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  • 301.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Domestication of dogs2007In: The Behavioural Biology of Dogs, CABI Publishing, 2007, p. 21-37Chapter in book (Refereed)
  • 302.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Extensive phenotypic diversity among South Chinese dogs2013In: ISRN Evolutionary Biology, ISSN 2314-4033, Vol. 2013, no Article ID 621836Article in journal (Refereed)
    Abstract [en]

    We describe here a broad diversity in phenotype among dogs in southern China’s rural areas, previously relatively unknown outside of China. These dogs display a much broader spectrum of diversity than is observed for the Indian Pariah Dog and the Australian Dingo, which are of a more uniform type and popularly thought to be typical for South Asian dogs and to represent the primitive morphology of the earliest domestic dogs. We show here that the village dog population of southern China harbors a broad diversity of morphological features, for color, body structure and size, coat texture, ear, and tail set, that are otherwise typically associated with the wide variety of Western dog breeds and assumed to be the result of intense selective breeding. The diversity of southern China’s dogs is cast in the light of mtDNA and Y-chromosome DNA studies showing that the genetic diversity is distinctly higher in southern East Asia than in the rest of the world, indicating that this was the geographical origins of today’s dog. These data suggest that the diverse morphologies of European dogs may have been formed from genetic “building blocks" still present in the dog population of rural southern China.

  • 303.
    Savolainen, Peter
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Fitzsimmons, C.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Numerical Analysis and Computer Science, NADA.
    Andersson, L.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    ESTs from brain and testis of White Leghorn and red junglefowl: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels2005In: Cytogenetic and Genome Research, ISSN 1424-8581, E-ISSN 1424-859X, Vol. 111, no 1, p. 79-87Article in journal (Refereed)
    Abstract [en]

    We report the generation, assembly and annotation of expressed sequence tags (ESTs) from four chicken cDNA libraries, constructed from brain and testis tissue dissected from red junglefowl and White Leghorn. 21,285 5'-end ESTs were generated and assembled into 2,813 contigs and 9,737 singletons, giving 12,549 tentative unique transcripts. The transcripts were annotated using BLAST by matching to known chicken genes or to putative homologues in other species using the major gene/protein databases. The results for these similarity searches are available on www.sbc.su.se/ -arve/chicken. 4,129 (32.9%) of the transcripts remained without a significant match to gene/protein databases, a proportion of unmatched transcripts similar to earlier non-mammalian EST studies. To estimate how many of these transcripts may represent novel genes, they were studied for the presence of coding sequence. It was shown that most of the unique chicken transcripts do not contain coding parts of genes, but it was estimated that at least 400 of the transcripts contain coding sequence, indicating that 3.2% of avian genes belong to previously unknown gene families. Further BLAST search against dbEST left 1,649 (13.1 %) of the transcripts unmatched to any library. The number of completely unmatched transcripts containing coding sequence was estimated at 180, giving a measure of the number of putative novel chicken genes identified in this study. 84.3 % of the identified transcripts were found only in testis tissue, which has been poorly studied in earlier chicken EST studies. Large differences in expression levels were found between the brain and testis libraries for a large number of transcripts, and among the 525 most frequently represented transcripts, there were at least 20 transcripts with significant difference in expression levels between red junglefowl and White Leghorn

  • 304. Seidel, Sascha
    et al.
    Garvalov, Boyan K.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology.
    von Stechow, Louise
    Schaenzer, Anne
    Meletis, Konstantinos
    Wolter, Marietta
    Sommerlad, Daniel
    Henze, Anne-Theres
    Nister, Monica
    Reifenberger, Guido
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisen, Jonas
    Acker, Till
    A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha2010In: Brain, ISSN 0006-8950, E-ISSN 1460-2156, Vol. 133, p. 983-995Article in journal (Refereed)
    Abstract [en]

    Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2 alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.

  • 305. Serang, O.
    et al.
    Cansizoglu, A. E.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Steen, H.
    Steen, J. A.
    Nonparametric bayesian evaluation of differential protein quantification2013In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 12, no 10, p. 4556-4565Article in journal (Refereed)
    Abstract [en]

    Arbitrary cutoffs are ubiquitous in quantitative computational proteomics: maximum acceptable MS/MS PSM or peptide q value, minimum ion intensity to calculate a fold change, the minimum number of peptides that must be available to trust the estimated protein fold change (or the minimum number of PSMs that must be available to trust the estimated peptide fold change), and the "significant" fold change cutoff. Here we introduce a novel experimental setup and nonparametric Bayesian algorithm for determining the statistical quality of a proposed differential set of proteins or peptides. By comparing putatively nonchanging case-control evidence to an empirical null distribution derived from a control-control experiment, we successfully avoid some of these common parameters. We then apply our method to evaluating different fold-change rules and find that for our data a 1.2-fold change is the most permissive of the plausible fold-change rules.

  • 306. Serang, Oliver
    et al.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Solution to Statistical Challenges in Proteomics Is More Statistics, Not Less2015In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 14, no 10, p. 4099-4103Article in journal (Other academic)
    Abstract [en]

    In any high-throughput scientific study, it is often essential to estimate the percent of findings that are actually incorrect. This percentage is called the false discovery rate (abbreviated "FDR"), and it is an invariant (albeit, often unknown) quantity for any well-formed study. In proteomics, it has become common practice to incorrectly conflate the protein FDR (the percent of identified proteins that are actually absent) with protein-level target-decoy, a particular method for estimating the protein-level FDR. In this manner, the challenges of one approach have been used as the basis for an argument that the field should abstain from protein-level FDR analysis altogether or even the suggestion that the very notion of a protein FDR is flawed. As we demonstrate in simple but accurate simulations, not only is the protein-level FDR an invariant concept, when analyzing large data sets, the failure to properly acknowledge it or to correct for multiple testing can result in large, unrecognized errors, whereby thousands of absent proteins (and, potentially every protein in the FASTA database being considered) can be incorrectly identified.

  • 307. Serang, Oliver
    et al.
    Moruz, Luminita
    Hoopmann, Michael R.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Recognizing Uncertainty Increases Robustness and Reproducibility of Mass Spectrometry-based Protein Inferences2012In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 11, no 12, p. 5586-5591Article in journal (Refereed)
    Abstract [en]

    Parsimony and protein grouping are widely employed to enforce economy in the number of identified proteins, with the goal of increasing the quality and reliability of protein identifications; however, in a counterintuitive manner, parsimony and protein grouping may actually decrease the reproducibility and interpretability of protein identifications. We present a simple illustration demonstrating ways in which parsimony and protein grouping may lower the reproducibility or interpretability of results. We then provide an example of a data set where a probabilistic method increases the reproducibility and interpretability of identifications made on replicate analyses of Human Du145 prostate cancer cell lines.

  • 308. Shirley, B. C.
    et al.
    Mucaki, E. J.
    Whitehead, T.
    Costea, Paul Igor
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rogan, P. K.
    Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences2013In: Genomics, Proteomics and Bioinformatics, ISSN 1672-0229, Vol. 11, no 2, p. 77-85Article in journal (Refereed)
    Abstract [en]

    Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.

  • 309. Sievertzon, M.
    et al.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Improving reliability and performance of DNA microarrays2006In: Expert Review of Molecular Diagnostics, ISSN 1473-7159, Vol. 6, no 3, p. 481-492Article, review/survey (Refereed)
    Abstract [en]

    A great many platforms and versions of the microarray technology, with different characteristics and applications, have been developed. This review will describe some key issues in reliability and performance with the two most commonly used platforms for gene expression analysis, in situ-synthesized oligonucleotide microarrays or GeneChip((R)) and spotted microarrays. Some recent advances and new applications within the field will be mentioned briefly.

  • 310.
    Sievertzon, Maria
    et al.
    KTH, School of Biotechnology (BIO).
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO).
    Mercer, Alex
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation2005In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, p. 55-Article in journal (Refereed)
    Abstract [en]

    Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.

    Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.

    Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

  • 311.
    Sigurgeirsson, Benjamin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Emanuelsson, Olof
    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.
    Analysis of stranded information using an automated procedure for strand specific RNA sequencing2014In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 15, no 1, article id 631Article in journal (Refereed)
    Abstract [en]

    Background: Strand specific RNA sequencing is rapidly replacing conventional cDNA sequencing as an approach for assessing information about the transcriptome. Alongside improved laboratory protocols the development of bioinformatical tools is steadily progressing. In the current procedure the Illumina TruSeq library preparation kit is used, along with additional reagents, to make stranded libraries in an automated fashion which are then sequenced on Illumina HiSeq 2000. By the use of freely available bioinformatical tools we show, through quality metrics, that the protocol is robust and reproducible. We further highlight the practicality of strand specific libraries by comparing expression of strand specific libraries to non-stranded libraries, by looking at known antisense transcription of pseudogenes and by identifying novel transcription. Furthermore, two ribosomal depletion kits, RiboMinus and RiboZero, are compared and two sequence aligners, Tophat2 and STAR, are also compared. Results: The, non-stranded, Illumina TruSeq kit can be adapted to generate strand specific libraries and can be used to access detailed information on the transcriptome. The RiboZero kit is very effective in removing ribosomal RNA from total RNA and the STAR aligner produces high mapping yield in a short time. Strand specific data gives more detailed and correct results than does non-stranded data as we show when estimating expression values and in assembling transcripts. Even well annotated genomes need improvements and corrections which can be achieved using strand specific data. Conclusions: Researchers in the field should strive to use strand specific data; it allows for more confidence in the data analysis and is less likely to lead to false conclusions. If faced with analysing non-stranded data, researchers should be well aware of the caveats of that approach.

  • 312.
    Sigurgeirsson, Benjamin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Emanuelsson, Olof
    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.
    Sequencing Degraded RNA Addressed by 3' Tag Counting2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 3, p. e91851-Article in journal (Refereed)
    Abstract [en]

    RNA sequencing has become widely used in gene expression profiling experiments. Prior to any RNA sequencing experiment the quality of the RNA must be measured to assess whether or not it can be used for further downstream analysis. The RNA integrity number (RIN) is a scale used to measure the quality of RNA that runs from 1 (completely degraded) to 10 (intact). Ideally, samples with high RIN (>8) are used in RNA sequencing experiments. RNA, however, is a fragile molecule which is susceptible to degradation and obtaining high quality RNA is often hard, or even impossible when extracting RNA from certain clinical tissues. Thus, occasionally, working with low quality RNA is the only option the researcher has. Here we investigate the effects of RIN on RNA sequencing and suggest a computational method to handle data from samples with low quality RNA which also enables reanalysis of published datasets. Using RNA from a human cell line we generated and sequenced samples with varying RINs and illustrate what effect the RIN has on the basic procedure of RNA sequencing; both quality aspects and differential expression. We show that the RIN has systematic effects on gene coverage, false positives in differential expression and the quantification of duplicate reads. We introduce 3' tag counting (3TC) as a computational approach to reliably estimate differential expression for samples with low RIN. We show that using the 3TC method in differential expression analysis significantly reduces false positives when comparing samples with different RIN, while retaining reasonable sensitivity.

  • 313.
    Sigurgeirsson, Benjamin
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Åmark, Hanna
    Karolinska Inst, Dept Clin Sci & Educ, Unit Obstet & Gynecol, Sjukhusbacken 10, S-11883 Stockholm, Sweden.
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ujvari, Dorina
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden.
    Westgren, Magnus
    Karolinska Inst, Dept Clin Sci Intervent & Technol, Stockholm, Sweden.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gidlöf, Sebastian
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden;Karolinska Inst, Dept Clin Sci Intervent & Technol, Stockholm, Sweden;Karolinska Univ Hosp Huddinge, Dept Obstet & Gynecol, Stockholm, Sweden.
    Comprehensive RNA sequencing of healthy human endometrium at two time points of the menstrual cycle2017In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 96, no 1, p. 24-33Article in journal (Refereed)
    Abstract [en]

    Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from being understood. In this paper, we comprehensively present the transcriptome of the human endometrium by comparing endometrial biopsies from proliferative phase with consecutive biopsies 7-9 days after ovulation. The results show a clear difference in expression between the two time points using both total and small RNA sequencing. A total of 3,297 messenger RNAs (mRNAs), 516 long noncoding RNAs (lncRNAs), and 102 small noncoding RNAs were identified as statistically differentially expressed between the two time points. We show a thorough description of the change in mRNA between the two time points and display lncRNAs, small nucleolar RNAs, and small nuclear RNAs not previously reported in the healthy human endometrium. In conclusion, this paper reports in detail the shift in RNA expression from the proliferative to receptive endometrium. Summary Sentence Messenger RNA, sncRNA, and lncRNA show a clear difference in expression between proliferative phase and 7-9 days after ovulation, thoroughly described together with lncRNA, snoRNA, and snRNA not previously reported in healthy human endometrium.

  • 314.
    Sigurgeirsson, Benjamín
    KTH, School of Biotechnology (BIO), Gene Technology.
    Analysis of RNA and DNA sequencing data: Improved bioinformatics applications2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Massively parallel sequencing has rapidly revolutionized DNA and RNA research. Sample preparations are steadfastly advancing, sequencing costs have plummeted and throughput is ever growing. This progress has resulted in exponential growth in data generation with a corresponding demand for bioinformatic solutions. This thesis addresses methodological aspects of this sequencing revolution and applies it to selected biological topics.

    Papers I and II are technical in nature and concern sample preparation and data anal- ysis of RNA sequencing data. Paper I is focused on RNA degradation and paper II on generating strand specific RNA-seq libraries.

    Paper III and IV deal with current biological issues. In paper III, whole exomes of cancer patients undergoing chemotherapy are sequenced and their genetic variants associ- ated to their toxicity induced adverse drug reactions. In paper IV a comprehensive view of the gene expression of the endometrium is assessed from two time points of the menstrual cycle.

    Together these papers show relevant aspects of contemporary sequencing technologies and how it can be applied to diverse biological topics. 

  • 315.
    Sigurgeirsson, Benjamín
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Svedberg, Anna
    Björn, Niclas
    Pradhananga, Sailendra
    Brandén, Eva
    Koyi, Hirsh
    Lewensohn, Rolf
    DePetris, Luigi
    Lundeberg, Joakim
    Gréen, Henrik
    Genetic association of gemcitabine/carboplatin induced myelosuppression in patients with non-small cell lung cancer using whole exome sequencingManuscript (preprint) (Other academic)
    Abstract [en]

    Purpose: Chemotherapy induced myelosuppression is a recurrent problem in cancer treatment, both for the patients’ quality of life and response. Severe hematological toxicities lead to dose reduction, postponed or ceased treatment, affecting the treatment effect. Identifying genetic markers associated with toxicity is an important factor for individualized chemotherapy and might increase the overall effect of the treatment.

    Material and methods: Non-small cell lung cancer patients undergoing gemcitabine/carboplatin chemotherapy were included and their exomes were sequenced. Genetic variants from 212 exomes were correlated to thrombocytopenia, leukopenia, and neutropenia on single nucleotide and gene level. Results were processed through enrichment analysis and variants were validated using externally available datasets.

    Results: SNV analysis identified 103, 131 and 112 variants to be associated with thrombocytopenia, leukopenia and neutropenia, respectively. Gene based analysis identified 21, 54 and 31 genes to be associated with thrombocytopenia, leukopenia and neutropenia, respectively. Using external data sets 8, 26 and 9 SNVs were validated through linkage disequilibrium for thrombocytopenia, leukopenia and neutropenia, respectively.

    The variant rs61739531 (CADD = 25.7) in the gene MYO1G was identified to be associated with high toxicity in all forms of myelosuppression. Validated variants include rs6118 (CADD = 22.3) in SERPINA5, rs16910526 (CADD = 35.0) in CLEC7A and rs79350244 (CADD = 24.2) in DNAH2. Enrichment analysis of associated genes identified the pathways hemostasis, HIF-1 alpha transcription factor network and vitamin B12 metabolism to be involved in thrombocytopenia, leukopenia and neutropenia, respectively.

    Factors involved in megakaryocyte development and platelet production, was also associated with thrombocytopenia for three genes JMJD1C with the variant rs34491125 (CADD = 22.1), DOCK8 with the variant rs10491684 (CADD = 11.7) and CAPZA2 based on three variants in the gene based analysis.

    Conclusion: The results highlight genetic markers and relevant pathways associated with chemotherapy induced myelosuppression and form a strong foundation for further investigation into toxicity induced myelosuppression. 

  • 316. Sigurgeirsson, Benjamín
    et al.
    Åmark, Hanna
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ujvari, Dorina
    Westgren, Magnus
    Lundeberg, Joakim
    Gidlöf, Sebastian
    Comprehensive RNA sequencing of healthy human endometrium at two time points of the menstrual cycle2016Manuscript (preprint) (Other academic)
    Abstract [en]

    Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from understood. In this paper we comprehensively present the transcriptome of the human endometrium by comparing endometrial biopsies from proliferative phase with consecutive biopsies 7-9 days after ovulation. The results show a clear difference in expression between the two time points using both total and small RNA sequencing.  3297 mRNAs, 516 long non-coding RNAs and 102 small non-coding RNAs were identified as statistically differentially expressed between the two time points. We show a thorough description of the change in mRNA between the two time points and display lncRNAs, snoRNAs and snRNAs not previously reported in the healthy human endometrium. In conclusion this paper reports in detail the shift in RNA expression from the proliferative to receptive endometrium.

  • 317.
    Sillén, Anna
    et al.
    Karolinska Institutet.
    Andrade, Jorge
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lilius, Lena
    Karolinska Institutet.
    Forsell, Charlotte
    Karolinska Institutet.
    Axelman, Karin
    Karolinska Institutet.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Winblad, Bengt
    Karolinska Institutet.
    Graff, Caroline
    Karolinska Institutet.
    Expanded high-resolution genetic study of 109 Swedish families with Alzheimer's disease2008In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 16, no 2, p. 202-208Article in journal (Refereed)
    Abstract [en]

    Alzheimer's disease (AD) is a neurodegenerative disease that affects approximately 20 million persons all over the world. There are both sporadic and familial forms of AD. We have previously reported a genome-wide linkage analysis on 71 Swedish AD families using 365 genotyped microsatellite markers. In this study, we increased the number of individuals included in the original 71 analysed families besides adding 38 new families. These 109 families were genotyped for 1100 novel microsatellite markers. The present study reports on the linkage data generated from the non-overlapping genotypes from the first genome scan and the genotypes of the present scan, which results in a total of 1289 successfully genotyped markers at an average density of 2.85 cM on 468 individuals from 109 AD families. Non-parametric linkage analysis yielded a significant multipoint LOD score in chromosome 19q13, the region harbouring the major susceptibility gene APOE, both for the whole set of families (LOD = 5.0) and the APOE epsilon 4-positive subgroup made up of 63 families (LOD = 5.3). Other suggestive linkage peaks that were observed in the original genome scan of 71 Swedish AD families were not detected in this extended analysis, and the previously reported linkage signals in chromosomes 9, 10 and 12 were not replicated.

  • 318. Sillén, Anna
    et al.
    Brohede, Jesper
    Lilius, Lena
    Forsell, Charlotte
    Andrade, Jorge
    KTH, School of Biotechnology (BIO), Gene Technology.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ebise, Hayao
    Winblad, Bengt
    Graff, Caroline
    Linkage to 20p13 including the ANGPT4 gene in families with mixed Alzheimer's disease and vascular dementia2010In: Journal of Human Genetics, ISSN 1434-5161, E-ISSN 1435-232X, Vol. 55, no 10, p. 649-655Article in journal (Refereed)
    Abstract [en]

    This study aimed at identifying novel susceptibility genes for a mixed phenotype of Alzheimer's disease and vascular dementia. Results from a genome scan showed strongest linkage to 20p13 in 18 families, and subsequent fine mapping was performed with both microsatellites and single-nucleotide polymorphisms in 18 selected candidate transcripts in an extended sample set of 30 families. The multipoint linkage peak was located at marker rs2144151 in the ANGPT4 gene, which is a strong candidate gene for vascular disease because of its involvement in angiogenesis. Although the significance of the linkage decreased, we find this result intriguing, considering that we included additional families, and thus the reduced linkage signal may be caused by genetic heterogeneity.

  • 319. Sivik, T.
    et al.
    Vikingsson, S.
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jansson, A.
    Expression patterns of 17β-hydroxysteroid dehydrogenase 14 in human tissues2012In: Hormone and Metabolic Research, ISSN 0018-5043, E-ISSN 1439-4286, Vol. 44, no 13, p. 949-956Article in journal (Refereed)
    Abstract [en]

    17βHSD enzymes catalyze the stereospecific oxidation/reduction at carbon 17β of androgens and estrogens, and are important players in intracrine sex hormone synthesis. The biological relevance of 17βHSD14, first named retSDR3, is largely unknown. We generated and validated an antibody targeting the 17βHSD14 antigen and used this for immunohistochemical evaluation of expression patterns in 33 healthy human tissues. Furthermore, sex steroid conversional activity in HSD17B14 overexpressing HEK293 and MCF10A cells was investigated by assessing interconversion products of estrone, estradiol, androstenedione, testosterone, and dehydroepiandrosterone. Immunohistochemical staining patterns of 17βHSD14 with the enzyme being primarily expressed in glandular epithelial tissue reveal an enzyme with possible implications in the secretion or conversion of externally derived compounds. A role for 17βHSD14 in sex steroid metabolism is supported by the finding that 17HSD14 oxidizes both estradiol and testosterone into less bioactive steroid metabolites estrone and androstenedione, respectively.

  • 320. Snir, O.
    et al.
    Widhe, M.
    von Spee, C.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Padyukov, L.
    Lundberg, K.
    Engstrom, A.
    Venables, P. J.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Holmdahl, R.
    Klareskog, L.
    Malmstrom, V.
    Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles2009In: Annals of the Rheumatic Diseases, ISSN 0003-4967, E-ISSN 1468-2060, Vol. 68, no 5, p. 736-743Article in journal (Refereed)
    Abstract [en]

    Background: Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis ( RA), and associate with HLA-DRB1 shared epitope (SE) alleles. Objective: To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles. Methods: Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, alpha-enolase peptide-1 and the C1-epitope of type II collagen (C1(III)). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed. Results: 72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients' sera and in 41% for both citrullinated alpha-enolase peptide-1 and citrullinated C1(III). These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles. Conclusions: Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.

  • 321.
    Solnestam, Beata W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Elin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, A. O.
    Laddach, N.
    Sahlén, Pellin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoringManuscript (preprint) (Other academic)
  • 322.
    Song, Yajing
    KTH, School of Biotechnology (BIO), Gene Technology.
    Advances in DNA Detection2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA detection technologies have an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis, food safety evaluation, and environmental monitoring. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used in research laboratories, in clinical and forensic practice.

    The first aim of this thesis is to unravel the potential of DNA detection on cellulose filter paper and further investigate the filter paper as a viable candidate for DNA array support. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on the active paper using fluorescence. In Paper II, we addressed visual detection with magnetic beads and increased the detection throughput on the active filter paper, which required no instrumentation. Second, in pursuit of a rapid, sensitive and specific pathogen diagnosis in bloodstream infection (BSI), we explored the possibility of rare DNA detection in the presence of a high amount of background DNA by an enzymatic reaction, which can remove background DNA while enriching the rare DNA fraction. In order to overcome the challenge of the second objective, we developed a chemical fragmentation method to increase the efficiency of enzymatic digestion and hybridization. In addition, DNA library preparation for massively parallel sequencing may benefit from the chemical fragmentation. Paper III and Paper IV introduce this work.

    The findings in Paper I showed that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups, while preserving the base pairing ability of the oligonucleotides. In Paper II, visual detection of DNA on active paper was achieved without instrumentation, based on the natural colour of magnetic beads. Furthermore, the possibility to increase the throughput of DNA detection on active paper was demonstrated by successful multiplex detection. In Paper III, the developed chemical fragmentation was verified to be suitable for DNA library preparation in massively parallel sequencing. The fragmentation technique is simple to perform, cost-effective and amenable to automation. In Paper IV, a limited amount of E.coli DNA was detected amid a much larger amount of human background DNA in a BSI model, which comprises of human and E.coli amplicons with an abundance ratio of 108. Human β-actin amplicons were suppressed 105-fold, whereas the E.coli amplicons remained unaffected. The model system was applied to and improved with clinical plasma and blood samples from septic patients.

  • 323.
    Song, Yajing
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Advances in DNA Detection on Paper Chips2013Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice.

    The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner).

    The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers.

    In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample.

  • 324.
    Song, Yajing
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Giske, Christian G.
    Gille-Johnson, P.
    Emanuelsson, Olof
    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.
    Gyarmati, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nuclease-Assisted Suppression of Human DNA Background in Sepsis2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, p. e103610-Article in journal (Refereed)
    Abstract [en]

    Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C(0)t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C(0)t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.

  • 325.
    Song, Yajing
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gyarmati, Péter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Araújo, Ana Catarina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brumer, Harry, III
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Ståhl, Patrik L.
    Visual detection of DNA on paper chips2014In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 86, no 3, p. 1575-1582Article in journal (Refereed)
    Abstract [en]

    On-site DNA analysis for diagnostic or forensic purposes is much anticipated in the future of molecular testing. Yet the challenges to achieve this goal remain large with rapid and inexpensive detection and visualization being key factors for any portable analysis system. We have developed a filter paper-based nucleic acid assay, which is able to identify and distinguish dog and human genomic and mitochondrial samples in a forensic setting. The filter paper material allows for transport by capillary force of the sample DNA through the detection surface, allowing the targets to hybridize specifically to their complementary capture sequences. Coupling micrometer-sized beads to DNA allows the results to be visualized by the naked eye, enabling instant, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation.

  • 326.
    Stahl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Salmen, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundmark, Anna
    KTH, School of Biotechnology (BIO), Gene Technology.
    Navarro, Jose Fernandez
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnusson, Jens
    Giacomello, Stefania
    KTH, School of Biotechnology (BIO), Gene Technology.
    Asp, Michaela
    Westholm, Jakub O.
    Huss, Mikael
    Mollbrink, Annelie
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linnarsson, Sten
    Codeluppi, Simone
    Borg, Ake
    Ponten, Fredrik
    Costea, Paul Igor
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlen, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mulder, Jan
    Bergmann, Olaf
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisen, Jonas
    Visualization and analysis of gene expression in tissue sections by spatial transcriptomics2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 353, no 6294, p. 78-82Article in journal (Refereed)
    Abstract [en]

    Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

  • 327.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Enabling massive genomic and transcriptomic analysis2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid advances have enormously expanded sequencing opportunities and applications, but also imposed heavy strains on steps prior to sequencing, as well as the subsequent handling and analysis of the massive amounts of sequence data that are generated, in order to exploit the full capacity of these novel platforms. The work presented in this thesis (based on six appended papers) has contributed to balancing the sequencing process by developing techniques to accelerate the rate-limiting steps prior to sequencing, facilitating sequence data analysis and applying the novel techniques to address biological questions.

     

    Papers I and II describe techniques to eliminate expensive and time-consuming preparatory steps through automating library preparation procedures prior to sequencing. The automated procedures were benchmarked against standard manual procedures and were found to substantially increase throughput while maintaining high reproducibility. In Paper III, a novel algorithm for fast classification of sequences in complex datasets is described. The algorithm was first optimized and validated using a synthetic metagenome dataset and then shown to enable faster analysis of an experimental metagenome dataset than conventional long-read aligners, with similar accuracy. Paper IV, presents an investigation of the molecular effects on the p53 gene of exposing human skin to sunlight during the course of a summer holiday. There was evidence of previously accumulated persistent p53 mutations in 14% of all epidermal cells. Most of these mutations are likely to be passenger events, as the affected cell compartments showed no apparent growth advantage. An annual rate of 35,000 novel sun-induced persistent p53 mutations was estimated to occur in sun-exposed skin of a human individual.  Paper V, assesses the effect of using RNA obtained from whole cell extracts (total RNA) or cytoplasmic RNA on quantifying transcripts detected in subsequent analysis. Overall, more differentially detected genes were identified when using the cytoplasmic RNA. The major reason for this is related to the reduced complexity of cytoplasmic RNA, but also apparently due (at least partly) to the nuclear retention of transcripts with long, structured 5’- and 3’-untranslated regions or long protein coding sequences. The last paper, VI, describes whole-genome sequencing of a large, consanguineous family with a history of Leber hereditary optic neuropathy (LHON) on the maternal side. The analysis identified new candidate genes, which could be important in the aetiology of LHON. However, these candidates require further validation before any firm conclusions can be drawn regarding their contribution to the manifestation of LHON.

  • 328.
    Stranneheim, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bjursell, Magnus
    Naess, Karin
    Engvall, Martin
    Lesko, Nicole
    Wibom, Rolf
    Von Döbeln, Ulrika
    Tigerschiöld, Stephanie
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Holmberg, Kristina
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Wedell, Anna
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    ­A strategy for identifying nuclear modifier genes by massively parallel whole-genome sequencingManuscript (preprint) (Other academic)
    Abstract [en]

    Leber hereditary optic neuropathy (LHON) results from mutations in mtDNA, butadditional factors are required for disease expression. LHON is thus a model for theconcept of modifiers affecting expression of single gene diseases. No modifier factorhas yet been clearly identified. Here we describe a large, consanguineous familyaffected by LHON with offspring showing variable disease expression. This providesan opportunity to investigate the presence of nuclear modifiers in homozygousgenomic regions. We analyzed genomes from six members, parents and foursiblings. Each genome was sequenced to >23x coverage and approximately 3.8million single nucleotide variants and small indels per individual were called, where17,000‐20,000 were located in the exome. As a first step, we hypothesize that amodifier gene affecting penetrance of the LHON mutation, and another modifiergene predisposing to an aggravated phenotype, are located in the protein‐codingparts of the genome (the exome). As we gain experience in data analysis, this can befollowed by extended analyses of additional genomic regions. Our initial, simplehypothesis generated five lists of candidate modifier genes, conforming to fivedifferent models of inheritance. In total, 86 candidate genes were identified and 11of these genes contained 14 variants that were further validated by Sangersequencing. Additional Sanger validation in another two affected siblings reducedthe number of candidate genes to two potential disease‐causing variants.

  • 329. Stranneheim, Henrik
    et al.
    Engvall, Martin
    Naess, Karin
    Lesko, Nicole
    Larsson, Pontus
    Dahlberg, Mats
    Andeer, Robin
    Wredenberg, Anna
    Freyer, Chris
    Barbaro, Michela
    Bruhn, Helene
    Emahazion, Tesfail
    Magnusson, Måns
    Wibom, Rolf
    Zetterström, Rolf H.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    von Döbeln, Ulrika
    Wedell, Anna
    Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism2014In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 15, p. 1090-Article in journal (Refereed)
    Abstract [en]

    Background: Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine. Results: We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome. Conclusions: We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

  • 330.
    Stranneheim, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kaller, Max
    Allander, Tobias
    Andersson, Björn
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Classification of DNA sequences using Bloom filters2010In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 26, no 13, p. 1595-1600Article in journal (Refereed)
    Abstract [en]

    Motivation: New generation sequencing technologies producing increasingly complex datasets demand new efficient and specialized sequence analysis algorithms. Often, it is only the 'novel' sequences in a complex dataset that are of interest and the superfluous sequences need to be removed. Results: A novel algorithm, fast and accurate classification of sequences (FACSs), is introduced that can accurately and rapidly classify sequences as belonging or not belonging to a reference sequence. FACS was first optimized and validated using a synthetic metagenome dataset. An experimental metagenome dataset was then used to show that FACS achieves comparable accuracy as BLAT and SSAHA2 but is at least 21 times faster in classifying sequences.

  • 331.
    Stranneheim, Henrik
    et al.
    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.
    Stepping stones in DNA sequencing2012In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 7, no 9, p. 1063-1073Article, review/survey (Refereed)
    Abstract [en]

    In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid technological advances have enormously expanded sequencing opportunities and applications, but also imposed strains and challenges on steps prior to sequencing and in the downstream process of handling and analysis of these massive amounts of sequence data. Traditionally, sequencing has been limited to small DNA fragments of approximately one thousand bases (derived from the organism's genome) due to issues in maintaining a high sequence quality and accuracy for longer read lengths. Although many technological breakthroughs have been made, currently the commercially available massively parallel sequencing methods have not been able to resolve this issue. However, recent announcements in nanopore sequencing hold the promise of removing this read-length limitation, enabling sequencing of larger intact DNA fragments. The ability to sequence longer intact DNA with high accuracy is a major stepping stone towards greatly simplifying the downstream analysis and increasing the power of sequencing compared to today. This review covers some of the technical advances in sequencing that have opened up new frontiers in genomics.

  • 332.
    Stranneheim, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Orre, Lukas M.
    Lehtio, Janne
    Flygare, Jenny
    A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells2009In: Proteome Science, ISSN 1477-5956, E-ISSN 1477-5956, Vol. 7, p. 43-Article in journal (Refereed)
    Abstract [en]

    Background: B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL) is presumed to derive predominantly from naive, pre-germinal centre (pre-GC) B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods: Subpopulations of B cells representing the germinal centre (GC), the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS) of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results: Quantitative MS data of significant protein peaks (p-value < 0.05) separating the three B-cell subpopulations were generated. Together, hierarchical clustering and principal component analysis (PCA) showed that the primary MCL samples clustered together with the pre- and post-GC subpopulations. Both primary MCL cells and MCL cell lines were clearly separated from the B cells representing the GC compartment. Conclusion: AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.

  • 333.
    Stranneheim, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Werne, Beata
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sherwood, Ellen
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Scalable Transcriptome Preparation for Massive Parallel Sequencing2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 7, p. e21910-Article in journal (Refereed)
    Abstract [en]

    Background: The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity. Methodology: In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation. Conclusion/Significance: The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.

  • 334.
    Stranneheim, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Proteomics.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Transcript nuclear retention effects quantification of gene expression levelsManuscript (preprint) (Other academic)
    Abstract [en]

    The majority of published differential gene expression studies have used RNA isolated from whole cell extracts (total RNA), overlooking the potential impact of including the nuclear transcriptome in the analyses. It has not been firmly established that the contribution of nuclear RNA is negligible or how the inclusion of it affects quantification of gene expression. Previous studies have estimated that the nuclear transcriptome is five to ten times more complex than the cytoplasmic [1]. Hence, RNA purified solely from the cytoplasm should have fewer unique transcripts, resulting in more sequence counts per transcript and resulting in increased power to detect remaining transcripts. In this study, cytoplasmic and total mRNA have been prepared from three human cell‐lines and sequenced using massive parallel sequencing. The resulting sequence data was analyzed regarding the effect of number of biological replicates, read length and transcripts fractionation on calling differentially detected genes. In addition, the impact of length and secondary structure of mRNAs un‐translated regions (UTRs), and coding sequence length on nucleus to cytoplasm transportation rates of mRNAs was studied. We observe that the number of differentially detected genes was not significantly increased by adding more than three biological replicates or by increasing the sequence read length > 35bp. More differentially detected genes were found in the cytoplasmic RNA compared to the total RNA and a nuclear retention effect was observed for transcripts with long and structured 5’‐ and 3’‐UTR or long protein coding sequences.

  • 335. Stromberg, Sara
    et al.
    Bjorklund, Marcus Gry
    Asplund, Anna
    Rimini, Rebecca
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Ponten, Fredrik
    Olsson, Mats J.
    Transcriptional profiling of melanocytes from patients with vitiligo vulgaris2008In: Pigment Cell & Melanoma Research, ISSN 1755-1471, Vol. 21, no 2, p. 162-171Article in journal (Refereed)
    Abstract [en]

    Vitiligo is a complex, polygenic disorder characterized by patchy loss of skin pigmentation due to abnormal melanocyte function. Both genetic and environmental etiological factors have been proposed for vitiligo and lack of molecular markers renders difficulties to predict development and progression of the disease. Identification of dysregulated genes has the potential to unravel biological pathways involved in vitiligo pathogenesis, facilitating discovery of potential biomarkers and novel therapeutic approaches. In this study, we characterized the transcriptional profile of melanocytes from vitiligo patients. Oligonucleotide microarrays containing similar to 16 000 unique genes were used to analyse mRNA expression in melanocytes from vitiligo patients and age-matched healthy controls. In total, 859 genes were identified as differentially expressed. A substantial number of these genes were involved in (i) melanocyte development, (ii) intracellular processing and trafficking of tyrosinase gene family proteins, (iii) packing and transportation of melanosomes, (iv) cell adhesion and (v) antigen processing and presentation. In conclusion, our results show a significantly different transcription profile in melanocytes from vitiligo patients compared with controls. Several genes of potential importance for the pathogenesis and development of vitiligo were identified. Our data indicate that autoimmunity involving melanocytes may be a secondary event in vitiligo patients caused by abnormal melanocyte function.

  • 336.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Methods for Analyzing Genomes2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human genome reference sequence has given us a two‐dimensional blueprint of our inherited code of life, but we need to employ modern‐day technology to expand our knowledge into a third dimension. Inter‐individual and intra‐individual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNA‐sequencing platforms has increased a thousand‐fold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions.

    Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and cost‐efficient method for enrichment and titration of clonally amplified DNA‐libraries on beads. The method uses fluorescent labeling and a flow‐cytometer to separate DNA‐beads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sun‐exposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its well‐documented involvement in the disease progression of cancers, an estimated 35,000 novel sun‐induced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor protein‐altering mutations in multiple individuals.

  • 337.
    Ståhl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bjursell, Magnus K.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mahdessian, Hovsep
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Jirström, Karin
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Discovery of breast cancer biomarker candidates by translational database selection and multiplex enrichment strategiesManuscript (preprint) (Other academic)
  • 338.
    Ståhl, Patrik L.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Bjursell, Magnus K.
    Mahdessian, Hovsep
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Jirström, Karin
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates2011In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 6, p. e20794-Article in journal (Refereed)
    Abstract [en]

    Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.

  • 339.
    Ståhl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Natanaelsson, Christian
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson-Svahn, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Visual DNA: Identification of DNA sequence variations by bead trapping2007In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 90, p. 741-745Article in journal (Refereed)
    Abstract [en]

    In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with ail ordinary mobile phone equipped with a built-in camera.

  • 340. Ståhl, Patrik L.
    et al.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Toward the Single-Hour High-Quality Genome2012In: ANNUAL REVIEW OF BIOCHEMISTRY, VOL 81, Annual Reviews , 2012, p. 359-378Chapter in book (Refereed)
    Abstract [en]

    Today, resequencing of a human genome can be performed in approximately a week using a single instrument. Thanks to a steady logarithmic rate of increase in performance for DNA sequencing platforms over the past seven years, DNA sequencing is one of the fastest developing technology fields. As the process becomes faster, it opens up possibilities within health care, diagnostics, and entirely new fields of research. Immediate genetic characterization of contagious outbreaks has been exemplified, and with such applications for the direct benefit of human health, expectations of future sensitive, rapid, high-throughput, and cost-effective technologies are steadily growing. Simultaneously, some of the limitations of a rapidly growing field have become apparent, and questions regarding the quality of some of the data deposited into databases have been raised. A human genome sequenced in only an hour is likely to become a reality in the future, but its definition may not be as certain.

  • 341.
    Ståhl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stranneheim, Hanrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Asplund, Anna
    Berglund, Lisa
    KTH, School of Biotechnology (BIO), Proteomics.
    Pontén, Fredrik
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sun‐induced Missense Mutations Are Extensively Accumulated and Tolerated in Phenotypically Intact Stem Cell Compartments of Human SkinArticle in journal (Refereed)
    Abstract [en]

    Here we demonstrate that intermittently sun‐exposed human skin contains an extensive number of phenotypically intact stem cell compartments bearing missense mutations in the p53 tumor suppressor gene. Deep sequencing of sun‐exposed and shielded microdissected skin from mid‐life individuals revealed that persistent p53 mutations had accumulated in 14% of all epidermal cells, with no apparent signs of a growth advantage of the affected cell compartments. Furthermore, 6% of the mutated epidermal cells encoded a truncated protein. The abundance of these events, not taking into account intron mutations and mutations in other genes that also may have functional implications, suggests an extensive tolerance of human cells to severe genetic alterations caused by ultraviolet light, with an estimated annual rate of accumulation of approximately 35,000 new persistent protein altering p53 mutations in sun exposed skin of a human individual.

  • 342.
    Ståhl, Patrik L.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Stranneheim, Henrik
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Asplund, Anna
    Berglund, Lisa
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Pontén, Fredrik
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Sun-Induced Nonsynonymous p53 Mutations Are Extensively Accumulated and Tolerated in Normal Appearing Human Skin2011In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 131, no 2, p. 504-508Article in journal (Refereed)
    Abstract [en]

    Here we demonstrate that intermittently sun-exposed human skin contains an extensive number of phenotypically intact cell compartments bearing missense and nonsense mutations in the p53 tumor suppressor gene. Deep sequencing of sun-exposed and shielded microdissected skin from mid-life individuals revealed that persistent p53 mutations had accumulated in 14% of all epidermal cells, with no apparent signs of a growth advantage of the affected cell compartments. Furthermore, 6% of the mutated epidermal cells encoded a truncated protein. The abundance of these events, not taking into account intron mutations and mutations in other genes that also may have functional implications, suggests an extensive tolerance of human cells to severe genetic alterations caused by UV light, with an estimated annual rate of accumulation of similar to 35,000 new persistent protein-altering p53 mutations in sun-exposed skin of a human individual.

  • 343. Stöck, Matthias
    et al.
    Dubey, Sylvain
    Klütsch, Cornelya
    KTH, School of Biotechnology (BIO), Gene Technology.
    Litvinchuk, Spartak N.
    Scheidt, Ulrich
    Perrin, Nicolas
    Mitochondrial and nuclear phylogeny of circum-Mediterranean tree frogs from the Hyla arborea group2008In: Molecular Phylogenetics and Evolution, ISSN 1055-7903, E-ISSN 1095-9513, Vol. 49, no 3, p. 1019-1024Article in journal (Refereed)
  • 344. Stödberg, T
    et al.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kurian, Manju A.
    et al.,
    Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 8038Article in journal (Refereed)
    Abstract [en]

    The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

  • 345. Sundberg, Carl Johan B.
    et al.
    Lindholm, Malene
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fischer, Helene
    Variation of global mRNA expression in biopsies from the same human muscle2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26Article in journal (Other academic)
  • 346. Sundberg, Erik
    et al.
    Grundtman, Cecilia
    Klint, Erik A. F.
    Lindberg, Johan
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ernestam, Sofia
    Ulfgren, Ann-Kristin
    Erlandsson Harris, Helena
    Andersson, Ulf
    Systemic TNF blockade does not modulate synovial expression of the pro-inflammatory mediator HMGB1 in rheumatoid arthritis patients: a prospective clinical study2008In: Arthritis Research & Therapy, ISSN 1478-6354, E-ISSN 1478-6362, Vol. 10, no 2, p. R33-Article in journal (Refereed)
    Abstract [en]

    Introduction High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an endogenous mediator of arthritis. TNF and IL-1 beta, pivotal cytokines in arthritis pathogenesis, both have the ability to induce the release of HMGB1 from myeloid and dendritic cells. It was, therefore, decided to investigate whether treatment based on TNF blockade in rheumatoid arthritis (RA) affects the expression of synovial HMGB1. Methods Repeated arthroscopy-guided sampling of synovial tissue was performed in nine patients with RA before and nine weeks after initiation of anti-TNF mAb (infliximab) therapy. Synovial biopsy specimens were analysed for HMGB1 protein by immunohistochemical staining and for HMGB1 mRNA expression by real-time reverse transcriptase PCR (RT-PCR). Statistical evaluations were based on Wilcoxon's signed rank tests or Spearman rank sum tests. Results Aberrant, extranuclear HMGB1 and constitutive nuclear HMGB1 expression, with histological signs of inflammation, were evident in all biopsies obtained before infliximab therapy. Signs of inflammation were still evident in the second biopsies obtained nine weeks after initiation of infliximab therapy. The cytoplasmic and extracellular expression of HMGB1 decreased in five patients, remained unchanged in one patient and increased in three patients, making the overall change in HMGB1 protein expression not significant. No correlation between the clinical response, as measured by disease activity score calculated for 28 joints (DAS28) or the American College of Rheumatology response criteria (ACR 20, 50, and 70), and the direction of change of HMGB1 expression in individual patients could be discerned. In addition, infliximab therapy did not alter HMGB1 mRNA synthesis. Conclusion Pro-inflammatory HMGB1 expression during rheumatoid synovitis was not consistently influenced by TNF-blocking therapy with infliximab. This suggests that TNF is not the main inducer of extranuclear HMGB1 during synovitis and that HMGB1 may represent a TNF-independent molecule that could be considered as a possible target for future therapeutic intervention in RA.

  • 347. Suri, Fatemeh
    et al.
    Kalhor, Reza
    Zargar, Seyed Jalal
    Nilforooshan, Navid
    Yazdani, Shahin
    Nezari, Hossein
    Paylakhi, Seyed Hassan
    Narooie-Nejhad, Mehrnaz
    Bayat, Behnaz
    Sedaghati, Tina
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Elahi, Elahe
    Screening of common CYP1B1 mutations in Iranian POAG patients using a microarray-based PrASE protocol2008In: Molecular Vision, ISSN 1090-0535, E-ISSN 1090-0535, Vol. 14, no 271-74, p. 2349-2356Article in journal (Refereed)
    Abstract [en]

    Purpose: The gene coding cytochrome P4501B1 (CYP1B1) has been shown to be a major cause of primary congenital glaucoma in the Iranian population. More recently it was shown to also be important in juvenile-onset open angle glaucoma (JOAG). We aimed to further investigate the role of CYP1B1 in a larger cohort of primary open angle glaucoma (POAG) patients which included late-onset patients. We also aimed to set up a microarray based protocol for mutation screening with an intent of using the protocol in a future population level screening program. Methods: Sixty three POAG patients, nine affected family members, and thirty three previously genotyped primary congenital glaucoma (PCG) patients were included in the study. Clinical examination included slit lamp biomicroscopy, IOP measurement, gonioscopic evaluation, fundus examination, and measurement of perimetry. G61E, R368H, R390H, and R469W were screened by a protocol that included multiplexed allele specific amplification in the presence of a protease (PrASE), use of sequence tagged primers, and hybridization to generic arrays on microarray slides. The entire coding sequences of CYP1B1 and myocilin (MYOC) genes were sequenced in all individuals assessed by the microarray assay to carry a mutation. Intragenic single nucleotide polymorphism (SNP) haplotpes were determined for mutated alleles. Results: Genotypes assessed by the array- based PrASE methodology were in 100% concordance with sequencing results. Seven mutation carrying POAG patients (11.1%) were identified, and their distribution was quite skewed between the juvenile-onset individuals (5/21) as compared to late-onset cases (2/42). Four of the seven mutation carrying Iranian patients harbored two mutated alleles. CYP1B1 mutated alleles in Iranian PCG and POAG patients shared common haplotypes. MYOC mutations were not observed in any of the patients. Conclusions: The PrASE approach allowed reliable simultaneous genotyping of many individuals. It can be an appropriate tool for screening common mutations in large sample sizes. The results suggest that CYP1B1 is implicated in POAG among Iranians, notably in the juvenile-onset form. Contrary to POAG patients studied in other populations, many mutation harboring Iranian patients carry two mutated alleles. We propose an explanation for this observation.

  • 348.
    Svartström, Olov
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Terrapon, Nicolas
    Lombard, Vincent
    de Bruijn, Ino
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Malmsten, Jonas
    Dalin, Ann-Marie
    El Muller, Emilie
    Shah, Pranjul
    Wilmes, Paul
    Henrissat, Bernard
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ninety-nine de novo assembled genomes from the moose (Alces alces) rumen microbiome provide new insights into microbial plant biomass degradation2017In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 11, no 11, p. 2538-2551Article in journal (Refereed)
    Abstract [en]

    The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

  • 349. Svedberg, Anna
    et al.
    Green, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Linkoping Univ, Dept Med & Hlth Sci, Div Drug Res, Clin Pharmacol, SE-58185 Linkoping, Sweden.
    Vikström, Anders
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vikingsson, Svante
    A validated liquid chromatography tandem mass spectrometry method for quantification of erlotinib, OSI-420 and didesmethyl erlotinib and semi-quantification of erlotinib metabolites in human plasma2015In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 107, p. 186-195Article in journal (Refereed)
    Abstract [en]

    A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of erlotinib and its metabolites in human plasma. The method is suitable for therapeutic drug monitoring and pharmacokinetic studies. The substances were extracted using protein precipitation, separated on a BEH XBridge C18 column (100 x 2.1 mm, 1.7 mu m) by gradient elution at 0.7 mL/min of acetonitrile and 5 mM ammonium acetate. The concentration was determined using a Waters Xevo triple quadrupole mass spectrometer in a multi reaction monitoring mode. The total run time was 7 min. Deuterated erlotinib and OSI-597 were used as internal standard for erlotinib and its metabolites, respectively. Erlotinib, OSI-420 and didesmethyl erlotinib were quantified in the concentration range 25-5000 ng/mL, 0.5-500 ng/mL and 0.15-10 ng/mL, respectively. Precision and accuracy was <14% except for OSI-420 at LLOQ (17%). Extraction recovery was above 89%, 99% and 89% for erlotinib, OSI-420 and didesmethyl erlotinib, respectively. The human liver microsomes generated 14 metabolites, three of them not previously reported. Twelve metabolites were measured semi-quantitatively and validated with respect to selectivity, precision and stability.

  • 350. Svensson, Per
    et al.
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ryden, Patrik
    Bergqvist, Ingela
    Edlund, Helena
    Gene array identification of Ipf1/Pdx1(-/-) regulated genes in pancreatic progenitor cells2007In: BMC Developmental Biology, ISSN 1471-213X, E-ISSN 1471-213X, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Background: The homeodomain transcription factor IPF1/PDX1 exerts a dual role in the pancreas; Ipf1/Pdx1 global null mutants fail to develop a pancreas whereas conditional inactivation of Ipf1/Pdx1 in beta-cells leads to impaired beta-cell function and diabetes. Although several putative target genes have been linked to the beta-cell function of Ipf1/Pdx1, relatively little is known with respect to genes regulated by IPF1/PDX1 in early pancreatic progenitor cells. Results: Microarray analyses identified a total of III genes that were differentially expressed in e10.5 pancreatic buds of Ipf1/Pdx1(-/-) embryos. The expression of one of these, Spondin 1, which encodes an extracellular matrix protein, has not previously been described in the pancreas. Quantitative real-time RT-PCR analyses and immunohistochemical analyses also revealed that the expression of FgfR2IIIb, that encodes the receptor for FGF10, was down-regulated in Ipf1/Pdx1(-/-) pancreatic progenitor cells. Conclusion: This microarray analysis has identified a number of candidate genes that are differentially expressed in Ipf1/Pdx1(-/-) pancreatic buds. Several of the differentially expressed genes were known to be important for pancreatic progenitor cell proliferation and differentiation whereas others have not previously been associated with pancreatic development.

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