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  • 201. Lazar, Vladimir
    et al.
    Suo, Chen
    Orear, Cedric
    van den Oord, Joost
    Balogh, Zsofia
    Guegan, Justine
    Job, Bastien
    Meurice, Guillaume
    Ripoche, Hugues
    Calza, Stefano
    Hasmats, Johanna
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lacroix, Ludovic
    Vielh, Philippe
    Dufour, Fabienne
    Lehtio, Janne
    Napieralski, Rudolf
    Eggermont, Alexander
    Schmitt, Manfred
    Cadranel, Jacques
    Besse, Benjamin
    Girard, Philippe
    Blackhall, Fiona
    Validire, Pierre
    Soria, Jean-Charles
    Dessen, Philippe
    Hansson, Johan
    Pawitan, Yudi
    Integrated molecular portrait of non-small cell lung cancers2013In: BMC Medical Genomics, ISSN 1755-8794, E-ISSN 1755-8794, Vol. 6, no 1, p. 53-Article in journal (Refereed)
    Abstract [en]

    Background: Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. Methods: Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. Results: At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of similar to 800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. Conclusions: Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

  • 202. Leandro-Garcia, Luis J.
    et al.
    Inglada-Perez, Lucia
    Pita, Guillermo
    Hjerpe, Elisabet
    Leskelae, Susanna
    Jara, Carlos
    Mielgo, Xabier
    Gonzalez-Neira, Anna
    Robledo, Mercedes
    Avall-Lundqvist, Elisabeth
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rodriguez-Antona, Cristina
    Genome-wide association study identifies ephrin type A receptors implicated in paclitaxel induced peripheral sensory neuropathy2013In: Journal of Medical Genetics, ISSN 0022-2593, E-ISSN 1468-6244, Vol. 50, no 9, p. 599-605Article in journal (Refereed)
    Abstract [en]

    Background Peripheral neuropathy is the dose limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat solid tumours. This toxicity exhibits great inter-individual variability of unknown origin. The present study aimed to identify genetic variants associated with paclitaxel induced neuropathy via a whole genome approach. Methods A genome-wide association study (GWAS) was performed in 144 white European patients uniformly treated with paclitaxel/carboplatin and for whom detailed data on neuropathy was available. Per allele single nucleotide polymorphism (SNP) associations were assessed by Cox regression, modelling the cumulative dose of paclitaxel up to the development of grade 2 sensory neuropathy. Results The strongest evidence of association was observed for the ephrin type A receptor 4 (EPHA4) locus (rs17348202, p=1.0x10(-6)), and EPHA6 and EPHA5 were among the top 25 and 50 hits (rs301927, p=3.4x10(-5) and rs1159057, p=6.8x10(-5)), respectively. A meta-analysis of EPHA5-rs7349683, the top marker for paclitaxel induced neuropathy in a previous GWAS (r(2)=0.79 with rs1159057), gave a hazard ratio (HR) estimate of 1.68 (p=1.4x10(-9)). Meta-analysis of the second hit of this GWAS, XKR4-rs4737264, gave a HR of 1.71 (p=3.1x10(-8)). Imputed SNPs at LIMK2 locus were also strongly associated with this toxicity (HR=2.78, p=2.0x10(-7)). Conclusions This study provides independent support of EPHA5-rs7349683 and XKR4-rs4737264 as the first markers of risk of paclitaxel induced neuropathy. In addition, it suggests that other EPHA genes also involved in axonal guidance and repair following neural injury, as well as LIMK2 locus, may play an important role in the development of this toxicity. The identified SNPs could form the basis for individualised paclitaxel chemotherapy.

  • 203. Leandro-Garcia, Luis J.
    et al.
    Leskelä, Susanna
    Jara, Carlos
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Åvall-Lundqvist, Elisabeth
    Wheeler, Heather E.
    Dolan, M. Eileen
    Inglada-Perez, Lucia
    Maliszewska, Agnieszka
    de Cubas, Aguirre A.
    Comino-Mendez, Inaki
    Mancikova, Veronika
    Cascon, Alberto
    Robledo, Mercedes
    Rodriguez-Antona, Cristina
    Regulatory Polymorphisms in beta-Tubulin IIa Are Associated with Paclitaxel-Induced Peripheral Neuropathy2012In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 18, no 16, p. 4441-4448Article in journal (Refereed)
    Abstract [en]

    Purpose: Peripheral neuropathy is the dose-limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat several solid tumors such as breast, lung, and ovary. The cytotoxic effect of paclitaxel is mediated through beta-tubulin binding in the cellular microtubules. In this study, we investigated the association between paclitaxel neurotoxicity risk and regulatory genetic variants in beta-tubulin genes. Experimental Design: We measured variation in gene expression of three beta-tubulin isotypes (I, IVb, and IIa) in lymphocytes from 100 healthy volunteers, sequenced the promoter region to identify polymorphisms putatively influencing gene expression and assessed the transcription rate of the identified variants using luciferase assays. To determine whether the identified regulatory polymorphisms were associated with paclitaxel neurotoxicity, we genotyped them in 214 patients treated with paclitaxel. In addition, paclitaxel-induced cytotoxicity in lymphoblastoid cell lines was compared with beta-tubulin expression as measured by Affymetrix exon array. Results: We found a 63-fold variation in beta-tubulin IIa gene (TUBB2A) mRNA content and three polymorphisms located at -101, -112, and -157 in TUBB2A promoter correlated with increased mRNA levels. The -101 and -112 variants, in total linkage disequilibrium, conferred TUBB2A increased transcription rate. Furthermore, these variants protected from paclitaxel-induced peripheral neuropathy [HR, 0.62; 95% confidence interval (CI), 0.42-0.93; P = 0.021, multivariable analysis]. In addition, an inverse correlation between TUBB2A and paclitaxel-induced apoptosis (P = 0.001) in lymphoblastoid cell lines further supported that higher TUBB2A gene expression conferred lower paclitaxel sensitivity. Conclusions: This is the first study showing that paclitaxel neuropathy risk is influenced by polymorphisms regulating the expression of a beta-tubulin gene.

  • 204.
    Lee, Sunjae
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Korea Adv Inst Sci & Technol, South Korea.
    Mardinoglu, Adil
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers, Sweden.
    Zhang, Cheng
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lee, Doheon
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers, Sweden.
    Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 12, p. 5529-5539Article in journal (Refereed)
    Abstract [en]

    Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies.

  • 205.
    Lee, Sunjae
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhang, Cheng
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kilicarslan, Murat
    Piening, Brian D.
    Björnson, Elias
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Groen, Albert K.
    Ferrannini, Ele
    Laakso, Markku
    Snyder, Michael
    Bluher, Matthias
    Uhlèn, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. Chalmers, Sweden.
    Smith, Ulf
    Serlie, Mireille J.
    Boren, Jan
    Mardinoglu, Adil
    Integrated Network Analysis Reveals an Association between Plasma Mannose Levels and Insulin Resistance2016In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 24, no 1, p. 172-184Article in journal (Refereed)
    Abstract [en]

    To investigate the biological processes that are altered in obese subjects, we generated cell-specific integrated networks (INs) by merging genome-scale metabolic, transcriptional regulatory and protein-protein interaction networks. We performed genome-wide transcriptomics analysis to determine the global gene expression changes in the liver and three adipose tissues from obese subjects undergoing bariatric surgery and integrated these data into the cell-specific INs. We found dysregulations in mannose metabolism in obese subjects and validated our predictions by detecting mannose levels in the plasma of the lean and obese subjects. We observed significant correlations between plasma mannose levels, BMI, and insulin resistance (IR). We also measured plasma mannose levels of the subjects in two additional different cohorts and observed that an increased plasma mannose level was associated with IR and insulin secretion. We finally identified mannose as one of the best plasma metabolites in explaining the variance in obesity-independent IR.

  • 206. Lehours, P.
    et al.
    Vale, F. F.
    Bjursell, M. K.
    Melefors, O.
    Advani, R.
    Glavas, S.
    Guegueniat, J.
    Gontier, E.
    Lacomme, S.
    Alves Matos, A.
    Menard, A.
    Megraud, F.
    Engstrand, L.
    Andersson, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome sequencing reveals a phage in Helicobacter pylori2011In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 2, no 6, p. e00239-Article in journal (Refereed)
    Abstract [en]

     Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H pylori.

  • 207. Lehtio, J.
    et al.
    Branca, M.
    Johansson, H.
    Orre, M.
    Granholm, Viktor
    KTH.
    Forshed, J.
    Perez-Bercoff, M.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, SeRC - Swedish e-Science Research Centre. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome Wide Proteomics Using Peptide High Resolution Isoelectric Focusing Hirief-Ms Allows Detection New Human Gene Models2012In: Annals of Oncology, ISSN 0923-7534, E-ISSN 1569-8041, Vol. 23, p. 33-34Article in journal (Other academic)
  • 208.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Transcriptional patterns in inflammatory disease2008Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    In the studies this thesis is based upon, microarrays were applied to profilemRNA populations in biological samples to gain insights into transcriptionalpatterns and their relation to inflammatory disease.Rheumatoid arthritis (RA) is a chronic inflammatory disease, which leads todegradation of cartilage and bone. RA is characterized by synovial inflammationwith varying levels of tissue heterogeneity. This was confirmed by microarrayanalyses of multiple biopsies from the joints of 13 patients, which showed interindividualvariation in transcript populations to be higher than intra‐individualvariationTherapeutic antibodies targeting TNF‐α have revolutionized treatment of RA,although some patients do not respond well. Identification of non‐responders isimportant, not only because anti‐TNF treatment elevates the risk of infections,but also because of the cost of treatment. A proof‐of‐concept study to investigatetranscriptional effects of anti‐TNF treatment demonstrated that differencesbetween response groups could be identified and that these differences revealedbiological themes related to inflammatory disease.A subsequent study was therefore initiated with a larger cohort of 62 patients toinvestigate gene expression patterns in the synovium prior to anti‐TNFtreatment. Here, the heterogeneity was even more pronounced, thetranscriptional patterns were confounded by the presence of synovial aggregatesand only a weak therapy‐correlated signature was detected. The presence oflymphocyte aggregates was found to correlate to response to therapy, which isconsistent with previous findings indicating a higher level of inflammation ingood responding patients.Periodontitis is an inflammatory disease with many similarities to RA. Both areincurable chronic auto‐immune diseases, characterized by tissue destructionwith common genetic associations. Individuals with RA are at higher risk ofaccumulating significant periodontal problems than the general population. PGE2(prostaglandin E2) is known to stimulate inflammation and bone resorption inperiodontitis. In further studies, microarrays were applied in a time seriesdesign on human gingival fibroblats to explore the signal transduction pathwayscontrolling TNF‐α induced PGE2 synthesis in order to identify novel therapeutictargets. The JNK and NF‐kb pathways were identified as being differentiallyaffected by TNF‐a treatment. The transcriptional patterns were further verifiedusing antibodies against phosphorylated JNK/NF‐kb molecules and specificinhibitors of the JNK and NF‐kb signaling cascades.

  • 209.
    Lindberg, Johan
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    af Klint, Erik
    Department of Medicine, Karolinska Institute, Karolinska University Hospital.
    Catrina, Anca Irinel
    Department of Medicine, Karolinska Institute, Karolinska University Hospital.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klareskog, Lars
    Department of Medicine, Karolinska Institute, Karolinska University Hospital.
    Ulfgren, Ann-Kristin
    Department of Medicine, Karolinska Institute, Karolinska University Hospital.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients2006In: Arthritis Research & Therapy, ISSN 1478-6354, E-ISSN 1478-6362, Vol. 8, no 6, p. R179-Article in journal (Refereed)
    Abstract [en]

    We examined the gene expression profiles in arthroscopic biopsies retrieved from 10 rheumatoid arthritis patients before and after anti-TNF treatment with infliximab to investigate whether such profiles can be used to predict responses to the therapy, and to study effects of the therapy on the profiles. Responses to treatment were assessed using European League Against Rheumatism response criteria. Three patients were found to be good responders, five patients to be moderate responders and two patients to be nonresponders. The TNF-alpha status of the biopsies from each of the patients before treatment was also investigated immunohistochemically, and it was detected in biopsies from four of the patients, including all three of the good responders. The gene expression data demonstrate that all patients had unique gene expression signatures, with low intrapatient variability between biopsies. The data also revealed significant differences between the good responding and nonresponding patients (279 differentially expressed genes were detected, with a false discovery rate < 0.025). Among the identified genes we found that MMP-3 was significantly upregulated in good responders (log(2) fold change, 2.95) compared with nonresponders, providing further support for the potential of MMP-3 as a marker for good responses to therapy. An even more extensive list of 685 significantly differentially expressed genes was found between patients in whom TNF-alpha was found and nonresponders, indicating that TNF-alpha could be an important biomarker for successful infliximab treatment. Significant differences were also observed between biopsies taken before and after anti-TNF treatment, including 115 differentially expressed genes in the good responding group. Interestingly, the effect was even stronger in the group in which TNF-a was immunohistochemically detected before therapy. Here, 1,058 genes were differentially expressed, including many that were novel in this context (for example, CXCL3 and CXCL14). Subsequent Gene Ontology analysis revealed that several 'themes' were significantly over-represented that are known to be affected by anti-TNF treatment in inflammatory tissue; for example, immune response (GO:0006955), cell communication (GO:0007154), signal transduction (GO:0007165) and chemotaxis (GO:0006935). No genes reached statistical significance in the moderately responding or nonresponding groups. In conclusion, this pilot study suggests that further investigation is warranted on the usefulness of gene expression profiling of synovial tissue to predict and monitor the outcome of rheumatoid arthritis therapies.

  • 210.
    Lindberg, Johan
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    af Klint, Erik
    Department of Rheumatology, Karolinska Institutet, Karolinska University Hospital.
    Ulfgren, Ann-Kristin
    Department of Rheumatology, Karolinska Institutet, Karolinska University Hospital.
    Stark, André
    Department of Orthopedics, Karolinska University Hospital.
    Andersson, Tove
    KTH, School of Biotechnology (BIO), Gene Technology.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klareskog, Lars
    Department of Rheumatology, Karolinska Institutet, Karolinska University Hospital.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Variability in synovial inflammation in rheumatoid arthritis investigated by microarray technology2006In: Arthritis Research & Therapy, ISSN 1478-6354, E-ISSN 1478-6362, Vol. 8, no 2Article in journal (Refereed)
    Abstract [en]

    In recent years microarray technology has been used increasingly to acquire knowledge about the pathogenic processes involved in rheumatoid arthritis. The present study investigated variations in gene expression in synovial tissues within and between patients with rheumatoid arthritis. This was done by applying microarray technology on multiple synovial biopsies obtained from the same knee joints. In this way the relative levels of intra-patient and inter-patient variation could be assessed. The biopsies were obtained from 13 different patients: 7 by orthopedic surgery and 6 by rheumatic arthroscopy. The data show that levels of heterogeneity varied substantially between the biopsies, because the number of genes found to be differentially expressed between pairs of biopsies from the same knee ranged from 6 to 2,133. Both arthroscopic and orthopedic biopsies were examined, allowing us to compare the two sampling methods. We found that the average number of differentially expressed genes between biopsies from the same patient was about three times larger in orthopedic than in arthroscopic biopsies. Using a parallel analysis of the tissues by immunohistochemistry, we also identified orthopedic biopsies that were unsuitable for gene expression analysis of synovial inflammation due to sampling of non-inflamed parts of the tissue. Removing these biopsies reduced the average number of differentially expressed genes between the orthopedic biopsies from 455 to 171, in comparison with 143 for the arthroscopic biopsies. Hierarchical clustering analysis showed that the remaining orthopedic and arthroscopic biopsies had gene expression signatures that were unique for each patient, apparently reflecting patient variation rather than tissue heterogeneity. Subsets of genes found to vary between biopsies were investigated for overrepresentation of biological processes by using gene ontology. This revealed representative 'themes' likely to vary between synovial biopsies affected by inflammatory disease.

  • 211.
    Lindberg, Johan
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    The plasticity of the mammalian transcriptome2010In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 95, no 1, p. 1-6Article, review/survey (Refereed)
    Abstract [en]

    The dogmatic view of RNA as a mere necessity in the transfer of information between DNA and proteins has during recent years come into question. Novel approaches and new technology has revealed an unprecedented level of inherent complexity in the mammalian transcriptome. Here, the majority of nucleotides are expressed, in sharp contrast to the similar to 1.2% of the human genome harboring protein coding information. Also, >50% of genomic loci contain antisense and interleaved transcription, a conservative estimate since non-coding RNA is highly regulated between tissues and developmental stages, which has only been investigated to a limited extent. Subsequent focus on RNA with no coding potential has revealed numerous species with novel functions, and deep sequencing studies imply that many remain to be discovered. This review gives an overview of the plasticity and dynamics of the mammalian transcriptome and the prevailing interpretation of its effect on the complexity of species.

  • 212.
    Lindberg, Johan
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wijbrandts, Carla A.
    Univ Amsterdam, Acad Med Ctr, Div Clin Immunol & Rheumatol.
    van Baarsen, Lisa G.
    Univ Amsterdam, Acad Med Ctr, Div Clin Immunol & Rheumatol.
    Nader, Gustavo
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Rheumatol Unit.
    Klareskog, Lars
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Rheumatol Unit.
    Catrina, Anca Irinel
    Karolinska Univ Hosp, Karolinska Inst, Dept Med, Rheumatol Unit.
    Thurlings, Rogier
    Univ Amsterdam, Acad Med Ctr, Div Clin Immunol & Rheumatol.
    Vervoordeldonk, Margriet
    Univ Amsterdam, Acad Med Ctr, Div Clin Immunol & Rheumatol.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tak, Paul P.
    Univ Amsterdam, Acad Med Ctr, Div Clin Immunol & Rheumatol.
    Gene expression analysis of synovial tissue biopsies obtained from rheumatoid arthritis patients prior to infliximab treatmentManuscript (Other academic)
  • 213.
    Lindberg, Johan
    et al.
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Wijbrandts, Carla A.
    van Baarsen, Lisa G.
    Nader, Gustavo
    Klareskog, Lars
    Catrina, Anca
    Thurlings, Rogier
    Vervoordeldonk, Margriet
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Tak, Paul P.
    The Gene Expression Profile in the Synovium as a Predictor of the Clinical Response to Infliximab Treatment in Rheumatoid Arthritis2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 6, p. e11310-Article in journal (Refereed)
    Abstract [en]

    Background: Although the use of TNF inhibitors has fundamentally changed the way rheumatoid arthritis (RA) is treated, not all patients respond well. It is desirable to facilitate the identification of responding and non-responding patients prior to treatment, not only to avoid unnecessary treatment but also for financial reasons. In this work we have investigated the transcriptional profile of synovial tissue sampled from RA patients before anti-TNF treatment with the aim to identify biomarkers predictive of response. Methodology/Principal Findings: Synovial tissue samples were obtained by arthroscopy from 62 RA patients before the initiation of infliximab treatment. RNA was extracted and gene expression profiling was performed using an in-house spotted long oligonucleotide array covering 17972 unique genes. Tissue sections were also analyzed by immunohistochemistry to evaluate cell infiltrates. Response to infliximab treatment was assessed according to the EULAR response criteria. The presence of lymphocyte aggregates dominated the expression profiles and a significant overrepresentation of lymphocyte aggregates in good responding patients confounded the analyses. A statistical model was set up to control for the effect of aggregates, but no differences could be identified between responders and non-responders. Subsequently, the patients were split into lymphocyte aggregate positive-and negative patients. No statistically significant differences could be identified except for 38 transcripts associated with differences between good- and non-responders in aggregate positive patients. A profile was identified in these genes that indicated a higher level of metabolism in good responding patients, which indirectly can be connected to increased inflammation. Conclusions/Significance: It is pivotal to account for the presence of lymphoid aggregates when studying gene expression patterns in rheumatoid synovial tissue. In spite of our original hypothesis, the data do not support the notion that microarray analysis of whole synovial biopsy specimens can be used in the context of personalized medicine to identify non-responders to anti-TNF therapy before the initiation of treatment.

  • 214. Linde, C.
    et al.
    Eriksson, M. J.
    Hage, C.
    Wallén, H.
    Persson, B.
    Corbascio, M.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maret, E.
    Ugander, M.
    Persson, H.
    Rationale and design of the PREFERS (Preserved and Reduced Ejection Fraction Epidemiological Regional Study) Stockholm heart failure study: an epidemiological regional study in Stockholm county of 2.1 million inhabitants2016In: European Journal of Heart Failure, ISSN 1388-9842, E-ISSN 1879-0844, Vol. 18, no 10, p. 1287-1297Article in journal (Refereed)
    Abstract [en]

    Aims: Heart failure (HF) with preserved (HFpEF) or reduced (HFrEF) ejection fraction is associated with poor prognosis and quality of life. While the incidence of HFrEF is declining and HF treatment is effective, HFpEF is increasing, with no established therapy. PREFERS Stockholm is an epidemiological study with the aim of improving clinical care and research in HF and to find new targets for drug treatment in HFpEF ( https://internwebben.ki.se/sites/default/files/20150605_4d_research_appendix_final.pdf). Methods: Patients with new-onset HF (n = 2000) will be characterized at baseline and after 1-year follow-up by standardized protocols for clinical evaluation, echocardiography, and ECG. In one subset undergoing elective coronary bypass surgery (n = 100) and classified according to LV function, myocardial biopsies will be collected during surgery, and cardiac magnetic resonance (CMR) imaging will be performed at baseline and after 1 year. Blood and tissue samples will be stored in a biobank. We will characterize and compare new-onset HFpEF and HFrEF patients regarding clinical findings and cardiac imaging, genomics, proteomics, and transcriptomics from blood and cardiac biopsies, and by established biomarkers of fibrosis, inflammation, haemodynamics, haemostasis, and thrombosis. The data will be explored by state-of-the-art bioinformatics methods to investigate gene expression patterns, sequence variation, DNA methylation, and post-translational modifications, and using systems biology approaches including pathway and network analysis. Conclusions: In this epidemiological HF study with biopsy studies in a subset of patients, we aim to identify new biomarkers of disease progression and to find pathophysiological mechanisms to support explorations of new treatment regimens for HFpEF. © 2016 The Authors European Journal of Heart Failure © 2016 European Society of Cardiology.

  • 215. Lindh, Markus V.
    et al.
    Sjostedt, Johanna
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Baltar, Federico
    Hugerth, Luisa W.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Daniel
    Muthusamy, Saraladevi
    Legrand, Catherine
    Pinhassi, Jarone
    Disentangling seasonal bacterioplankton population dynamics by high-frequency sampling2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 7, p. 2459-2476Article in journal (Refereed)
    Abstract [en]

    Multiyear comparisons of bacterioplankton succession reveal that environmental conditions drive community shifts with repeatable patterns between years. However, corresponding insight into bacterioplankton dynamics at a temporal resolution relevant for detailed examination of variation and characteristics of specific populations within years is essentially lacking. During 1 year, we collected 46 samples in the Baltic Sea for assessing bacterial community composition by 16S rRNA gene pyrosequencing (nearly twice weekly during productive season). Beta-diversity analysis showed distinct clustering of samples, attributable to seemingly synchronous temporal transitions among populations (populations defined by 97% 16S rRNA gene sequence identity). A wide spectrum of bacterioplankton dynamics was evident, where divergent temporal patterns resulted both from pronounced differences in relative abundance and presence/absence of populations. Rates of change in relative abundance calculated for individual populations ranged from 0.23 to 1.79 day(-1). Populations that were persistently dominant, transiently abundant or generally rare were found in several major bacterial groups, implying evolution has favoured a similar variety of life strategies within these groups. These findings suggest that high temporal resolution sampling allows constraining the timescales and frequencies at which distinct populations transition between being abundant or rare, thus potentially providing clues about physical, chemical or biological forcing on bacterioplankton community structure.

  • 216. Lindholm, M. E.
    et al.
    Huss, M.
    Solnestam, Beata Werne
    KTH, School of Biotechnology (BIO), Gene Technology.
    Kjellqvist, S.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sundberg, C. J.
    The human skeletal muscle transcriptome: sex differences, alternative splicing and tissue homogeneity assessed with RNA sequencing2014In: Acta Physiologica, ISSN 1748-1708, E-ISSN 1748-1716, Vol. 211, p. 38-38Article in journal (Other academic)
  • 217. Lindholm, Malene E.
    et al.
    Giacomello, Stefania
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Solnestam, Beata W.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fischer, H.
    Kjellqvist, Sanela
    Huss, Mikael
    Sundberg, Carl J.
    Long-­‐term endurance training induces global isoform changes in human skeletal muscleManuscript (preprint) (Other academic)
  • 218. Lindholm, Malene E.
    et al.
    Giacomello, Stefania
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Solnestam, Beata Werne
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fischer, Helene
    Huss, Mikael
    Kjellqvist, Sanela
    Sundberg, Carl Johan
    The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts2016In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 9, article id e1006294Article in journal (Refereed)
    Abstract [en]

    Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

  • 219. Lindholm, Malene E.
    et al.
    Huss, Mikael
    Solnestam, Beata W.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kjellqvist, Sanela
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sundberg, Carl J.
    The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing2014In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 10, p. 4571-4581Article in journal (Refereed)
    Abstract [en]

    Human skeletal muscle health is important for quality of life and several chronic diseases, including type II diabetes, heart disease, and cancer. Skeletal muscle is a tissue widely used to study mechanisms behind different diseases and adaptive effects of controlled interventions. For such mechanistic studies, knowledge about the gene expression profiles in different states is essential. Since the baseline transcriptome has not been analyzed systematically, the purpose of this study was to provide a deep reference profile of female and male skeletal muscle. RNA sequencing data were analyzed from a large set of 45 resting human muscle biopsies. We provide extensive information on the skeletal muscle transcriptome, including 5 previously unannotated protein-coding transcripts. Global transcriptional tissue homogeneity was strikingly high, within both a specific muscle and the contralateral leg. We identified >23,000 known isoforms and found >5000 isoforms that differ between the sexes. The female and male transcriptome was enriched for genes associated with oxidative metabolism and protein catabolic processes, respectively. The data demonstrate remarkably high tissue homogeneity and provide a deep and extensive baseline reference for the human skeletal muscle transcriptome, with regard to alternative splicing, novel transcripts, and sex differences in functional ontology.

  • 220. Lindqvist, Christina
    et al.
    Janczak, Andrew M.
    Natt, Daniel
    Baranowska, Izabella
    Lindqvist, Niclas
    Wichman, Anette
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Torjesen, Peter A.
    Jensen, Per
    Transmission of Stress-Induced Learning Impairment and Associated Brain Gene Expression from Parents to Offspring in Chickens2007In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 2, no 4, p. e364-Article in journal (Refereed)
    Abstract [en]

    Background. Stress influences many aspects of animal behaviour and is a major factor driving populations to adapt to changing living conditions, such as during domestication. Stress can affect offspring through non-genetic mechanisms, but recent research indicates that inherited epigenetic modifications of the genome could possibly also be involved. Methodology/Principal Findings. Red junglefowl (RJF, ancestors of modern chickens) and domesticated White Leghorn (WL) chickens were raised in a stressful environment (unpredictable light-dark rhythm) and control animals in similar pens, but on a 12/12 h light-dark rhythm. WL in both treatments had poorer spatial learning ability than RJF, and in both populations, stress caused a reduced ability to solve a spatial learning task. Offspring of stressed WL, but not RJF, raised without parental contact, had a reduced spatial learning ability compared to offspring of non-stressed animals in a similar test as that used for their parents. Offspring of stressed WL were also more competitive and grew faster than offspring of non-stressed parents. Using a whole-genome cDNA microarray, we found that in WL, the same changes in hypothalamic gene expression profile caused by stress in the parents were also found in the offspring. In offspring of stressed WL, at least 31 genes were up- or down-regulated in the hypothalamus and pituitary compared to offspring of non-stressed parents. Conclusions/Significance. Our results suggest that, in WL the gene expression response to stress, as well as some behavioural stress responses, were transmitted across generations. The ability to transmit epigenetic information and behaviour modifications between generations may therefore have been favoured by domestication. The mechanisms involved remain to be investigated; epigenetic modifications could either have been inherited or acquired de novo in the specific egg environment. In both cases, this would offer a novel explanation to rapid evolutionary adaptation of a population.

  • 221.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Eriksson, M.
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology. National Institute of Health, United States .
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisén, J.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    A microwell chip for parallel culture and analysis of stem cells2009In: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Chemical and Biological Microsystems Society , 2009, p. 1309-1311Conference paper (Refereed)
    Abstract [en]

    With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to maintain the cells as stem/progenitor cells over time was shown, as was isolation and clonal analysis.

  • 222.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Eriksson, Malin
    Vazin, Tandis
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisén, Jonas
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO).
    High-Density Microwell Chip for Culture and Analysis of Stem Cells2009In: PLos ONE, ISSN 1932-6203, Vol. 4, no 9, p. e6997-Article in journal (Refereed)
    Abstract [en]

    With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

  • 223.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    PCR amplification and genetic analysis in a microwell cell culturing chip2009In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, p. 3465-3471Article in journal (Refereed)
    Abstract [en]

    We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity

  • 224.
    Lindström, Sara
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Hammond, Maria
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    PCR amplification and genetic analysis in a microwell cell cultivation chip2008In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference, Chemical and Biological Microsystems Society , 2008, p. 576-578Conference paper (Refereed)
    Abstract [en]

    We present a method for long-term single cell/clone cultivation followed by cell lysis, DNA amplification and detection of PCR product in a chip containing 672 individual microwells. By performing all steps on-chip in microwells, the proliferation and cell morphology of every single cell or clone can be linked to its genetic information. In this study two mammalian cell lines (mutated A431 vs. wild type U-2 OS) were used as a model system for mutation screening in the p53 gene. The presented method could improve the sensitivity in mutation frequency analysis of heterogeneous tumor samples.

  • 225. Liu, Tao
    et al.
    Laurell, Cecilia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Selivanova, Galina
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wiman, Klas
    Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis2007In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 14, no 3, p. 411-421Article in journal (Refereed)
    Abstract [en]

    p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/ GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.

  • 226. Ljungberg, Liza U.
    et al.
    Persson, Karin
    Eriksson, Andreas C.
    Green, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Whiss, Per A.
    Effects of nicotine, its metabolites and tobacco extracts on human platelet function in vitro2013In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 27, no 2, p. 932-938Article in journal (Refereed)
    Abstract [en]

    Cigarette smoking is a leading cause of cardiovascular disease. The cardiovascular effects of smoking are probably multifactorial, including effects on platelets. Previous reports investigating the effects of nicotine and tobacco on platelet function are inconsistent. The present study investigated in vitro effects of nicotine, its major metabolites, tobacco extracts and extract of tobacco-free snuff on human platelets. None of the metabolites cotinine, cotinine-N-oxide, nicotine-1'-N-oxide or trans-3'-hydroxycotinine (0.1-10 mu M) affected platelet aggregation or P-selectin expression. Nicotine (10 mu M) weakly increased platelet aggregation, whereas trans-3'-hydroxycotinine (0.1 mu M) and nicotine-1'-N-oxide (1-10 mu M) weakly inhibited adhesion to fibrinogen. To elucidate the influence of other tobacco compounds, we investigated the impact of moist tobacco and smoke extracts on platelet function. Filtered extracts of oral snuff, cigarette smoke and tobacco free snuff inhibited platelet adhesion concentration-dependently. The inhibitory effects of tobacco extracts on platelet adhesion were independent of nicotine content and the nitric-oxide-pathway and not mediated through a platelet-nicotine-receptor. Taken together, tobacco extracts inhibit platelet activation during short-term in vitro challenge. As only limited effects of nicotine and nicotine metabolites were seen, the tobacco-induced platelet inhibition are likely induced by other compounds present in tobacco and tobacco free snuff.

  • 227. Logue, Jurg B.
    et al.
    Stedmon, Colin A.
    Kellerman, Anne M.
    Nielsen, Nikoline J.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Laudon, Hjalmar
    Lindstrom, Eva S.
    Kritzberg, Emma S.
    Experimental insights into the importance of aquatic bacterial community composition to the degradation of dissolved organic matter2016In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 10, no 3, p. 533-545Article in journal (Refereed)
    Abstract [en]

    Bacteria play a central role in the cycling of carbon, yet our understanding of the relationship between the taxonomic composition and the degradation of dissolved organic matter (DOM) is still poor. In this experimental study, we were able to demonstrate a direct link between community composition and ecosystem functioning in that differently structured aquatic bacterial communities differed in their degradation of terrestrially derived DOM. Although the same amount of carbon was processed, both the temporal pattern of degradation and the compounds degraded differed among communities. We, moreover, uncovered that low-molecular-weight carbon was available to all communities for utilisation, whereas the ability to degrade carbon of greater molecular weight was a trait less widely distributed. Finally, whereas the degradation of either low-or high-molecular-weight carbon was not restricted to a single phylogenetic clade, our results illustrate that bacterial taxa of similar phylogenetic classification differed substantially in their association with the degradation of DOM compounds. Applying techniques that capture the diversity and complexity of both bacterial communities and DOM, our study provides new insight into how the structure of bacterial communities may affect processes of biogeochemical significance.

  • 228.
    Lundberg, Emma
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Matic, Ivan
    Geiger, Tamar
    Cox, Juergen
    Älgenäs, Cajsa
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Mann, Matthias
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Defining the transcriptome and proteome in three functionally different human cell lines2010In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 6, p. 450-Article in journal (Refereed)
    Abstract [en]

    An essential question in human biology is how cells and tissues differ in gene and protein expression and how these differences delineate specific biological function. Here, we have performed a global analysis of both mRNA and protein levels based on sequence-based transcriptome analysis (RNA-seq), SILAC-based mass spectrometry analysis and antibody-based confocal microscopy. The study was performed in three functionally different human cell lines and based on the global analysis, we estimated the fractions of mRNA and protein that are cell specific or expressed at similar/different levels in the cell lines. A highly ubiquitous RNA expression was found with > 60% of the gene products detected in all cells. The changes of mRNA and protein levels in the cell lines using SILAC and RNA ratios show high correlations, even though the genome-wide dynamic range is substantially higher for the proteins as compared with the transcripts. Large general differences in abundance for proteins from various functional classes are observed and, in general, the cell-type specific proteins are low abundant and highly enriched for cell-surface proteins. Thus, this study shows a path to characterize the transcriptome and proteome in human cells from different origins.

  • 229.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Intense parallel sequencing and high sensitivity identification of mutations, transcriptomes and genomes2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, p. 19-19Article in journal (Other academic)
  • 230.
    Lundin, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Poole, Anthony M.
    Sjöberg, Britt-Marie
    Högbom, Martin
    Use of Structural Phylogenetic Networks for Classification of the Ferritin-like Superfamily2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 24, p. 20565-20575Article in journal (Refereed)
    Abstract [en]

    In the postgenomic era, bioinformatic analysis of sequence similarity is an immensely powerful tool to gain insight into evolution and protein function. Over long evolutionary distances, however, sequence-based methods fail as the similarities become too low for phylogenetic analysis. Macromolecular structure generally appears better conserved than sequence, but clear models for how structure evolves over time are lacking. The exponential growth of three-dimensional structural information may allow novel structure-based methods to drastically extend the evolutionary time scales amenable to phylogenetics and functional classification of proteins. To this end, we analyzed 80 structures from the functionally diverse ferritin-like superfamily. Using evolutionary networks, we demonstrate that structural comparisons can delineate and discover groups of proteins beyond the "twilight zone" where sequence similarity does not allow evolutionary analysis, suggesting that considerable and useful evolutionary signal is preserved in three-dimensional structures.

  • 231.
    Lundin, Daniel
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Severin, I.
    Logue, J. B.
    Östman, O.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lindström, E. S.
    Which sequencing depth is sufficient to describe patterns in bacterial alpha- and beta-diversity?2012In: Environmental Microbiology Reports, ISSN 1758-2229, E-ISSN 1758-2229, Vol. 4, no 3, p. 367-372Article in journal (Refereed)
    Abstract [en]

    The vastness of microbial diversity implies that an almost infinite number of individuals needs to be identified to accurately describe such communities. Practical and economical constraints may therefore prevent appropriate study designs. However, for many questions in ecology it is not essential to know the actual diversity but rather the trends among samples thereof. It is, hence, important to know to what depth microbial communities need to be sampled to accurately measure trends in diversity. We used three data sets of freshwater and sediment bacteria, where diversity was explored using 454 pyrosequencing. Each data set contained 615 communities from which 15 00020 000 16S rRNA gene sequences each were obtained. These data sets were subsampled repeatedly to 10 different depths down to 200 sequences per community. Diversity estimates varied with sequencing depth, yet, trends in diversity among samples were less sensitive. We found that 1000 denoised sequences per sample explained to 90% the trends in beta-diversity (Bray-Curtis index) among samples observed for 15 00020 000 sequences. Similarly, 5000 denoised sequences were sufficient to describe trends in a-diversity (Shannon index) with the same accuracy. Further, 5000 denoised sequences captured to more than 80% the trends in Chao1 richness and Pielou's evenness.

  • 232.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology.
    Methods to Prepare DNA for Efficient Massive Sequencing2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity.

    The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding.

    Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments.

  • 233.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gruselius, Joel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystedt, Björn
    Science for Life Laboratory, Stockholm University, Department of Biochemistry and Biophysics, Stockholm.
    Lexow, Preben
    Käller, Max
    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.
    Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, p. 1186-Article in journal (Refereed)
    Abstract [en]

    Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.

  • 234.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Finn, Terje-Hegge
    Foam, Napoleon
    Pettersson, Erik
    Käller, Max
    Wirta, Valtteri
    Lexow, Preben
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Endonuclease specificity and sequence dependence of Type IIS restriction enzymes2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 1, article id e0117059Article in journal (Refereed)
    Abstract [en]

    Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  • 235.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Pettersson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Increased Throughput by Parallelization of Library Preparation for Massive Sequencing2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 3, p. e10029-Article in journal (Refereed)
    Abstract [en]

    Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

  • 236. Lundmark, Anna
    et al.
    Davanian, Haleh
    Bage, Tove
    Johannsen, Gunnar
    Koro, Catalin
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Yucel-Lindberg, Tulay
    Transcriptome analysis reveals mucin 4 to be highly associated with periodontitis and identifies pleckstrin as a link to systemic diseases2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 18475Article in journal (Refereed)
    Abstract [en]

    The multifactorial chronic inflammatory disease periodontitis, which is characterized by destruction of tooth-supporting tissues, has also been implicated as a risk factor for various systemic diseases. Although periodontitis has been studied extensively, neither disease-specific biomarkers nor therapeutic targets have been identified, nor its link with systemic diseases. Here, we analyzed the global transcriptome of periodontitis and compared its gene expression profile with those of other inflammatory conditions, including cardiovascular disease (CVD), rheumatoid arthritis (RA), and ulcerative colitis (UC). Gingival biopsies from 62 patients with periodontitis and 62 healthy subjects were subjected to RNA sequencing. The up-regulated genes in periodontitis were related to inflammation, wounding and defense response, and apoptosis, whereas down-regulated genes were related to extracellular matrix organization and structural support. The most highly up-regulated gene was mucin 4 (MUC4), and its protein product was confirmed to be over-expressed in periodontitis. When comparing the expression profile of periodontitis with other inflammatory diseases, several gene ontology categories, including inflammatory response, cell death, cell motion, and homeostatic processes, were identified as common to all diseases. Only one gene, pleckstrin (PLEK), was significantly overexpressed in periodontitis, CVD, RA, and UC, implicating this gene as an important networking link between these chronic inflammatory diseases.

  • 237.
    Mardinoglu, Adil
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Heiker, John T.
    Gaertner, Daniel
    Bjornson, Elias
    Schoen, Michael R.
    Flehmig, Gesine
    Kloeting, Nora
    Krohn, Knut
    Fasshauer, Mathias
    Stumvoll, Michael
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Blueher, Matthias
    Extensive weight loss reveals distinct gene expression changes in human subcutaneous and visceral adipose tissue2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 14841Article in journal (Refereed)
    Abstract [en]

    Weight loss has been shown to significantly improve Adipose tissue (AT) function, however changes in AT gene expression profiles particularly in visceral AT (VAT) have not been systematically studied. Here, we tested the hypothesis that extensive weight loss in response to bariatric surgery (BS) causes AT gene expression changes, which may affect energy and lipid metabolism, inflammation and secretory function of AT. We assessed gene expression changes by whole genome expression chips in AT samples obtained from six morbidly obese individuals, who underwent a two step BS strategy with sleeve gastrectomy as initial and a Roux-en-Y gastric bypass as second step surgery after 12 +/- 2 months. Global gene expression differences in VAT and subcutaneous (S) AT were analyzed through the use of genome-scale metabolic model (GEM) for adipocytes. Significantly altered gene expressions were PCR-validated in 16 individuals, which also underwent a two-step surgery intervention. We found increased expression of cell death-inducing DFFA-like effector a (CIDEA), involved in formation of lipid droplets in both fat depots in response to significant weight loss. We observed that expression of the genes associated with metabolic reactions involved in NAD+, glutathione and branched chain amino acid metabolism are significantly increased in AT depots after surgery-induced weight loss.

  • 238. Mardinoglu, Adil
    et al.
    Kampf, Caroline
    Asplund, Anna
    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.
    Edlund, Karolina
    Blüher, Matthias
    Pontén, Fredrik
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Defining the Human Adipose Tissue Proteome To Reveal Metabolic Alterations in Obesity2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 11, p. 5106-5119Article in journal (Refereed)
    Abstract [en]

    White adipose tissue (WAT) has a major role in the progression of obesity. Here, we combined data from RNA-Seq and antibody-based immunohistochemistry to describe the normal physiology of human WAT obtained from three female subjects and explored WAT-specific genes by comparing WAT to 26 other major human tissues. Using the protein evidence in WAT, we validated the content of a genome-scale metabolic model for adipocytes. We employed this high-quality model for the analysis of subcutaneous adipose tissue (SAT) gene expression data obtained from subjects included in the Swedish Obese Subjects Sib Pair study to reveal molecular differences between lean and obese individuals. We integrated SAT gene expression and plasma metabolomics data, investigated the contribution of the metabolic differences in the mitochondria of SAT to the occurrence of obesity, and eventually identified cytosolic branched-chain amino acid (BCAA) transaminase 1 as a potential target that can be used for drug development. We observed decreased glutaminolysis and alterations in the BCAAs metabolism in SAT of obese subjects compared to lean subjects. We also provided mechanistic explanations for the changes in the plasma level of BCAAs, glutamate, pyruvate, and alpha-ketoglutarate in obese subjects. Finally, we validated a subset of our model-based predictions in 20 SAT samples obtained from 10 lean and 10 obese male and female subjects.

  • 239.
    Mardinoglu, Adil
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Editorial: The Impact of Systems Medicine on Human Health and Disease2016In: Frontiers in Physiology, ISSN 1664-042X, E-ISSN 1664-042X, Vol. 7, article id 552Article in journal (Other academic)
  • 240.
    Mardinoglu, Adil
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Shoaie, Saeed
    Bergentall, Mattias
    Ghaffari, Pouyan
    Zhang, Cheng
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Larsson, Erik
    Backhed, Fredrik
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    The gut microbiota modulates host amino acid and glutathione metabolism in mice2015In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 11, no 10, article id 834Article in journal (Refereed)
    Abstract [en]

    The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV-R) and germ-free (GF) mice using gene expression data and tissue-specific genome-scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue-specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV-R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N-acetylated AAs in the hepatic portal vein of CONV-R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV-R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice.

  • 241. Mardinoglu, Adil
    et al.
    Ågren, Rasmus
    Kampf, Caroline
    Asplund, Anna
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in patients with non-alcoholic fatty liver disease2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 3083-Article in journal (Refereed)
    Abstract [en]

    Several liver disorders result from perturbations in the metabolism of hepatocytes, and their underlying mechanisms can be outlined through the use of genome-scale metabolic models (GEMs). Here we reconstruct a consensus GEM for hepatocytes, which we call iHepatocytes2322, that extends previous models by including an extensive description of lipid metabolism. We build iHepatocytes2322 using Human Metabolic Reaction 2.0 database and proteomics data in Human Protein Atlas, which experimentally validates the incorporated reactions. The reconstruction process enables improved annotation of the proteomics data using the network centric view of iHepatocytes2322. We then use iHepatocytes2322 to analyse transcriptomics data obtained from patients with non-alcoholic fatty liver disease. We show that blood concentrations of chondroitin and heparan sulphates are suitable for diagnosing non-alcoholic steatohepatitis and for the staging of non-alcoholic fatty liver disease. Furthermore, we observe serine deficiency in patients with NASH and identify PSPH, SHMT1 and BCAT1 as potential therapeutic targets for the treatment of non-alcoholic steatohepatitis.

  • 242. McCormack, T.
    et al.
    Frings, O.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sonnhammer, E. L. L.
    Statistical Assessment of Crosstalk Enrichment between Gene Groups in Biological Networks2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 1, p. e54945-Article in journal (Refereed)
    Abstract [en]

    Motivation: Analyzing groups of functionally coupled genes or proteins in the context of global interaction networks has become an important aspect of bioinformatic investigations. Assessing the statistical significance of crosstalk enrichment between or within groups of genes can be a valuable tool for functional annotation of experimental gene sets. Results: Here we present CrossTalkZ, a statistical method and software to assess the significance of crosstalk enrichment between pairs of gene or protein groups in large biological networks. We demonstrate that the standard z-score is generally an appropriate and unbiased statistic. We further evaluate the ability of four different methods to reliably recover crosstalk within known biological pathways. We conclude that the methods preserving the second-order topological network properties perform best. Finally, we show how CrossTalkZ can be used to annotate experimental gene sets using known pathway annotations and that its performance at this task is superior to gene enrichment analysis (GEA). Availability and Implementation: CrossTalkZ (available at http://sonnhammer.sbc.su.se/download/software/CrossTalkZ/) is implemented in C++, easy to use, fast, accepts various input file formats, and produces a number of statistics. These include z-score, p-value, false discovery rate, and a test of normality for the null distributions.

  • 243. McIlwain, Sean
    et al.
    Tamura, Kaipo
    Kertesz-Farkas, Attila
    Grant, Charles E.
    Diament, Benjamin
    Frewen, Barbara
    Howbert, J. Jeffry
    Hoopmann, Michael R.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, SeRC - Swedish e-Science Research Centre. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Eng, Jimmy K.
    MacCoss, Michael J.
    Noble, William Stafford
    Crux: Rapid Open Source Protein Tandem Mass Spectrometry Analysis2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 10, p. 4488-4491Article in journal (Refereed)
    Abstract [en]

    Efficiently and accurately analyzing big protein tandem mass spectrometry data sets requires robust software that incorporates state-of-the-art computational, machine learning, and statistical methods. The Crux mass spectrometry analysis software toolkit (http://cruxtoolkit.sourceforge.net) is an open source project that aims to provide users with a cross-platform suite of analysis tools for interpreting protein mass spectrometry data.

  • 244. Milani, Lili
    et al.
    Gupta, Manu
    Andersen, Malin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Dhar, Sumeer
    Fryknäs, Mårten
    Isaksson, Anders
    Larsson, Rolf
    Syvänen, Ann-Christine
    Allelic imbalance in gene expression as a guide to cis-acting regulatory single nucleotide polymorphisms in cancer cells2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 5, p. E34-Article in journal (Refereed)
    Abstract [en]

    Using the relative expression levels of two SNIP alleles of a gene in the same sample is an effective approach for identifying cis-acting regulatory SNPs (rSNPs). In the current study, we established a process for systematic screening for cis-acting rSNPs using experimental detection of Al as an initial approach. We selected 160 expressed candidate genes that are involved in cancer and anticancer drug resistance for analysis of All in a panel of cell lines that represent different types of cancers and have been well characterized for their response patterns against anticancer drugs. Of these genes, 60 contained heterozygous SNPs in their coding regions, and 41 of the genes displayed imbalanced expression of the two cSNP alleles. Genes that displayed Al were subjected to bioinformatics-assisted identification of rSNPs that alter the strength of transcription factor binding. rSNPs in 15 genes were subjected to electrophoretic mobility shift assay, and in eight of these genes (APC, BCL2, CCND2, MLH1, PARP1, SLIT2, YES1, XRCC1) we identified differential protein binding from a nuclear extract between the SNIP alleles. The screening process allowed us to zoom in from 160 candidate genes to eight genes that may contain functional rSNPs in their promoter regions.

  • 245. Mok, Bobo W.
    et al.
    Ribacke, Ulf
    Rasti, Niloofar
    Kironde, Fred
    Chen, Qijun
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wahlgren, Mats
    Default Pathway of var2csa Switching and Translational Repression in Plasmodium falciparum2008In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 3, no 4, p. e1982-Article in journal (Refereed)
    Abstract [en]

    Antigenic variation is a subtle process of fundamental importance to the survival of a microbial pathogen. In Plasmodium falciparum malaria, PfEMP1 is the major variable antigen and adhesin expressed at the surface of the infected erythrocyte, which is encoded for by members of a family of 60 var-genes. Peri-nuclear repositioning and epigenetic mechanisms control their mono-allelic expression. The switching of PfEMP1 depends in part on variable transition rates and short-lived immune responses to shared minor epitopes. Here we show var-genes to switch to a common gene that is highly transcribed, but sparsely translated into PfEMP1 and not expressed at the erythrocyte surface. Highly clonal and adhesive P. falciparum, which expressed distinct var-genes and the corresponding PfEMP1s at onset, were propagated without enrichment or panning. The parasites successively and spontaneously switched to transcribe a shared var-gene (var2csa) matched by the loss of PfEMP1 surface expression and host cell-binding. The var2csa gene repositioned in the peri-nuclear area upon activation, away from the telomeric clusters and heterochromatin to transcribe spliced, full-length RNA. Despite abundant transcripts, the level of intracellular PfEMP1 was low suggesting post-transcriptional mechanisms to partake in protein expression. In vivo, off-switching and translational repression may constitute one pathway, among others, coordinating PfEMP1 expression.

  • 246. Moreno, Samuel Boiso
    et al.
    Zackrisson, Anna-Lena
    Falk, Ingrid Jakobsen
    Karlsson, Louise
    Carlsson, Björn
    Tillmar, Andreas
    Kugelberg, Fredrik C.
    Ahlner, Johan
    Hägg, Staffan
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    ABCB1 gene polymorphisms are associated with suicide in forensic autopsies2013In: Pharmacogenetics & Genomics, ISSN 1744-6872, E-ISSN 1744-6880, Vol. 23, no 9, p. 463-469Article in journal (Refereed)
    Abstract [en]

    Background Polymorphisms in ABCB1 have the ability to affect both the function and the expression of the transporter protein P-glycoprotein and may lead to an altered response for many drugs including some antidepressants and antipsychotics.Objective The aim of this study was to examine the impact of the ABCB1 polymorphisms 1199G>A, 1236C>T, 2677G>T/A, and 3435C>T in deaths by suicide.Patients and methods A total of 998 consecutive Swedish forensic autopsies performed in 2008 in individuals 18 years of age or older, where femoral blood was available and a toxicological screening had been performed, were investigated. Genotypes were assessed with pyrosequencing and information on the cause and manner of each death was obtained from the forensic pathology and toxicology databases.Results There was a significantly higher frequency of the T allele at positions 1236, 2677, and 3435 among the suicide cases compared with the nonsuicide cases.Conclusion Our result from forensic cases suggests that ABCB1 polymorphisms are associated with an increased risk for completed suicides. The biological mechanisms involved and the clinical implications for these findings are largely unknown and need to be examined further.

  • 247. Moruz, Luminita
    et al.
    Hoopmann, Michael R.
    Rosenlund, Magnus
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Granholm, Viktor
    Moritz, Robert L.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    Mass Fingerprinting of Complex Mixtures: Protein Inference from High-Resolution Peptide Masses and Predicted Retention Times2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 12, p. 5730-5741Article in journal (Refereed)
    Abstract [en]

    In typical shotgun experiments, the mass spectrometer records the masses of a large set of ionized analytes but fragments only a fraction of them. In the subsequent analyses, normally only the fragmented ions are used to compile a set of peptide identifications, while the unfragmented ones are disregarded. In this work, we show how the unfragmented ions, here denoted MS1-features, can be used to increase the confidence of the proteins identified in shotgun experiments. Specifically, we propose the usage of in silico mass tags, where the observed MS1-features are matched against de novo predicted masses and retention times for all peptides derived from a sequence database. We present a statistical model to assign protein-level probabilities based on the MS1-features and combine this data with the fragmentation spectra. Our approach was evaluated for two triplicate data sets from yeast and human, respectively, leading to up to 7% more protein identifications at a fixed protein-level false discovery rate of 1%. The additional protein identifications were validated both in the context of the mass spectrometry data and by examining their estimated transcript levels generated using RNA-Seq. The proposed method is reproducible, straightforward to apply, and can even be used to reanalyze and increase the yield of existing data sets.

  • 248. Moruz, Luminita
    et al.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    GradientOptimizer: An open-source graphical environment for calculating optimized gradients in reversed-phase liquid chromatography2014In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 14, no 12, p. 1464-1466Article in journal (Refereed)
    Abstract [en]

    We here present GradientOptimizer, an intuitive, lightweight graphical user interface to design nonlinear gradients for separation of peptides by reversed-phase liquid chromatography. The software allows to calculate three types of nonlinear gradients, each of them optimizing a certain retention time distribution of interest. GradientOptimizer is straightforward to use, requires minimum processing of the input files, and is supported under Windows, Linux, and OS X platforms. The software is open-source and can be downloaded under an Apache 2.0 license at https://github.com/statisticalbiotechnology/NonlinearGradientsUI.

  • 249. Moruz, Luminita
    et al.
    Pichler, Peter
    Stranzl, Thomas
    Mechtler, Karl
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    Optimized Nonlinear Gradients for Reversed-Phase Liquid Chromatography in Shotgun Proteomics2013In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 16, p. 7777-7785Article in journal (Refereed)
    Abstract [en]

    Reversed-phase liquid chromatography has become the preferred method for separating peptides in most of the mass spectrometry-based proteomics workflows of today. In the way the technique is typically applied, the peptides are released from the chromatography column by the gradual addition of an organic buffer according to a linear function. However, when applied to complex peptide mixtures, this approach leads to unequal spreads of the peptides over the chromatography time. To address this, we investigated the use of nonlinear gradients, customized for each setup at hand. We developed an algorithm to generate optimized gradient functions for shotgun proteomics experiments and evaluated it for two data sets consisting each of four replicate runs of a human complex sample. Our results show that the optimized gradients produce a more even spread of the peptides over the chromatography run, while leading to increased numbers of confident peptide identifications. In addition, the list of peptides identified using nonlinear gradients differed considerably from those found with the linear ones, suggesting that such gradients can be a valuable tool for increasing the proteome coverage of mass spectrometry-based experiments.

  • 250. Moruz, Luminita
    et al.
    Staes, An
    Foster, Joseph M.
    Hatzou, Maria
    Timmerman, Evy
    Martens, Lennart
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chromatographic retention time prediction for posttranslationally modified peptides2012In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 12, no 8, p. 1151-1159Article in journal (Refereed)
    Abstract [en]

    Retention time prediction of peptides in liquid chromatography has proven to be a valuable tool for mass spectrometry-based proteomics, especially in designing more efficient procedures for state-of-the-art targeted workflows. Additionally, accurate retention time predictions can also be used to increase confidence in identifications in shotgun experiments. Despite these obvious benefits, the use of such methods has so far not been extended to (posttranslationally) modified peptides due to the absence of efficient predictors for such peptides. We here therefore describe a new retention time predictor for modified peptides, built on the foundations of our existing Elude algorithm. We evaluated our software by applying it on five types of commonly encountered modifications. Our results show that Elude now yields equally good prediction performances for modified and unmodified peptides, with correlation coefficients between predicted and observed retention times ranging from 0.93 to 0.98 for all the investigated datasets. Furthermore, we show that our predictor handles peptides carrying multiple modifications as well. This latest version of Elude is fully portable to new chromatographic conditions and can readily be applied to other types of posttranslational modifications. Elude is available under the permissive Apache2 open source License at or can be run via a web-interface at.

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