRs of protein coding genes. Alu repeats are also common in other primates, and similar inverted repeats can frequently be found in non-primates, although the latter are less prone to duplex formation. In human, as many as 700,000 Alu elements have been identified as substrates for RNA editing, of which many are edited at several sites. In fact, recent advancements in transcriptome sequencing techniques and bioinformatics have revealed that the human editome comprises at least a hundred million adenosine to inosine (A-to-I) editing sites in Alu sequences. Although substantial additional efforts are required in order to map the editome, already present knowledge provides an excellent starting point for studying cis-regulation of editing. In this review, we will focus on editing of long stem loop structures in the human transcriptome and how it can effect gene expression.

  • 207. Danielsson, Angelika
    et al.
    Pontén, Fredrik
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Korsgren, Olle
    Lindskog, Cecilia
    The Human Pancreas Proteome Defined by Transcriptomics and Antibody-Based Profiling2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, p. e115421-Article in journal (Refereed)
    Abstract [en]

    The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects.

  • 208.
    Danielsson, Frida
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fasterius, Erik
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Sullivan, Devin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hases, Linnea
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute, Huddinge, Sweden.
    Sanli, Kemal
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Huss, M.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Hørsholm, Denmark.
    Williams, Cecilia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute, Huddinge, Sweden.
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Transcriptome profiling of the interconnection of pathways involved in malignant transformation and response to hypoxia2018In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 9, no 28, p. 19730-19744Article in journal (Refereed)
    Abstract [en]

    In tumor tissues, hypoxia is a commonly observed feature resulting from rapidly proliferating cancer cells outgrowing their surrounding vasculature network. Transformed cancer cells are known to exhibit phenotypic alterations, enabling continuous proliferation despite a limited oxygen supply. The four-step isogenic BJ cell model enables studies of defined steps of tumorigenesis: the normal, immortalized, transformed, and metastasizing stages. By transcriptome profiling under atmospheric and moderate hypoxic (3% O2) conditions, we observed that despite being highly similar, the four cell lines of the BJ model responded strikingly different to hypoxia. Besides corroborating many of the known responses to hypoxia, we demonstrate that the transcriptome adaptation to moderate hypoxia resembles the process of malignant transformation. The transformed cells displayed a distinct capability of metabolic switching, reflected in reversed gene expression patterns for several genes involved in oxidative phosphorylation and glycolytic pathways. By profiling the stage-specific responses to hypoxia, we identified ASS1 as a potential prognostic marker in hypoxic tumors. This study demonstrates the usefulness of the BJ cell model for highlighting the interconnection of pathways involved in malignant transformation and hypoxic response.

  • 209.
    Danielsson, Frida
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlen, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gad, A. K.
    Profiling the Molecular changes during malignant transformation and response to different oxygen levels, using a combined transcriptomics and proteomics approach2014In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 25, article id P1845Article in journal (Other academic)
  • 210.
    Danielsson, Frida
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Huss, Mikael
    Rexhepaj, Elton
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    O'Hurley, Gillian
    Klevebring, Daniel
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, Fredrik
    Gad, Annica K. B.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 17, p. 6853-6858Article in journal (Refereed)
    Abstract [en]

    The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.

  • 211.
    Danielsson, Frida
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Wiking, Mikaela
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mahdessian, Diana
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Skogs, Marie
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ait Blal, Hammou
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hjelmare, Martin
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stadler, Charlotte
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    RNA Deep Sequencing as a Tool for Selection of Cell Lines for Systematic Subcellular Localization of All Human Proteins2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 1, p. 231-239Article in journal (Refereed)
    Abstract [en]

    One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.

  • 212. Darmanis, Spyros
    et al.
    Cui, Tao
    Drobin, Kimi
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Li, Su-Chen
    Öberg, Kjell
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Giandomenico, Valeria
    Identification of Candidate Serum Proteins for Classifying Well-Differentiated Small Intestinal Neuroendocrine Tumors2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. e81712-Article in journal (Refereed)
    Abstract [en]

    Background: Patients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NETs) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required. Materials and Methods: Suspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases. Results: A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1 alpha, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification. Conclusions: We propose new potential protein biomarker candidates for classifying WD-SI-NETs at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NETs and their eventual use in diagnostics.

  • 213.
    Das, Sarbashis
    et al.
    Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, S-75124 Uppsala, Sweden..
    Frisk, Christoffer
    Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, S-75124 Uppsala, Sweden..
    Eriksson, Maria J.
    Karolinska Univ Hosp, Dept Clin Physiol, S-17176 Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, S-17177 Stockholm, Sweden..
    Walentinsson, Anna
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab Dis, S-43183 Gothenburg, Sweden..
    Corbascio, Matthias
    Karolinska Inst, Dept Mol Med & Surg, S-17177 Stockholm, Sweden.;Karolinska Univ Hosp, Dept Thorac Surg, S-17176 Stockholm, Sweden..
    Hage, Camilla
    Karolinska Inst, Dept Med, S-17177 Stockholm, Sweden.;Karolinska Univ Hosp, Heart & VascularTheme, S-17176 Stockholm, Sweden..
    Kumar, Chanchal
    AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab Dis, S-43183 Gothenburg, Sweden.;Karolinska Inst, ICMC, Dept Med, S-14157 Huddinge, Sweden..
    Asp, Michaela
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Maret, Eva
    Karolinska Univ Hosp, Dept Clin Physiol, S-17176 Stockholm, Sweden.;Karolinska Inst, Dept Mol Med & Surg, S-17177 Stockholm, Sweden..
    Persson, Hans
    Karolinska Inst, Danderyd Hosp, Dept Clin Sci, S-18288 Stockholm, Sweden.;Danderyd Hosp, Dept Cardiol, S-18288 Stockholm, Sweden..
    Linde, Cecilia
    Karolinska Inst, Dept Med, S-17177 Stockholm, Sweden.;Karolinska Univ Hosp, Heart & VascularTheme, S-17176 Stockholm, Sweden..
    Persson, Bengt
    Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, S-75124 Uppsala, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, S-17177 Stockholm, Sweden..
    Transcriptomics of cardiac biopsies reveals differences in patients with or without diagnostic parameters for heart failure with preserved ejection fraction2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 3179Article in journal (Refereed)
    Abstract [en]

    Heart failure affects 2-3% of adult Western population. Prevalence of heart failure with preserved left ventricular (LV) ejection fraction (HFpEF) increases. Studies suggest HFpEF patients to have altered myocardial structure and functional changes such as incomplete relaxation and increased cardiac stiffness. We hypothesised that patients undergoing elective coronary bypass surgery (CABG) with HFpEF characteristics would show distinctive gene expression compared to patients with normal LV physiology. Myocardial biopsies for mRNA expression analysis were obtained from sixteen patients with LV ejection fraction >= 45%. Five out of 16 patients (31%) had echocardiographic characteristics and increased NTproBNP levels indicative of HFpEF and this group was used as HFpEF proxy, while 11 patients had Normal LV physiology. Utilising principal component analysis, the gene expression data clustered into two groups, corresponding to HFpEF proxy and Normal physiology, and 743 differentially expressed genes were identified. The associated top biological functions were cardiac muscle contraction, oxidative phosphorylation, cellular remodelling and matrix organisation. Our results also indicate that upstream regulatory events, including inhibition of transcription factors STAT4, SRF and TP53, and activation of transcription repressors HEY2 and KDM5A, could provide explanatory mechanisms to observed gene expression differences and ultimately cardiac dysfunction in the HFpEF proxy group.

  • 214. Davanian, H.
    et al.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Båge, T.
    Lagervall, M.
    Jansson, L.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Yucel-Lindberg, T.
    Gene Expression Profiles in Paired Gingival Biopsies from Periodontitis-Affected and Healthy Tissues Revealed by Massively Parallel Sequencing2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, p. e46440-Article in journal (Refereed)
    Abstract [en]

    Periodontitis is a chronic inflammatory disease affecting the soft tissue and bone that surrounds the teeth. Despite extensive research, distinctive genes responsible for the disease have not been identified. The objective of this study was to elucidate transcriptome changes in periodontitis, by investigating gene expression profiles in gingival tissue obtained from periodontitis-affected and healthy gingiva from the same patient, using RNA-sequencing. Gingival biopsies were obtained from a disease-affected and a healthy site from each of 10 individuals diagnosed with periodontitis. Enrichment analysis performed among uniquely expressed genes for the periodontitis-affected and healthy tissues revealed several regulated pathways indicative of inflammation for the periodontitis-affected condition. Hierarchical clustering of the sequenced biopsies demonstrated clustering according to the degree of inflammation, as observed histologically in the biopsies, rather than clustering at the individual level. Among the top 50 upregulated genes in periodontitis-affected tissues, we investigated two genes which have not previously been demonstrated to be involved in periodontitis. These included interferon regulatory factor 4 and chemokine (C-C motif) ligand 18, which were also expressed at the protein level in gingival biopsies from patients with periodontitis. In conclusion, this study provides a first step towards a quantitative comprehensive insight into the transcriptome changes in periodontitis. We demonstrate for the first time site-specific local variation in gene expression profiles of periodontitis-affected and healthy tissues obtained from patients with periodontitis, using RNA-seq. Further, we have identified novel genes expressed in periodontitis tissues, which may constitute potential therapeutic targets for future treatment strategies of periodontitis.

  • 215. Davanian, Haleh
    et al.
    Båge, Tove
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Concha, Hernan Q.
    Chen, Margaret Sallberg
    Yucel-Lindberg, Tulay
    Signaling pathways involved in the regulation of TNF alpha-induced toll-like receptor 2 expression in human gingival fibroblasts2012In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 57, no 3, p. 406-416Article in journal (Refereed)
    Abstract [en]

    Periodontitis is a chronic inflammatory disease characterized by a host inflammatory response against bacteria that leads to destruction of the supporting structures of the teeth. Bacterial components of pathogens in the periodontal pocket are recognized by toll-like receptors (TLRs) that trigger an inflammatory response. In this study, we investigated the effects of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha) on TLR2 expression in human gingival fibroblasts. In addition, we examined the signaling pathways involved in the regulation of TNF alpha-induced TLR2 expression. Our results showed that TNF alpha increased TLR2 mRNA and protein expression. Microarray analysis and the inhibition of specific signaling pathways demonstrated that c-Jun N-terminal kinases (JNK) and nuclear factor kappa B (NF-kappa B) were involved in the regulation of INF alpha-induced TLR2 expression in gingival fibroblasts. Furthermore, the prostaglandin E-2 (PGE(2)) regulatory enzyme cytosolic phospholipase A(2) (cPLA(2)) and the anti-inflammatory prostaglandin 15-deoxy-Delta(12.14)-prostaglandin J(2) (15d-PGJ(2)), were found to regulate TLR2 mRNA expression stimulated by TNF alpha. Our findings suggest that these pathways and mediators, through the regulation of TLR2 expression in gingival fibroblasts, may be involved in the pathogenesis of periodontitis. The study provides new insights into the molecular mechanisms underlying the regulation of TLR2, implicated in the chronic inflammatory disease periodontitis.

  • 216.
    Davidsson, Marcus
    et al.
    Lund Univ, Mol Neuromodulat, Lund, Sweden..
    Aldrin-Kirk, Patrick
    Lund Univ, Mol Neuromodulat, Lund, Sweden..
    Cardoso, Tiago
    Lund Univ, Dev & Regenerat Neurobiol, Lund, Sweden..
    Hartnor, Morgan
    Lund Univ, Mol Neuromodulat, Lund, Sweden..
    Heuer, Andreas
    Lund Univ, Mol Neuromodulat, Lund, Sweden..
    Mollbrink, Annelie
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Parmar, Malin
    Lund Univ, Dev & Regenerat Neurobiol, Lund, Sweden..
    Bjorklund, Tomas
    Lund Univ, Mol Neuromodulat, Lund, Sweden..
    Mapping the Connectome Using Novel AAV Vectors, DNA Barcoding and Spatial Transcriptomics2018In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 26, no 5, p. 319-319Article in journal (Other academic)
  • 217. Dawed, A. Y.
    et al.
    Mari, A.
    McDonald, T. J.
    Hong, Mun-Gwan
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sharma, S.
    Robertson, N. R.
    Mahajan, A.
    Walker, M.
    Gough, S.
    Zhou, K.
    Forgie, I.
    Ruetten, H.
    Jones, A. G.
    Pearson, E. R.
    GLP-1 receptor variants markedly differentiate glycaemic response to GLP-1 receptor agonists: a DIRECT study2017In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 60, p. S393-S393Article in journal (Refereed)
  • 218.
    Dawed, A. Y.
    et al.
    Univ Dundee, Mol & Clin Med, Dundee, Scotland..
    Mari, A.
    CNR, Inst Neurosci, Padua, Italy..
    McDonald, T. J.
    Royal Devon & Exeter Hosp, NIHR Exeter Clin Res Facil, Exeter, Devon, England..
    Hong, Mun-Gwan
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Sharma, S.
    Helmholtz Zentrum Muenchen, Inst Epidemiol 2, Res Unit Mol Epidemiol, Munich, Germany..
    Robertson, N. R.
    Univ Oxford, Wellcome Trust Ctr Human Genet, Oxford, England..
    Mahajan, A.
    Univ Oxford, Wellcome Trust Ctr Human Genet, Oxford, England..
    Walker, M.
    Newcastle Univ, Inst Cellular Med, Newcastle Upon Tyne, Tyne & Wear, England..
    Gough, S.
    NIHR Oxford Biomed Res Ctr, Oxford Ctr Diabet Endocrinol & Metab, Oxford, England..
    Zhou, K.
    Univ Dundee, Mol & Clin Med, Dundee, Scotland..
    Forgie, I
    Univ Dundee, Mol & Clin Med, Dundee, Scotland..
    Ruetten, H.
    Sanofi Aventis Deutschland GmbH, TMED, Frankfurt, Germany..
    Jones, A. G.
    Royal Devon & Exeter Hosp, NIHR Exeter Clin Res Facil, Exeter, Devon, England..
    Pearson, E. R.
    Univ Dundee, Mol & Clin Med, Dundee, Scotland..
    GLP-1 RECEPTOR VARIANTS MARKEDLY DIFFERENTIATE GLYCAEMIC RESPONSE TO GLP-1 RECEPTOR AGONISTS: A DIRECT STUDY2018In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 123, p. 13-14Article in journal (Other academic)
  • 219.
    de Melo, Fernando Menegatti
    et al.
    Univ Sao Paulo, Supramol Nanotech Lab, Dept Chem, BR-05508000 Sao Paulo, SP, Brazil..
    Grasseschi, Daniel
    Univ Prebiteriana Mackenzie, Machg Graphene & Nanomat Res Ctr, BR-01303907 Sao Paulo, SP, Brazil..
    Brandao, Bruno B. N. S.
    Univ Sao Paulo, Supramol Nanotech Lab, Dept Chem, BR-05508000 Sao Paulo, SP, Brazil..
    Fu, Ying
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Toma, Henrique E.
    Univ Sao Paulo, Supramol Nanotech Lab, Dept Chem, BR-05508000 Sao Paulo, SP, Brazil..
    Superparamagnetic Maghemite-Based CdTe Quantum Dots as Efficient Hybrid Nanoprobes for Water-Bath Magnetic Particle Inspection2018In: ACS Applied Nano Materials, ISSN 2574-0970, Vol. 1, no 6, p. 2858-2868Article in journal (Refereed)
    Abstract [en]

    Fluorescent water-based cadmium telluride quantum dots (QDs) and citrate-functionalized maghemite nanoparticles (MghNPs) were synthesized and assembled together (MghNPs@QDs) through electrostatic interactions by using cetyltrimethylammonium bromide (CTAB) as a linker and steric spacer to minimize the Forster resonance energy transfer (FRET) restriction. A whole family of hybrid and multifunctional nanoparticles has been successfully obtained, exhibiting good performance in nondestructive water-bath magnetic particle inspection (MPI) assays.

  • 220.
    Delemotte, Lucie
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Opening leads to closing: Allosteric crosstalk between the activation and inactivation gates in KcsA2018In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 215, no 10, p. 1356-1359Article in journal (Refereed)
  • 221. Dengjel, Joern
    et al.
    Hoyer-Hansen, Maria
    Nielsen, Maria O.
    Eisenberg, Tobias
    Harder, Lea M.
    Schandorff, Soren
    Farkas, Thomas
    Kirkegaard, Thomas
    Becker, Andrea C.
    Schroeder, Sabrina
    Vanselow, Katja
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Mogens M.
    Kristensen, Anders R.
    Akimov, Vyacheslav
    Bunkenborg, Jakob
    Madeo, Frank
    Jaattela, Marja
    Andersen, Jens S.
    Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome- associated proteins were dependent on stimulus, but a core set of proteins was stimulus- independent. Remarkably, proteasomal proteins were abundant among the stimulus- independent common autophagosome- associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome- associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

  • 222.
    Deutsch, Eric W.
    et al.
    Inst Syst Biol, Seattle, WA 98109 USA..
    Perez-Riverol, Yasset
    European Bioinformat Inst, European Mol Biol Lab, Wellcome Trust Genome Campus, Cambridge CB10 1SD, England..
    Chalkley, Robert J.
    Univ Calif San Francisco, San Francisco, CA 94158 USA..
    Wilhelm, Mathias
    Tech Univ Munich, Prote & Bioanalyt, D-85354 Freising Weihenstephan, Germany..
    Tate, Stephen
    SCIEX Ltd, Concord, ON L4K4 V8, Canada..
    Sachsenberg, Timo
    Univ Tubingen, Ctr Bioinformat, Dept Comp Sci, Sand 14, D-72076 Tubingen, Germany..
    Walzer, Mathias
    European Bioinformat Inst, European Mol Biol Lab, Wellcome Trust Genome Campus, Cambridge CB10 1SD, England..
    Käll, Lukas
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Delanghe, Bernard
    Thermo Fisher Sci Bremen, Hanna Kunath Str 11, D-28199 Bremen, Germany..
    Boecker, Sebastian
    Friedrich Schiller Univ Jena, Bioinformat, D-07743 Jena, Germany..
    Schymanski, Emma L.
    Univ Luxembourg, Luxembourg Ctr Syst Biomed, 6 Ave Swing, L-4367 Belvaux, Luxembourg..
    Wilmes, Paul
    Univ Luxembourg, Luxembourg Ctr Syst Biomed, 6 Ave Swing, L-4367 Belvaux, Luxembourg..
    Dorfer, Viktoria
    Univ Appl Sci Upper Austria, Bioinformat Res Grp, A-4232 Hagenberg, Austria..
    Kuster, Bernhard
    Tech Univ Munich, Prote & Bioanalyt, D-85354 Freising Weihenstephan, Germany.;Tech Univ Munich, Bavarian Biomol Mass Spectrometry Ctr, D-85354 Freising Weihenstephan, Germany..
    Volders, Pieter-Jan
    Univ Gent VIB, Ctr Med Biotechnol, B-9000 Ghent, Belgium..
    Jehmlich, Nico
    UFZ Helmholtz Ctr Environm Res, D-04318 Leipzig, Germany..
    Vissers, Johannes P. C.
    Waters Corp, Wilmslow SK9 4AX, Cheshire, England..
    Wolan, Dennis W.
    Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA..
    Wang, Ana Y.
    Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA..
    Mendoza, Luis
    Inst Syst Biol, Seattle, WA 98109 USA..
    Shofstahl, Jim
    Thermo Fisher Sci, 355 River Oaks Pkwy, San Jose, CA 95134 USA..
    Dowsey, Andrew W.
    Univ Bristol, Dept Populat Hlth Sci, Fac Hlth Sci, Bristol BS9 1BN, Avon, England.;Univ Bristol, Bristol Vet Sch, Fac Hlth Sci, Bristol BS9 1BN, Avon, England..
    Griss, Johannes
    Med Univ Vienna, Div Immunol Allergy & Infect Dis, Dept Dermatol, Wahringer Gurtel 18-20, A-1090 Vienna, Austria..
    Salek, Reza M.
    Int Agcy Res Canc, 150 Cours Albert Thomas, F-69372 Lyon 08, France..
    Neumann, Steffen
    Leibniz Inst Plant Biochem, Dept Stress & Dev Biol, D-06120 Halle, Germany.;German Ctr Integrat Biodivers Res IDiv, D-04103 Leipzig, Germany..
    Binz, Pierre-Alain
    CHU Vaudois, Clin Chem Serv, CH-1011 Lausanne, Switzerland..
    Lam, Henry
    Hong Kong Univ Sci & Technol, Dept Chem & Biol Engn, Clear Water Bay, Hong Kong 999077, Peoples R China..
    Vizcaino, Juan Antonio
    European Bioinformat Inst, European Mol Biol Lab, Wellcome Trust Genome Campus, Cambridge CB10 1SD, England..
    Bandeira, Nuno
    Univ Calif San Diego, Skaggs Sch Pharm & Pharmaceut Sci, Dept Comp Sci & Engn, Ctr Computat Mass Spectrometry, San Diego, CA 92093 USA..
    Rost, Hannes
    Univ Toronto, Donnelly Ctr, 160 Coll St, Toronto, ON M5S 3E1, Canada..
    Expanding the Use of Spectral Libraries in Proteomics2018In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 17, no 12, p. 4051-4060Article in journal (Refereed)
    Abstract [en]

    The 2017 Dagstuhl Seminar on Computational Proteomics provided an opportunity for a broad discussion on ABSTRACT: The 2017 Dagstuhl Seminar on Computational the current state and future directions of the generation and use of peptide tandem mass spectrometry spectral libraries. Their use in proteomics is growing slowly, but there are multiple challenges in the field that must be addressed to further increase the adoption of spectral libraries and related techniques. The primary bottlenecks are the paucity of high quality and comprehensive libraries and the general difficulty of adopting spectral library searching into existing workflows. There are several existing spectral library formats, but none captures a satisfactory level of metadata; therefore, a logical next improvement is to design a more advanced, Proteomics Standards Initiative-approved spectral library format that can encode all of the desired metadata. The group discussed a series of metadata requirements organized into three designations of completeness or quality, tentatively dubbed bronze, silver, and gold. The metadata can be organized at four different levels of granularity: at the collection (library) level, at the individual entry (peptide ion) level, at the peak (fragment ion) level, and at the peak annotation level. Strategies for encoding mass modifications in a consistent manner and the requirement for encoding high-quality and commonly seen but as-yet-unidentified spectra were discussed. The group also discussed related topics, including strategies for comparing two spectra, techniques for generating representative spectra for a library, approaches for selection of optimal signature ions for targeted workflows, and issues surrounding the merging of two or more libraries into one. We present here a review of this field and the challenges that the community must address in order to accelerate the adoption of spectral libraries in routine analysis of proteomics datasets.

  • 223. Dey, Prasenjit
    et al.
    Velazquez-Villegas, Laura A.
    Faria, Michelle
    Turner, Anthony
    Jonsson, Philp
    Webb, Paul
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. University of Houston, United States.
    Gustafsson, Jan-Åke
    Ström, Anders M.
    Estrogen Receptor beta 2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1 alpha in Prostate Cancer2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 5, article id e0128239Article in journal (Refereed)
    Abstract [en]

    The estrogen receptor (ER) beta variant ER beta 2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ER beta 2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ER beta 2 interacts with and stabilizes HIF-1 alpha protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1 alpha is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ER beta 2 interacts with HIF-1 alpha and piggybacks to the HIF-1 alpha response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ER beta 2 is mediated by HIF-1 alpha and that targeting of this ER beta 2 -HIF-1 alpha interaction may be a strategy to treat prostate cancer.

  • 224.
    Dezfouli, Mahya
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Barcoded DNA Sequencing for Parallel Protein Detection2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The work presented in this thesis describes methodologies developed for integration and accurate interpretation of barcoded DNA, to empower large-scale-omics analysis. The objectives mainly aim at enabling multiplexed proteomic measurements in high-throughput format through DNA barcoding and massive parallel sequencing. The thesis is based on four scientific papers that focus on three main criteria; (i) to prepare reagents for large-scale affinity-proteomics, (ii) to present technical advances in barcoding systems for parallel protein detection, and (iii) address challenges in complex sequencing data analysis.

    In the first part, bio-conjugation of antibodies is assessed at significantly downscaled reagent quantities. This allows for selection of affinity binders without restrictions to accessibility in large amounts and purity from amine-containing buffers or stabilizer materials (Paper I). This is followed by DNA barcoding of antibodies using minimal reagent quantities. The procedure additionally enables efficient purification of barcoded antibodies from free remaining DNA residues to improve sensitivity and accuracy of the subsequent measurements (Paper II). By utilizing a solid-phase approach on magnetic beads, a high-throughput set-up is ready to be facilitated by automation. Subsequently, the applicability of prepared bio-conjugates for parallel protein detection is demonstrated in different types of standard immunoassays (Papers I and II).

    As the second part, the method immuno-sequencing (I-Seq) is presented for DNAmediated protein detection using barcoded antibodies. I-Seq achieved the detection of clinically relevant proteins in human blood plasma by parallel DNA readout (Paper II). The methodology is further developed to track antibody-antigen interaction events on suspension bead arrays, while being encapsulated in barcoded emulsion droplets (Paper III). The method, denoted compartmentalized immuno-sequencing (cI-Seq), is potent to perform specific detections with paired antibodies and can provide information on details of joint recognition events.

    Recent progress in technical developments of DNA sequencing has increased the interest in large-scale studies to analyze higher number of samples in parallel. The third part of this thesis focuses on addressing challenges of large-scale sequencing analysis. Decoding of a huge DNA-barcoded data is presented, aiming at phase-defined sequence investigation of canine MHC loci in over 3000 samples (Paper IV). The analysis revealed new single nucleotide variations and a notable number of novel haplotypes for the 2nd exon of DLA DRB1.

    Taken together, this thesis demonstrates emerging applications of barcoded sequencing in protein and DNA detection. Improvements through the barcoding systems for assay parallelization, de-convolution of antigen-antibody interactions, sequence variant analysis, as well as large-scale data interpretation would aid biomedical studies to achieve a deeper understanding of biological processes. The future perspectives of the developed methodologies may therefore stem for advancing large-scale omics investigations, particularly in the promising field of DNA-mediated proteomics, for highly multiplex studies of numerous samples at a notably improved molecular resolution.

  • 225.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnetic bead assisted labeling of antibodies at nanogram scale2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 1, p. 14-18Article in journal (Refereed)
    Abstract [en]

    There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.

  • 226.
    Dezfouli, Mahya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Iglesias, Maria Jesus
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Parallel barcoding of antibodies for DNA-assisted proteomics2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 21-22, p. 2432-2436Article in journal (Refereed)
    Abstract [en]

    DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.

  • 227. Di Carlo, Dino
    et al.
    Huang, Yanyi
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Emerging investigators: new challenges spawn new innovations2014In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, no 15, p. 2599-2600Article in journal (Refereed)
  • 228.
    Dias, Jorge T.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lama, Lara
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Minimizing antibody cross-reactivity in multiplex detection of biomarkers in paper-based point-of-care assays2016In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 8, no 15, p. 8195-8201Article in journal (Refereed)
    Abstract [en]

    Highly multiplexed immunoassays could allow convenient screening of hundreds or thousands of protein biomarkers simultaneously in a clinical sample such as serum or plasma, potentially allowing improved diagnostic accuracy and clinical management of many conditions such as autoimmune disorders, infections, and several cancers. Currently, antibody microarray-based tests are limited in part due to cross reactivity from detection antibody reagents. Here we present a strategy that reduces the cross-reactivity between nanoparticle-bound reporter antibodies through the application of ultrasound energy. By this concept, it was possible to achieve a sensitivity 10(3)-fold (5 pg mL(-1)) lower than when no ultrasound was applied (50 ng mL(-1)) for the simultaneous detection of three different antigens. The detection limits and variability achieved with this technique rival those obtained with other types of multiplex sandwich assays.

  • 229.
    Dias, Jorge. T.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svedberg, Gustav
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystrand, M.
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis2018In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, Vol. 2018, no 133, article id e57297Article in journal (Refereed)
    Abstract [en]

    The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good's buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera. 

  • 230.
    Dias, Jorge T.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svedberg, Gustav
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystrand, Mats
    Thermo Fisher Sci IDD, Global Res & Dev, Uppsala, Sweden..
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rapid signal enhancement method for nanoprobe-based biosensing (vol 7, 2017)2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, no 1, article id 8184Article in journal (Refereed)
    Abstract [en]

    In the Methods section of this Article references 18 to 22 are incorrectly cited. The correct references were omitted from the reference list and appear below as references 1-5. References 18 to 22 are correctly cited in Introduction and Results and Discussion sections. "AuNPs of 10 nm in diameter were prepared following the protocol described by Bastus et al.18." should read: "AuNPs of 10 nm in diameter were prepared following the protocol described by Bastus et al.1." "AgNPs of 90 nm in diameter were prepared following the protocol described by Rivero et al.19." should read: "AgNPs of 90 nm in diameter were prepared following the protocol described by Rivero et al.2" "The size was determined by UV-Vis spectroscopy according to the AgNPs size theory demonstrated by Malynych20." should read: "The size was determined by UV-Vis spectroscopy according to the AgNPs size theory demonstrated by Malynych3." "The coupling of antibody to the NPs was prepared following a modified version of a protocol previously reported by Puertas et al.21." should read: "The coupling of antibody to the NPs was prepared following a modified version of a protocol previously reported by Puertas et al.4." "Microarrays were prepared as previously reported by our group22." should read: "Microarrays were prepared as previously reported by our group5.

  • 231. Djureinovic, D.
    et al.
    Dodig-Crnkovic, Tea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hellström, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Holgersson, G.
    Bergqvist, M.
    Mattsson, J. S. M.
    Pontén, F.
    Ståhle, E.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Micke, P.
    Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer2018In: Lung Cancer, ISSN 0169-5002, E-ISSN 1872-8332, Vol. 125, p. 157-163Article in journal (Refereed)
    Abstract [en]

    Objectives: Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. Materials and methods: To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. Results: Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. Conclusion: We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets. 

  • 232. Djureinovic, D.
    et al.
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Danielsson, A.
    Lindskog, C.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, F.
    The human testis-specific proteome defined by transcriptomics and antibody-based profiling2014In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 20, no 6, p. 476-488Article in journal (Refereed)
    Abstract [en]

    The testis' function is to produce haploid germ cells necessary for reproduction. Here we have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to characterize the molecular components of the testis. Deep sequencing (RNA-Seq) of normal human testicular tissue from seven individuals was performed and compared with 26 other normal human tissue types. All 20 050 putative human genes were classified into categories based on expression patterns. The analysis shows that testis is the tissue with the most tissue-specific genes by far. More than 1000 genes show a testis-enriched expression pattern in testis when compared with all other analyzed tissues. Highly testis enriched genes were further characterized with respect to protein localization within the testis, such as spermatogonia, spermatocytes, spermatids, sperm, Sertoli cells and Leydig cells. Here we present an immunohistochemistry-based analysis, showing the localization of corresponding proteins in different cell types and various stages of spermatogenesis, for 62 genes expressed at > 50-fold higher levels in testis when compared with other tissues. A large fraction of these genes were unexpectedly expressed in early stages of spermatogenesis. In conclusion, we have applied a genome-wide analysis to identify the human testis-specific proteome using transcriptomics and antibody-based protein profiling, providing lists of genes expressed in a tissue-enriched manner in the testis. The majority of these genes and proteins were previously poorly characterised in terms of localization and function, and our list provides an important starting point to increase our molecular understanding of human reproductive biology and disease.

  • 233. Djureinovic, Dijana
    et al.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Horie, Masafumi
    Mattsson, Johanna Sofia Margareta
    La Fleur, Linnea
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brunnstrom, Hans
    Lindskog, Cecilia
    Madjar, Katrin
    Rahnenfuehrer, Joerg
    Ekman, Simon
    Stahle, Elisabeth
    Koyi, Hirsh
    Branden, Eva
    Edlund, Karolina
    Hengstler, Jan G.
    Lambe, Mats
    Saito, Akira
    Botling, Johan
    Ponten, Fredrik
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Micke, Patrick
    Profiling cancer testis antigens in non-small-cell lung cancer2016In: JCI INSIGHT, ISSN 2379-3708, Vol. 1, no 10, article id e86837Article in journal (Refereed)
    Abstract [en]

    Cancer testis antigens (CTAs) are of clinical interest as biomarkers and present valuable targets for immunotherapy. To comprehensively characterize the CTA landscape of non-small-cell lung cancer (NSCLC), we compared RNAseq data from 199 NSCLC tissues to the normal transcriptome of 142 samples from 32 different normal organs. Of 232 CTAs currently annotated in the Caner Testis Database (CTdatabase), 96 were confirmed in NSCLC. To obtain an unbiased CTA profile of NSCLC, we applied stringent criteria on our RNAseq data set and defined 90 genes as CTAs, of which 55 genes were not annotated in the CTdatabase, thus representing potential new CTAs. Cluster analysis revealed that CTA expression is histology dependent and concurrent expression is common. IHC confirmed tissue-specific protein expression of selected new CTAs (TKTL1, TGIF2LX, VCX, and CXORF67). Furthermore, methylation was identified as a regulatory mechanism of CTA expression based on independent data from The Cancer Genome Atlas. The proposed prognostic impact of CTAs in lung cancer was not confirmed, neither in our RNAseq cohort nor in an independent meta-analysis of 1,117 NSCLC cases. In summary, we defined a set of 90 reliable CTAs, including information on protein expression, methylation, and survival association. The detailed RNAseq catalog can guide biomarker studies and efforts to identify targets for immunotherapeutic strategies.

  • 234. Djureinovic, Dijana
    et al.
    Hallström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mattsson, Johanna Sofia Margareta
    La Fleur, Linnea
    Botling, Johan
    Fagerberg, Linn
    Brunnstrom, Hans
    Ekman, Simon
    Stahle, Elisabeth
    Koyi, Hirsh
    Lambe, Mats
    Branden, Eva
    Lindskog, Cecilia
    Ponten, Fredrik
    Uhlen, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Micke, Patrick
    The Identification of Therapeutic Targets in Lung Cancer Based on Transcriptomic and Proteomic Characterization of Cancer-Testis Antigens2015In: Journal of Thoracic Oncology, ISSN 1556-0864, E-ISSN 1556-1380, Vol. 10, no 9, p. S256-S256Article in journal (Other academic)
  • 235.
    Dolan, R. T.
    et al.
    Univ Coll Dublin, Conway Inst, Sch Biomol & Biomed Sci, Dublin 2, Ireland..
    Brennan, D. J.
    Univ Coll Dublin, Conway Inst, Sch Biomol & Biomed Sci, Dublin 2, Ireland..
    Rexhepaj, E.
    Univ Coll Dublin, Conway Inst, Sch Biomol & Biomed Sci, Dublin 2, Ireland..
    Kelly, C. M.
    Univ Coll Dublin, Conway Inst, Sch Biomol & Biomed Sci, Dublin 2, Ireland..
    Penny, S.
    Univ Coll Dublin, Conway Inst, Sch Biomol & Biomed Sci, Dublin 2, Ireland..
    Jirstrom, K.
    Lund Univ, Dept Pathol, Lund, Sweden..
    Ponten, F.
    Uppsala Univ, Dept Pathol, Uppsala, Sweden..
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Gallagher, W. M.
    Univ Coll Dublin, Conway Inst, Sch Biomol & Biomed Sci, Dublin 2, Ireland..
    DEVELOPMENT OF IMMUNOHISTOCHEMICAL SURROGATES FOR PREDICTION OF BREAST CANCER PATIENT OUTCOME VIA HIGH-THROUGHPUT ANTIBODY GENERATION AND APPLICATION OF TISSUE MICROARRAY TECHNOLOGY: AN INITIAL REPORT2009In: Annals of Oncology, ISSN 0923-7534, E-ISSN 1569-8041, Vol. 20, p. 54-54Article in journal (Other academic)
  • 236. Doucet, M.
    et al.
    Becker, K. F.
    Björkman, J.
    Bonnet, J.
    Clément, B.
    Daidone, M. -G
    Duyckaerts, C.
    Erb, G.
    Haslacher, H.
    Hofman, P.
    Huppertz, B.
    Junot, C.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Metspalu, A.
    Lavitrano, M.
    Litton, J. -E
    Moore, H. M.
    Morente, M.
    Naimi, B. -Y
    Oelmueller, U.
    Ollier, B.
    Parodi, B.
    Ruan, L.
    Stanta, G.
    Turano, P.
    Vaught, J.
    Watson, P.
    Wichmann, H. -E
    Yuille, M.
    Zaomi, M.
    Zatloukal, K.
    Dagher, G.
    Quality Matters: 2016 Annual Conference of the National Infrastructures for Biobanking2017In: Biopreservation and Biobanking, ISSN 1947-5535, E-ISSN 1947-5543, Vol. 15, no 3, p. 270-276Article in journal (Refereed)
  • 237. Dragomir, Anca
    et al.
    de Wit, Meike
    Johansson, Christine
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, Fredrik
    The Role of SATB2 as a Diagnostic Marker for Tumors of Colorectal Origin2014In: American Journal of Clinical Pathology, ISSN 0002-9173, E-ISSN 1943-7722, Vol. 141, no 5, p. 630-638Article in journal (Refereed)
    Abstract [en]

    Objectives: Immunohistochemistry is an important extension to clinical information and morphology, and prevails as an invaluable tool for establishing a correct cancer diagnosis in clinical diagnostic pathology. The applicability of immunohistochemistry is limited by the availability of validated cell- and cancer-type specific antibodies, rendering an unmet need to discover, test, and validate novel markers. The SATB2 protein is selectively expressed in glandular cells from the lower gastrointestinal tract and expression is retained in a large majority of primary and metastatic colorectal cancers. Methods: We analyzed the expression of SATB2 in all clinical cases (n = 840), in which immunohistochemistry for detection of CK20 was deemed necessary for a final diagnosis. Results: SATB2 showed a high sensitivity (93%) and specificity (77%) to determine a cancer of colorectal origin and in combination with CK7 and CK20, the specificity increased to 100%. Conclusions: We conclude that SATB2 provides a new and advantageous supplement for clinical differential diagnostics.

  • 238.
    Dreier, Jes
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Castello, Marco
    Ist Italiano Tecnol, Mol Microscopy & Spect, Via Morego 30, I-16136 Genoa, Italy..
    Coceano, Giovanna
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Caceres, Rodrigo
    PSL Res Univ, CNRS, Inst Curie, Lab Phys Chim Curie, F-75005 Paris, France.;Sorbonne Univ, F-75005 Paris, France.;Univ Paris 05, F-75005 Paris, France..
    Plastino, Julie
    PSL Res Univ, CNRS, Inst Curie, Lab Phys Chim Curie, F-75005 Paris, France.;Sorbonne Univ, F-75005 Paris, France..
    Vicidomini, Giuseppe
    Ist Italiano Tecnol, Mol Microscopy & Spect, Via Morego 30, I-16136 Genoa, Italy..
    Testa, Ilaria
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 556Article in journal (Refereed)
    Abstract [en]

    RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70-90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20-30 mu m inside a living Caenorhabditis elegans worm.

  • 239.
    Drobin, Kimi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Assadi, Ghazaleh
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Hong, Mun-Gwan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Anggraeni Andersson, Margaretha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, KTH, Sch Biotechnol, Affin Prote,SciLifeLab, Stockholm, Sweden..
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Reznichenko, Anna
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Akhter, Tahmina
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Ek, Weronica E.
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;Uppsala Univ, Sci Life Lab, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Bonfiglio, Ferdinando
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;Biodonostia Hlth Res Inst, Dept Gastrointestinal & Liver Dis, San Sebastian, Spain..
    Hansen, Mark Berner
    AstraZeneca R&D, Innovat & Global Med, Molndal, Sweden.;Univ Copenhagen, Bispebjerg Hosp, Ctr Digest Dis, Copenhagen, Denmark..
    Sandberg, Kristian
    Uppsala Univ, Sci Life Lab, Drug Discovery & Dev Platform, Uppsala, Sweden.;Uppsala Univ, Uppsala Biomed Ctr, Dept Med Chem, Organ Pharmaceut Chem, Uppsala, Sweden.;Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Greco, Dario
    Univ Helsinki, Inst Biotechnol, Helsinki, Finland..
    Repsilber, Dirk
    Orebro Univ, Sch Med Sci, Orebro, Sweden..
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    D'Amato, Mauro
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;BioDonostia Hlth Res Inst, San Sebastian, Spain.;Ikerbasque, Basque Fdn Sci, Bilbao, Spain..
    Halfvarson, Jonas
    Orebro Univ, Fac Med & Hlth, Dept Gastroenterol, SE-70182 Orebro, Sweden..
    Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci2019In: Inflammatory Bowel Diseases, ISSN 1078-0998, E-ISSN 1536-4844, Vol. 25, no 2, p. 306-316Article in journal (Refereed)
    Abstract [en]

    Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential. Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohns disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping. By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity. Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.

  • 240.
    Drobin, Kimi
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Highly multiplexed antibody suspension bead arrays for plasma protein profiling2013In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1023, p. 137-145Article in journal (Refereed)
    Abstract [en]

    Alongside the increasing availability of affinity reagents, antibody microarrays have become a powerful tool to screen for target proteins in complex samples. Applying directly labeled samples onto arrays instead of using sandwich assays offers an approach to facilitate a systematic, high-throughput, and flexible exploration of protein profiles in body fluids such as serum or plasma. As an alternative to planar arrays, a system based on color-coded beads for the creation of antibody arrays in suspension has become available to offer a microtiter plate-based option for screening larger number of samples with variable sets of capture reagents. A procedure was established for analyzing biotinylated samples without the necessity to remove excess labeling substance. We have shown that this assay system allows detecting proteins down into lower pico-molar and higher pg/ml levels with dynamic ranges over three orders of magnitude. Presently, this workflow enables the profiling of 384 samples for up to 384 proteins per assay.

  • 241. Du, K.
    et al.
    Wu, Y. -W
    Lindroos, R.
    Liu, Y.
    Rózsa, B.
    Katona, G.
    Ding, J. B.
    Hellgren Kotaleski, Jeanette
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Computer Science and Communication (CSC). Stockholm Brain Institute, Karolinska Institute, 171 77 Solna, Sweden;bDepartment of Neuroscience, Karolinska Institute, 171 77 Solna.
    Cell-type–specific inhibition of the dendritic plateau potential in striatal spiny projection neurons2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 36, p. E7612-E7621Article in journal (Refereed)
    Abstract [en]

    Striatal spiny projection neurons (SPNs) receive convergent excitatory synaptic inputs from the cortex and thalamus. Activation of spatially clustered and temporally synchronized excitatory inputs at the distal dendrites could trigger plateau potentials in SPNs. Such supralinear synaptic integration is crucial for dendritic computation. However, how plateau potentials interact with subsequent excitatory and inhibitory synaptic inputs remains unknown. By combining computational simulation, two-photon imaging, optogenetics, and dual-color uncaging of glutamate and GABA, we demonstrate that plateau potentials can broaden the spatiotemporal window for integrating excitatory inputs and promote spiking. The temporal window of spiking can be delicately controlled by GABAergic inhibition in a cell-type–specific manner. This subtle inhibitory control of plateau potential depends on the location and kinetics of the GABAergic inputs and is achieved by the balance between relief and reestablishment of NMDA receptor Mg2+ block. These findings represent a mechanism for controlling spatiotemporal synaptic integration in SPNs.

  • 242. Dupont, Chris L.
    et al.
    Larsson, John
    Yooseph, Shibu
    Ininbergs, Karolina
    Goll, Johannes
    Asplund-Samuelsson, Johannes
    McCrow, John P.
    Celepli, Narin
    Allen, Lisa Zeigler
    Ekman, Martin
    Lucas, Andrew J.
    Hagström, Åke
    Thiagarajan, Mathangi
    Brindefalk, Björn
    Richter, Alexander R.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tenney, Aaron
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tovchigrechko, Andrey
    Nylander, Johan A. A.
    Brami, Daniel
    Badger, Jonathan H.
    Allen, Andrew E.
    Rusch, Douglas B.
    Hoffman, Jeff
    Norrby, Erling
    Friedman, Robert
    Pinhassi, Jarone
    Venter, J. Craig
    Bergman, Birgitta
    Functional Tradeoffs Underpin Salinity-Driven Divergence in Microbial Community Composition2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, p. e89549-Article in journal (Refereed)
    Abstract [en]

    Bacterial community composition and functional potential change subtly across gradients in the surface ocean. In contrast, while there are significant phylogenetic divergences between communities from freshwater and marine habitats, the underlying mechanisms to this phylogenetic structuring yet remain unknown. We hypothesized that the functional potential of natural bacterial communities is linked to this striking divide between microbiomes. To test this hypothesis, metagenomic sequencing of microbial communities along a 1,800 km transect in the Baltic Sea area, encompassing a continuous natural salinity gradient from limnic to fully marine conditions, was explored. Multivariate statistical analyses showed that salinity is the main determinant of dramatic changes in microbial community composition, but also of large scale changes in core metabolic functions of bacteria. Strikingly, genetically and metabolically different pathways for key metabolic processes, such as respiration, biosynthesis of quinones and isoprenoids, glycolysis and osmolyte transport, were differentially abundant at high and low salinities. These shifts in functional capacities were observed at multiple taxonomic levels and within dominant bacterial phyla, while bacteria, such as SAR11, were able to adapt to the entire salinity gradient. We propose that the large differences in central metabolism required at high and low salinities dictate the striking divide between freshwater and marine microbiomes, and that the ability to inhabit different salinity regimes evolved early during bacterial phylogenetic differentiation. These findings significantly advance our understanding of microbial distributions and stress the need to incorporate salinity in future climate change models that predict increased levels of precipitation and a reduction in salinity.

  • 243.
    Dusart, Philip
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Fagerberg, Linn
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Perisic, L.
    Civelek, M.
    Struck, Eike
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hedin, U.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Trégouët, D. -A
    Renné, T.
    Odeberg, Jacob
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO). Coagulation Unit, Centre for Hematology, Karolinska University Hospital, SE-171 76, Stockholm, Sweden.
    Butler, Lynn M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO). Clinical Chemistry and Blood Coagulation, Department of Molecular Medicine and Surgery, Karolinska Institute, SE-171 76, Stockholm, Sweden Institute for Clinical Chemistry and Laboratory Medicine, University Medical Centre Hamburg-Eppendorf, D-20246, Hamburg, Germany.
    A systems-approach reveals human nestin is an endothelial-enriched, angiogenesis-independent intermediate filament protein2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, no 1, article id 14668Article in journal (Refereed)
    Abstract [en]

    The intermediate filament protein nestin is expressed during embryonic development, but considered largely restricted to areas of regeneration in the adult. Here, we perform a body-wide transcriptome and protein-profiling analysis to reveal that nestin is constitutively, and highly-selectively, expressed in adult human endothelial cells (EC), independent of proliferative status. Correspondingly, we demonstrate that it is not a marker for tumour EC in multiple malignancy types. Imaging of EC from different vascular beds reveals nestin subcellular distribution is shear-modulated. siRNA inhibition of nestin increases EC proliferation, and nestin expression is reduced in atherosclerotic plaque neovessels. eQTL analysis reveals an association between SNPs linked to cardiovascular disease and reduced aortic EC nestin mRNA expression. Our study challenges the dogma that nestin is a marker of proliferation, and provides insight into its regulation and function in EC. Furthermore, our systems-based approach can be applied to investigate body-wide expression profiles of any candidate protein. 

  • 244. Dwivedi, Bhakti
    et al.
    Xue, Bingjie
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Edwards, Robert A.
    Breitbart, Mya
    A bioinformatic analysis of ribonucleotide reductase genes in phage genomes and metagenomes2013In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 13, p. 33-Article in journal (Refereed)
    Abstract [en]

    Background: Ribonucleotide reductase (RNR), the enzyme responsible for the formation of deoxyribonucleotides from ribonucleotides, is found in all domains of life and many viral genomes. RNRs are also amongst the most abundant genes identified in environmental metagenomes. This study focused on understanding the distribution, diversity, and evolution of RNRs in phages (viruses that infect bacteria). Hidden Markov Model profiles were used to analyze the proteins encoded by 685 completely sequenced double-stranded DNA phages and 22 environmental viral metagenomes to identify RNR homologs in cultured phages and uncultured viral communities, respectively. Results: RNRs were identified in 128 phage genomes, nearly tripling the number of phages known to encode RNRs. Class I RNR was the most common RNR class observed in phages (70%), followed by class II (29%) and class III (28%). Twenty-eight percent of the phages contained genes belonging to multiple RNR classes. RNR class distribution varied according to phage type, isolation environment, and the host's ability to utilize oxygen. The majority of the phages containing RNRs are Myoviridae (65%), followed by Siphoviridae (30%) and Podoviridae (3%). The phylogeny and genomic organization of phage and host RNRs reveal several distinct evolutionary scenarios involving horizontal gene transfer, co-evolution, and differential selection pressure. Several putative split RNR genes interrupted by self-splicing introns or inteins were identified, providing further evidence for the role of frequent genetic exchange. Finally, viral metagenomic data indicate that RNRs are prevalent and highly dynamic in uncultured viral communities, necessitating future research to determine the environmental conditions under which RNRs provide a selective advantage. Conclusions: This comprehensive study describes the distribution, diversity, and evolution of RNRs in phage genomes and environmental viral metagenomes. The distinct distributions of specific RNR classes amongst phages, combined with the various evolutionary scenarios predicted from RNR phylogenies suggest multiple inheritance sources and different selective forces for RNRs in phages. This study significantly improves our understanding of phage RNRs, providing insight into the diversity and evolution of this important auxiliary metabolic gene as well as the evolution of phages in response to their bacterial hosts and environments.

  • 245. Edberg, F.
    et al.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Holmström, S. J. M.
    Bacterial Community Composition in the Water Column of a Lake Formed by a Former Uranium Open Pit Mine2012In: Microbial Ecology, ISSN 0095-3628, E-ISSN 1432-184X, Vol. 64, no 4, p. 870-880Article in journal (Refereed)
    Abstract [en]

    Mining of pyrite minerals is a major environmental issue involving both biological and geochemical processes. Here we present a study of an artificial lake of a former uranium open pit mine with the aim to connect the chemistry and bacterial community composition (454-pyrosequencing of 16S rRNA genes) in the stratified water column. A shift in the water chemistry from oxic conditions in the epilimnion to anoxic, alkaline, and metal and sulfide-rich conditions in the hypolimnion was corresponded by a strong shift in the bacterial community, with few shared operational taxonomic units (OTU) between the water layers. The epilimnetic bacterial community of the lake (~20 years old) showed similarities to other temperate freshwater lakes, while the hypolimnetic bacterial community showed similarity to extreme chemical environments. The epilimnetic bacterial community had dominance of Actinobacteria and Betaproteobacteria. The hypolimnion displayed a higher bacterial diversity and was dominated by the phototrophic green sulphur bacterium of the genus Chlorobium (ca. 40 % of the total community). Deltaproteobacteria were only represented in the hypolimnion and the most abundant OTUs were affiliated with ferric iron and sulfate reducers of the genus Geobacter and Desulfobulbus, respectively. The chemistry is clearly controlling, especially the hypolimnetic, bacterial community but the community composition also indicates that the bacteria are involved in metal cycling in the lake.

  • 246.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Zeiler, Marlis
    Johansson, Henrik J.
    Karlinska Institute.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lehtiö, Janne
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mann, Matthias
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins2014In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, p. 1611-1624Article in journal (Refereed)
    Abstract [en]

    AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  • 247.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Danielsson, Frida
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Gene specific correlation of RNA and protein levels in human cells and tissues2016In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292Article in journal (Refereed)
    Abstract [en]

    An important issue for molecular biology is to establish if transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on Parallel Reaction Monitoring to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP) conversion factor independent of the tissue-type is introduced, thus significantly enhancing the predictability of protein copy numbers from RNA levels. The results show that the RTP-ratio varies significantly with a few hundred copies per mRNA molecule for some genes to several hundred thousands protein copies per mRNA molecule for others. In conclusion, our data suggests that transcriptome analysis can be used as a tool to predict the protein copy numbers per cell, thus forming an attractive link between the field of genomics and proteomics. 

  • 248.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Danielsson, Frida
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Emma
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ponten, Fredrik
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Technical University of Denmark, Denmark.
    Gene-specific correlation of RNA and protein levels in human cells and tissues2016In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 12, no 10, article id 883Article in journal (Refereed)
    Abstract [en]

    An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP) conversion factor independent of the tissue type is introduced, thus significantly enhancing the predictability of protein copy numbers from RNA levels. The results show that the RTP ratio varies significantly with a few hundred copies per mRNA molecule for some genes to several hundred thousands of protein copies per mRNA molecule for others. In conclusion, our data suggest that transcriptome analysis can be used as a tool to predict the protein copy numbers per cell, thus forming an attractive link between the field of genomics and proteomics.

  • 249.
    Edfors, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Boström, Tove
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. Atlas Antibodies AB.
    Maddalo, Gianluca
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svensson, Anne-Sophie
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Tegel, Hanna
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jochen, Schwenk
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. Technical University of Denmark, Denmark.
    A recombinant protein standard resource for targeted proteomicsManuscript (preprint) (Other academic)
    Abstract [en]

    Here, we have used a resource of 26,000 recombinant protein fragments to create custom libraries of standards for targeted proteomics based on parallel reaction monitoring (PRM). The recombinant fragments can be produced in a bacterial cell factory to generate heavy isotope labeled standards for absolute quantification of the corresponding protein targets and be used to produce high- quality spectral libraries. Altogether, coordinates for 25,684 unique proteotypic peptide assays have been experimentally defined covering 10,163 human proteins. The protocol allows for precise monitoring of digestion kinetics and thus enables to select peptides that behave quantitative during the sample preparation process. We show that the quantification tag of each recombinant protein fragment can be used for accurate retention time prediction and allows for assay standardization across different method parameters. The use of this resource was illustrated by determining the absolute concentrations of selected protein targets using multiplex targeted proteomics assays for determination of quantitative assessment of 49 protein targets in serum samples. 

  • 250.
    Edfors, Fredrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vunk, Helian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kotol, David
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svensson, Anne-Sophie
    KTH.
    Boström, Tove
    KTH.
    Tegel, Hanna
    KTH.
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Neurosci, SE-17165 Solna, Sweden.;Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics2019In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 7, p. 2706-2718Article in journal (Refereed)
    Abstract [en]

    The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

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