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  • 201. Hu, Yue O. O.
    et al.
    Ndegwa, Nelson
    Alneberg, Johannes
    Johansson, Sebastian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Logue, Jurg Brendan
    Huss, Mikael
    Käller, Max
    Lundeberg, Joakim
    Fagerberg, Jens
    Andersson, Anders F.
    Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systems2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 11907Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Urban sewer systems consist of wastewater and stormwater sewers, of which only wastewater is processed before being discharged. Occasionally, misconnections or damages in the network occur, resulting in untreated wastewater entering natural water bodies via the stormwater system. Cultivation of faecal indicator bacteria (e.g. Escherichia coli; E. coli) is the current standard for tracing wastewater contamination. This method is cheap but has limited specificity and mobility. Here, we compared the E. coli culturing approach with two sequencing-based methodologies (Illumina MiSeq 16S rRNA gene amplicon sequencing and Oxford Nanopore MinION shotgun metagenomic sequencing), analysing 73 stormwater samples collected in Stockholm. High correlations were obtained between E. coli culturing counts and frequencies of human gut microbiome amplicon sequences, indicating E. coli is indeed a good indicator of faecal contamination. However, the amplicon data further holds information on contamination source or alternatively how much time has elapsed since the faecal matter has entered the system. Shotgun metagenomic sequencing on a subset of the samples using a portable real-time sequencer, MinION, correlated well with the amplicon sequencing data. This study demonstrates the use of DNA sequencing to detect human faecal contamination in stormwater systems and the potential of tracing faecal contamination directly in the field.

  • 202. Hurst, Laurence D.
    et al.
    Ghanbarian, Avazeh T.
    Forrest, Alistair R. R.
    Huminiecki, Lukasz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden; Bioinformatics Infrastructure for Life Sciences (BILS), Sweden.
    The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome2015Ingår i: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 13, nr 12, artikel-id e1002315Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution.

  • 203. Hurst, Laurence D.
    et al.
    Sachenkova, Oxana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Daub, Carsten
    Forrest, Alistair R. R.
    Huminiecki, Lukasz
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden; BILS bioinformatics infrastructure for life sciences, Sweden; Uppsala University, Sweden.
    A simple metric of promoter architecture robustly predicts expression breadth of human genes suggesting that most transcription factors are positive regulators2014Ingår i: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 15, nr 7, s. 413-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Conventional wisdom holds that, owing to the dominance of features such as chromatin level control, the expression of a gene cannot be readily predicted from knowledge of promoter architecture. This is reflected, for example, in a weak or absent correlation between promoter divergence and expression divergence between paralogs. However, an inability to predict may reflect an inability to accurately measure or employment of the wrong parameters. Here we address this issue through integration of two exceptional resources: ENCODE data on transcription factor binding and the FANTOM5 high-resolution expression atlas. Results: Consistent with the notion that in eukaryotes most transcription factors are activating, the number of transcription factors binding a promoter is a strong predictor of expression breadth. In addition, evolutionarily young duplicates have fewer transcription factor binders and narrower expression. Nonetheless, we find several binders and cooperative sets that are disproportionately associated with broad expression, indicating that models more complex than simple correlations should hold more predictive power. Indeed, a machine learning approach improves fit to the data compared with a simple correlation. Machine learning could at best moderately predict tissue of expression of tissue specific genes. Conclusions: We find robust evidence that some expression parameters and paralog expression divergence are strongly predictable with knowledge of transcription factor binding repertoire. While some cooperative complexes can be identified, consistent with the notion that most eukaryotic transcription factors are activating, a simple predictor, the number of binding transcription factors found on a promoter, is a robust predictor of expression breadth.

  • 204. Högstrand, Kari
    et al.
    Lindvall, Jessica M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Sundblad, Anne
    Grandien, Alf
    Transformation of mature mouse B cells into malignant plasma cells in vitro via introduction of defined genetic elements2019Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 49, nr 3, s. 454-461Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An experimental system where defined alterations in gene function or gene expression levels in primary B cells would result in the development of transformed plasma cells in vitro would be useful in order to facilitate studies of the underlying molecular mechanisms of plasma cell malignancies. Here, such a system is described in which primary murine B cells rapidly become transformed into surface CD138(+), IgM(-/low), CD19(-) IgM-secreting plasma cells as a result of expression of the transcription factors IRF4 and MYC together with simultaneous expression of BMI1, mutated p53 or silencing of p19(Arf), and suppression of intrinsic apoptosis through expression of BCLXL. Analysis of gene expression patterns revealed that this combination of transforming genes resulted in expression of a number of genes previously associated with terminally differentiated B cells (plasma cells) and myeloma cells, whereas many genes associated with mature B cells and B-cell lymphomas were not expressed. Upon transplantation, the transformed cells preferentially localized to the bone marrow, presenting features of a plasma cell malignancy of the IgM isotype. The present findings may also be applicable in the development of novel methods for production of monoclonal antibodies.

  • 205. Idrees, M.
    et al.
    Sohail, Ayesha
    Javed, Sana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Comsats University Islamabad, Pakistan.
    Forecasting the critical role of intermittent therapies for the control of bone resorption2019Ingår i: Clinical Biomechanics, ISSN 0268-0033, E-ISSN 1879-1271, Vol. 68, s. 128-136Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Osteoporosis is a chronic metabolic disease characterized by an imbalance of bone resorption and formation, leading to bone fragility and increased susceptibility to fracture. Parathyroid hormone is approved therapy for the treatment of osteoporosis.

    Methods: The intermittent therapy of parathyroid hormone requires accurate administration. Meta-analysis is conducted to draw a clear picture of the impact of intermittent therapy and dose rates relative to time, on the osteoporotic patients. A novel mathematical model is presented in this article synchronised with the parametric values, depicted from meta-analysis.

    Findings: Results obtained from the mathematical model are in close agreement with the results obtained from the clinical trials. The model can be used to forecast the drug potency and dosage rates, to control the vicious cycle of osteoporosis.

    Interpretations: The intermittent administration of parathyroid hormone, rather than the continuous administration, is more effective, furthermore it is also concluded that a mathematical model, linked with the extensive literature of clinical trials, using meta-analysis can help in drug administration and future clinical studies of drug development.

  • 206.
    Ininbergs, Karolina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Bergman, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Larsson, John
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Linnaeus University, Sweden.
    Ekman, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Microbial metagenomics in the Baltic Sea: Recent advancements and prospects for environmental monitoring2015Ingår i: Ambio, ISSN 0044-7447, E-ISSN 1654-7209, Vol. 44, s. 439-450Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Metagenomics refers to the analysis of DNA from a whole community. Metagenomic sequencing of environmental DNA has greatly improved our knowledge of the identity and function of microorganisms in aquatic, terrestrial, and human biomes. Although open oceans have been the primary focus of studies on aquatic microbes, coastal and brackish ecosystems are now being surveyed. Here, we review so far published studies on microbes in the Baltic Sea, one of the world's largest brackish water bodies, using high throughput sequencing of environmental DNA and RNA. Collectively the data illustrate that Baltic Sea microbes are unique and highly diverse, and well adapted to this brackish-water ecosystem, findings that represent a novel base-line knowledge necessary for monitoring purposes and a sustainable management. More specifically, the data relate to environmental drivers for microbial community composition and function, assessments of the microbial biodiversity, adaptations and role of microbes in the nitrogen cycle, and microbial genome assembly from metagenomic sequences. With these discoveries as background, prospects of using metagenomics for Baltic Sea environmental monitoring are discussed.

  • 207.
    Ismail, Nurzian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hedman, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindén, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Charge-driven dynamics of nascent-chain movement through the SecYEG translocon2015Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 22, nr 2, s. 145-149Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.

  • 208.
    Ismail, Nurzian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hedman, Rickard
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schiller, Nina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    A biphasic pulling force acts on transmembrane helices during translocon mediated membrane integration2012Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 19, nr 10, s. 1018-1022Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane alpha-helical segments in the nascent chain exit the translocon through a lateral gate that opens toward the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration.

  • 209. Ivanov, Maxim
    et al.
    Kals, Mart
    Lauschke, Volker
    Barragan, Isabel
    Ewels, Philip
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Käller, Max
    Axelsson, Tomas
    Lehtiö, Janne
    Milani, Lili
    Ingelman-Sundberg, Magnus
    Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression2016Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, nr 14, s. 6756-6769Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene-and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications.

  • 210. James, Tojo
    et al.
    Lindén, Magdalena
    Morikawa, Hiromasa
    Fernandes, Sunjay Jude
    Ruhrmann, Sabrina
    Huss, Mikael
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brandi, Maya
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Piehl, Fredrik
    Jagodic, Maja
    Tegnér, Jesper
    Khademi, Mohsen
    Olsson, Tomas
    Gomez-Cabrero, David
    Kockum, Ingrid
    Impact of genetic risk loci for multiple sclerosis on expression of proximal genes in patients2018Ingår i: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 27, nr 5, s. 912-928Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Despite advancements in genetic studies, it is difficult to understand and characterize the functional relevance of disease-associated genetic variants, especially in the context of a complex multifactorial disease such as multiple sclerosis (MS). As a large proportion of expression quantitative trait loci (eQTLs) are context-specific, we performed RNA-Seq in peripheral blood mononuclear cells from MS patients (n = 145) to identify eQTLs in regions centered on 109 MS risk single nucleotide polymorphisms and 7 associated human leukocyte antigen variants. We identified 77 statistically significant eQTL associations, including pseudogenes and non-coding RNAs. Thirty-eight out of 40 testable eQTL effects were colocalized with the disease association signal. As many eQTLs are tissue specific, we aimed to detail their significance in different cell types. Approximately 70% of the eQTLs were replicated and characterized in at least one major peripheral blood mononuclear cell-derived cell type. Furthermore, 40% of eQTLs were found to be more pronounced in MS patients compared with non-inflammatory neurological diseases patients. In addition, we found two single nucleotide polymorphisms to be significantly associated with the proportions of three different cell types. Mapping to enhancer histone marks and predicted transcription factor binding sites added additional functional evidence for eight eQTL regions. As an example, we found that rs71624119, shared with three other autoimmune diseases and located in a primed enhancer (H3K4me1) with potential binding for STAT transcription factors, significantly associates with ANKRD55 expression. This study provides many novel and validated targets for future functional characterization of MS and other diseases.

  • 211.
    Javed, Sana
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Comsats University Islamabad, Pakistan.
    Younas, Muhammad
    Bhatti, M. Yousaf
    Sohail, Ayesha
    Sattar, Abdul
    Analytic approach to explore dynamical osteoporotic bone turnover2019Ingår i: Advances in Difference Equations, ISSN 1687-1839, E-ISSN 1687-1847, artikel-id 61Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The dynamics of the osteoporotic bone turnover is studied in this paper with the aid of stability analysis of the associated mathematical model. Osteoporosis, which is a common bone disorder, is studied in this papper in detail with an emphasis on the relative threshold values. We examine the expository signaling among the bone cells named osteoclast and osteoblast. Main functioning of osteoblasts is bone formation, whereas osteoclasts are bone removal cells. Mathematical framework for osteoporotic bone turnover comprising of the communication between osteoclasts and osteoblasts has been presented to exhibit the conditions for stability in bone turnover. The percentage ratios of the population of osteoblasts/osteoclasts have been determined via numerical simulations. The remedial upshots of targeting osteoporotic cells participating in such process are examined. From our analysis we have conclude that the role of external agents in treating the diseased bone can be better interpreted with the aid of a theoretical model.

  • 212. Johansson, Henrik J.
    et al.
    Sanchez, Betzabe C.
    Mundt, Filip
    Forshed, Jenny
    Kovacs, Aniko
    Panizza, Elena
    Hultin-Rosenberg, Lina
    Lundgren, Bo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Martens, Ulf
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Mathe, Gyongyver
    Yakhini, Zohar
    Helou, Khalil
    Krawiec, Kamilla
    Kanter, Lena
    Hjerpe, Anders
    Stal, Olle
    Linderholm, Barbro K.
    Lehtio, Janne
    Retinoic acid receptor alpha is associated with tamoxifen resistance in breast cancer2013Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 4, artikel-id 2175Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen.

  • 213. Johansson, Henrik J.
    et al.
    Socciarelli, Fabio
    Vacanti, Nathaniel M.
    Haugen, Mads H.
    Zhu, Yafeng
    Siavelis, Ioannis
    Fernandez-Woodbridge, Alejandro
    Aure, Miriam R.
    Sennblad, Bengt
    Vesterlund, Mattias
    Branca, Rui M.
    Orre, Lukas M.
    Huss, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Fredlund, Erik
    Beraki, Elsa
    Garred, Øystein
    Boekel, Jorrit
    Sauer, Torill
    Zhao, Wei
    Nord, Silje
    Höglander, Elen K.
    Jans, Daniel C.
    Brismar, Hjalmar
    Haukaas, Tonje H.
    Bathen, Tone F.
    Schlichting, Ellen
    Naume, Bjørn
    Geisler, Jürgen
    Hofvind, Solveig
    Engebråten, Olav
    Aarum Geitvik, Gry
    Langerød, Anita
    Kåresen, Rolf
    Mælandsmo, Gunhild Mari
    Sørlie, Therese
    Skjerven, Helle Kristine
    Park, Dæhoon
    Hartman-Johnsen, Olaf-Johan
    Luders, Torben
    Borgen, Elin
    Kristensen, Vessela N.
    Russnes, Hege G.
    Lingjærde, Ole Christian
    Mills, Gordon B.
    Sahlberg, Kristine K.
    Børresen-Dale, Anne-Lise
    Lehtiö, Janne
    Breast cancer quantitative proteome and proteogenomic landscape2019Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, artikel-id 1600Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.

  • 214. Johansson, Martin M.
    et al.
    Lundin, Elin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Qian, Xiaoyan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Mirzazadeh, Mohammadreza
    Halvardson, Jonatan
    Darj, Elisabeth
    Feuk, Lars
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Jazin, Elena
    Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development2016Ingår i: Biology of Sex Differences, ISSN 2042-6410, Vol. 7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Renewed attention has been directed to the functions of the Y chromosome in the central nervous system during early human male development, due to the recent proposed involvement in neurodevelopmental diseases. PCDH11Y and NLGN4Y are of special interest because they belong to gene families involved in cell fate determination and formation of dendrites and axon. Methods: We used RNA sequencing, immunocytochemistry and a padlock probing and rolling circle amplification strategy, to distinguish the expression of X and Y homologs in situ in the human brain for the first time. To minimize influence of androgens on the sex differences in the brain, we focused our investigation to human embryos at 8-11 weeks post-gestation. Results: We found that the X- and Y-encoded genes are expressed in specific and heterogeneous cellular sub-populations of both glial and neuronal origins. More importantly, we found differential distribution patterns of X and Y homologs in the male developing central nervous system. Conclusions: This study has visualized the spatial distribution of PCDH11X/Y and NLGN4X/Y in human developing nervous tissue. The observed spatial distribution patterns suggest the existence of an additional layer of complexity in the development of the male CNS.

  • 215.
    Jurkowski, Wiktor
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Yazdi, Samira
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ligand binding properties of human galanin receptors2013Ingår i: Molecular membrane biology, ISSN 0968-7688, E-ISSN 1464-5203, Vol. 30, nr 2, s. 206-216Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.

  • 216.
    Kaduk, Mateusz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Riegler, Christian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). FH OÖ - University of Applied Sciences Upper Austria, Austria.
    Lemp, Oliver
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). FH OÖ - University of Applied Sciences Upper Austria, Austria.
    Sonnhammer, Erik L. L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    HieranoiDB: a database of orthologs inferred by Hieranoid2017Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr D1, s. D687-D690Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    HieranoiDB (http://hieranoiDB.sbc.su.se) is a freely available on-line database for hierarchical groups of orthologs inferred by the Hieranoid algorithm. It infers orthologs at each node in a species guide tree with the InParanoid algorithm as it progresses from the leaves to the root. Here we present a database HieranoiDB with a web interface that makes it easy to search and visualize the output of Hieranoid, and to download it in various formats. Searching can be performed using protein description, identifier or sequence. In this first version, orthologs are available for the 66 Quest for Orthologs reference proteomes. The ortholog trees are shown graphically and interactively with marked speciation and duplication nodes that show the inferred evolutionary scenario, and allow for correct extraction of predicted orthologs from the Hieranoid trees.

  • 217.
    Kaduk, Mateusz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Sonnhammer, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Improved orthology inference with Hieranoid 22017Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 33, nr 8, s. 1154-1159Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: The initial step in many orthology inference methods is the computationally demanding establishment of all pairwise protein similarities across all analysed proteomes. The quadratic scaling with proteomes has become a major bottleneck. A remedy is offered by the Hieranoid algorithm which reduces the complexity to linear by hierarchically aggregating ortholog groups from InParanoid along a species tree. Results: We have further developed the Hieranoid algorithm in many ways. Major improvements have been made to the construction of multiple sequence alignments and consensus sequences. Hieranoid version 2 was evaluated with standard benchmarks that reveal a dramatic increase in the coverage/accuracy tradeoff over version 1, such that it now compares favourably with the best methods. The new parallelized cluster mode allows Hieranoid to be run on large data sets in a much shorter timespan than InParanoid, yet at similar accuracy.

  • 218.
    Kang, Wenjing
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Bang-Berthelsen, Claus Heiner
    Holm, Anja
    Houben, Anna J. S.
    Holt Müller, Anne
    Thymann, Thomas
    Pociot, Flemming
    Estivill, Xavier
    Friedländer, Marc R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Survey of 800+data sets from human tissue and body fluid reveals xenomiRs are likely artifacts2017Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 23, nr 4, s. 433-445Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.

  • 219. Karamyshev, Andrey L.
    et al.
    Patrick, Anna E.
    Karamysheva, Zemfira N.
    Griesemer, Dustin S.
    Hudson, Henry
    Tjon-Kon-Sang, Sandra
    Nilsson, IngMarie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Otto, Hendrik
    Liu, Qinghua
    Rospert, Sabine
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Johnson, Arthur E.
    Thomas, Philip J.
    Inefficient SRP Interaction with a Nascent Chain Triggers a mRNA Quality Control Pathway2014Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 156, nr 1-2, s. 146-157Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanismof translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).

  • 220. Karimi, Mohsen
    et al.
    Nilsson, Christer
    Dimitriou, Marios
    Jansson, Monika
    Matsson, Hans
    Unneberg, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Lehmann, Sören
    Kere, Juha
    Hellström-Lindberg, Eva
    High-throughput mutational screening adds clinically important information in myelodysplastic syndromes and secondary or therapy-related acute myeloid leukemia2015Ingår i: Haematologica (online), ISSN 0390-6078, E-ISSN 1592-8721, Vol. 100, nr 6, s. E223-E225Artikel i tidskrift (Refereegranskat)
  • 221. Kasimova, Marina A.
    et al.
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Delemotte, Lucie
    Determining the molecular basis of voltage sensitivity in membrane proteins2018Ingår i: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 150, nr 10, s. 1444-1458Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Voltage-sensitive membrane proteins are united by their ability to transform changes in membrane potential into mechanical work. They are responsible for a spectrum of physiological processes in living organisms, including electrical signaling and cell-cycle progression. Although the mechanism of voltage-sensing has been well characterized for some membrane proteins, including voltage-gated ion channels, even the location of the voltage-sensing elements remains unknown for others. Moreover, the detection of these elements by using experimental techniques is challenging because of the diversity of membrane proteins. Here, we provide a computational approach to predict voltage-sensing elements in any membrane protein, independent of its structure or function. It relies on an estimation of the propensity of a protein to respond to changes in membrane potential. We first show that this property correlates well with voltage sensitivity by applying our approach to a set of voltage-sensitive and voltage-insensitive membrane proteins. We further show that it correctly identifies authentic voltage-sensitive residues in the voltage-sensor domain of voltage-gated ion channels. Finally, we investigate six membrane proteins for which the voltage-sensing elements have not yet been characterized and identify residues and ions that might be involved in the response to voltage. The suggested approach is fast and simple and enables a characterization of voltage sensitivity that goes beyond mere identification of charges. We anticipate that its application before mutagenesis experiments will significantly reduce the number of potential voltage-sensitive elements to be tested.

  • 222. Kasmaei, Kamyar Mogodiniyai
    et al.
    Sundh, John
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Identification of Novel Putative Bacterial Feruloyl Esterases From Anaerobic Ecosystems by Use of Whole-Genome Shotgun Metagenomics and Genome Binning2019Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, artikel-id 2673Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Feruloyl esterases (FAEs) can reduce the recalcitrance of lignocellulosic biomass to enzymatic hydrolysis, thereby enhancing biorefinery potentials or animal feeding values of the biomass. In addition, ferulic acid, a product of FAE activity, has applications in pharmaceutical and food/beverage industries. It is therefore of great interest to identify new FAEs to enhance understanding about this enzyme family. For this purpose, we used whole-genome shotgun metagenomics and genome binning to explore rumens of dairy cows, large intestines of horses, sediments of freshwater and forest topsoils to identify novel prokaryotic FAEs and trace the responsible microorganisms. A number of prokaryotic genomes were recovered of which, genomes of Clostridiales order and Candidatus Rhabdochlamydia genus showed FAE coding capacities. In total, five sequences were deemed as putative FAE. The BLASTP search against non-redundant protein database of NCBI indicated that these putative FAEs represented novel sequences within this enzyme family. The phylogenetic analysis showed that at least three putative sequences shared evolutionary lineage with FAEs of type A and thus could possess specific activities similar to this type of FAEs, something that is not previously found outside fungal kingdom. We nominate Candidatus Rhabdochlamydia genus as a novel FAE producing taxonomic unit.

  • 223.
    Kasson, Peter M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Virginia. USA.
    Hess, Berk
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Probing microscopic material properties inside simulated membranes through spatially resolved three-dimensional local pressure fields and surface tensions2013Ingår i: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 169, s. 106-112Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cellular lipid membranes are spatially inhomogeneous soft materials. Materials properties such as pressure and surface tension thus show important microscopic-scale variation that is critical to many biological functions. We present a means to calculate pressure and surface tension in a 3D-resolved manner within molecular-dynamics simulations and show how such measurements can yield important insight. We also present the first corrections to local virial and pressure fields to account for the constraints typically used in lipid simulations that otherwise cause problems in highly oriented systems such as bilayers. Based on simulations of an asymmetric bacterial ion channel in a POPC bilayer, we demonstrate how 3D-resolved pressure can probe for both short-range and long-range effects from the protein on the membrane environment. We also show how surface tension is a sensitive metric for inter-leaflet equilibrium and can be used to detect even subtle imbalances between bilayer leaflets in a membrane-protein simulation. Since surface tension is known to modulate the function of many proteins, this effect is an important consideration for predictions of ion channel function. We outline a strategy by which our local pressure measurements, which we make available within a version of the GROMACS simulation package, may be used to design optimally equilibrated membrane-protein simulations.

  • 224. Ke, Rongqin
    et al.
    Mignardi, Marco
    Hauling, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Fourth Generation of Next-Generation Sequencing Technologies: Promise and Consequences2016Ingår i: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 37, nr 12, s. 1363-1367Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    In this review, we discuss the emergence of the fourth-generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution, thus enabling back mapping of sequencing reads to the original histological context. This information is used, for example, in two current large-scale projects that aim to unravel the function of the brain. Also in cancer research, fourth-generation sequencing has the potential to revolutionize the field. Cancer Research UK has named Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional next-generation sequencing (NGS) methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics.

  • 225.
    Ke, Rongqin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University .
    Mignardi, Marco
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University .
    Pacureanu, Alexandra
    Svedlund, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Botling, Johan
    Wählby, Carolina
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University.
    In situ sequencing for RNA analysis in preserved tissue and cells2013Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 10, nr 9, s. 857-+Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.

  • 226.
    Ke, Rongqin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Nong, Rachel Yuan
    Fredriksson, Simon
    Landegren, Ulf
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection2013Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 7, artikel-id e69813Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.

  • 227.
    Kemp, Grant
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kudva, Renuka
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    de la Rosa, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays2019Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, nr 6, s. 1308-1314Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have characterized the cotranslational folding of two small protein domains of different folds-the alpha-helical N-terminal domain of HemK and the beta-rich FLN5 filamin domain-by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (force-profile analysis, or FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, photo induced electron transfer, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.

  • 228. Khan, Mehmood Alam
    et al.
    Elias, Isaac
    Sjölund, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nylander, Kristina
    Guimera, Roman Valls
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Schobesberger, Richard
    Schmitzberger, Peter
    Lagergren, Jens
    Arvestad, Lars
    Stockholms universitet, Naturvetenskapliga fakulteten, Numerisk analys och datalogi (NADA).
    Fastphylo: Fast tools for phylogenetics2013Ingår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 14, s. 334-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Distance methods are ubiquitous tools in phylogenetics.Their primary purpose may be to reconstructevolutionary history, but they are also used as components in bioinformatic pipelines. However, poorcomputational efficiency has been a constraint on the applicability of distance methods on very largeproblem instances.

    RESULTS: We present fastphylo, a software package containing implementations of efficient algorithms for twocommon problems in phylogenetics: estimating DNA/protein sequence distances and reconstructing aphylogeny from a distance matrix. We compare fastphylo with other neighbor joining based methodsand report the results in terms of speed and memory efficiency.

    CONCLUSIONS: Fastphylo is a fast, memory efficient, and easy to use software suite. Due to its modular architecture,fastphylo is a flexible tool for many phylogenetic studies.

  • 229. Khan, Mehmood Alam
    et al.
    Mahmudi, Owais
    Ullah, Ikram
    Arvestad, Lars
    Stockholms universitet, Naturvetenskapliga fakulteten, Numerisk analys och datalogi (NADA). Stockholms universitet, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Centre, Sweden.
    Lagergren, Jens
    Probabilistic inference of lateral gene transfer events2016Ingår i: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 17, nr Suppl 14, artikel-id 431Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Lateral gene transfer (LGT) is an evolutionary process that has an important role in biology. It challenges the traditional binary tree-like evolution of species and is attracting increasing attention of the molecular biologists due to its involvement in antibiotic resistance. A number of attempts have been made to model LGT in the presence of gene duplication and loss, but reliably placing LGT events in the species tree has remained a challenge.

    Results: In this paper, we propose probabilistic methods that samples reconciliations of the gene tree with a dated species tree and computes maximum a posteriori probabilities. The MCMC-based method uses the probabilistic model DLTRS, that integrates LGT, gene duplication, gene loss, and sequence evolution under a relaxed molecular clock for substitution rates. We can estimate posterior distributions on gene trees and, in contrast to previous work, the actual placement of potential LGT, which can be used to, e.g., identify highways of LGT.

    Conclusions: Based on a simulation study, we conclude that the method is able to infer the true LGT events on gene tree and reconcile it to the correct edges on the species tree in most cases. Applied to two biological datasets, containing gene families from Cyanobacteria and Molicutes, we find potential LGTs highways that corroborate other studies as well as previously undetected examples.

  • 230.
    Kimanius, Dari
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Forsberg, Björn O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Scheres, Sjors H. W.
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-22016Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 5, artikel-id e18722Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    By reaching near-atomic resolution for a wide range of specimens, single-particle cryo-EM structure determination is transforming structural biology. However, the necessary calculations come at large computational costs, which has introduced a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the RELION image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure determination workflow. Both image classification and high-resolution refinement have been accelerated more than an order-of-magnitude, and template-based particle selection has been accelerated well over two orders-of-magnitude on desktop hardware. Memory requirements on GPUs have been reduced to fit widely available hardware, and we show that the use of single precision arithmetic does not adversely affect results. This enables high-resolution cryo-EM structure determination in a matter of days on a single workstation.

  • 231.
    Kimanius, Dari
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Andersson, M.
    Uptake dynamics in the Lactose permease (LacY) membrane protein transporter2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 14324Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 +/- 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.

  • 232.
    Kimanius, Dari
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Ingrid
    Schluckebier, Gerd
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Andersson, Magnus
    SAXS-Guided Metadynamics2015Ingår i: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 11, nr 7, s. 3491-3498Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The small-angle X-ray scattering (SAXS) methodology enables structural characterization of biological macromolecules in solution. However, because SAXS provides low-dimensional information, several potential structural configurations can reproduce the experimental scattering profile, which severely complicates the structural refinement process. Here, we present a bias-exchange metadynamics refinement protocol that incorporates SAXS data as collective variables and therefore tags all possible configurations with their corresponding free energies, which allows identification of a unique structural solution. The method has been implemented in PLUMED and combined with the GROMACS simulation package, and as a proof of principle, we explore the Trp-cage protein folding landscape.

  • 233.
    Kjellqvist, Sanela
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Klose, Christian
    Surma, Michal A.
    Hindy, George
    Mollet, Ines G.
    Johansson, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Chavaux, Patrick
    Gottfries, Johan
    Simons, Kai
    Melander, Olle
    Fernandez, Celine
    Identification of Shared and Unique Serum Lipid Profiles in Diabetes Mellitus and Myocardial Infarction2016Ingår i: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, ISSN 2047-9980, E-ISSN 2047-9980, Vol. 5, nr 12, artikel-id e004503Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background-Diabetes mellitus (DM) and cardiovascular disease are associated with dyslipidemia, but the detailed lipid molecular pattern in both diseases remains unknown. Methods and Results-We used shotgun mass spectrometry to determine serum levels of 255 molecular lipids in 316 controls, 171 DM, and 99 myocardial infarction (MI) events from a cohort derived from the Malmo Diet and Cancer study. Orthogonal projections to latent structures analyses were conducted between the lipids and clinical parameters describing DM or MI. Fatty acid desaturases (FADS) and elongation of very long chain fatty acid protein 5 (ELOVL5) activities were estimated by calculating product to precursor ratios of polyunsaturated fatty acids in complex lipids. FADS genotypes encoding these desaturases were then tested for association with lipid levels and ratios. Differences in the levels of lipids belonging to the phosphatidylcholine and triacylglyceride (TAG) classes contributed the most to separating DM from controls. TAGs also played a dominating role in discriminating MI from controls. Levels of C18:2 fatty acids in complex lipids were lower both in DM and MI versus controls (DM, P=0.004; MI, P=6.0E-06) at least due to an acceleration in the metabolic flux from C18: 2 to C20:4 (eg, increased estimated ELOVL5: DM, P=0.02; MI, P=0.04, and combined elongase-desaturase activities: DM, P=3.0E-06; MI, P=2.0E-06). Minor allele carriers of FADS genotypes were associated with increased levels of C18: 2 (P <= 0.007) and lower desaturase activity (P <= 0.002). Conclusions-We demonstrate a possible relationship between decreased levels of C18: 2 in complex lipids and DM or MI. We thereby highlight the importance of molecular lipids in the pathogenesis of both diseases.

  • 234. Klinter, Stefan
    et al.
    Bulone, Vincent
    Arvestad, Lars
    Stockholms universitet, Naturvetenskapliga fakulteten, Matematiska institutionen. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Diversity and evolution of chitin synthases in oomycetes (Straminipila: Oomycota)2019Ingår i: Molecular Phylogenetics and Evolution, ISSN 1055-7903, E-ISSN 1095-9513, Vol. 139, artikel-id 106558Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The oomycetes are filamentous eukaryotic microorganisms, distinct from true fungi, many of which act as crop or fish pathogens that cause devastating losses in agriculture and aquaculture. Chitin is present in all true fungi, but it occurs in only small amounts in some Saprolegniomycetes and it is absent in Peronosporomycetes. However, the growth of several oomycetes is severely impacted by competitive chitin synthase (CHS) inhibitors. Here, we shed light on the diversity, evolution and function of oomycete CHS proteins. We show by phylogenetic analysis of 93 putative CHSs from 48 highly diverse oomycetes, including the early diverging Ewychasma dicksonii, that all available oomycete genomes contain at least one putative CHS gene. All gene products contain conserved CHS motifs essential for enzymatic activity and form two Peronosporomycete-specific and six Saprolegniale-specific clades. Proteins of all clades, except one, contain an N-terminal microtubule interacting and trafficking (MIT) domain as predicted by protein domain databases or manual analysis, which is supported by homology modelling and comparison of conserved structural features from sequence logos. We identified at least three groups of CHSs conserved among all oomycete lineages and used phylogenetic reconciliation analysis to infer the dynamic evolution of CHSs in oomycetes. The evolutionary aspects of CHS diversity in modern-day oomycetes are discussed. In addition, we observed hyphal tip rupture in Phytophthora infestans upon treatment with the CHS inhibitor nikkomycin Z. Combining data on phylogeny, gene expression, and response to CHS inhibitors, we propose the association of different CHS clades with certain developmental stages.

  • 235. Koos, Björn
    et al.
    Kamali-Moghaddam, Masood
    David, Leonor
    Sobrinho-Simões, Manuel
    Dimberg, Anna
    Nilsson, Mats
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wählby, Carolina
    Söderberg, Ola
    Next-Generation Pathology-Surveillance of Tumor Microecology2015Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 11, s. 2013-2022Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

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

  • 236.
    Krautz, Robert
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Khalili, Dilan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Hauling, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Söll, Iris
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Hauptmann, Giselbert
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Theopold, Ulrich
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    An innate immune response against dysplasia in a secretory organManuskript (preprint) (Övrigt vetenskapligt)
  • 237.
    Krzewińska, Maja
    et al.
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kjellström, Anna
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur.
    Günther, Torsten
    Hedenstierna-Jonson, Charlotte
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur.
    Zachrisson, Torun
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur, Arkeologi.
    Omrak, Ayça
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur.
    Yaka, Reyhan
    Kılınç, Gülşah Merve
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur.
    Somel, Mehmet
    Sobrado, Veronica
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur.
    Evans, Jane
    Knipper, Conine
    Jakobsson, Mattias
    Storå, Jan
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur.
    Götherström, Anders
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Genomic and Strontium Isotope Variation Reveal Immigration Patterns in a Viking Age Town2018Ingår i: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 28, nr 17, s. 2730-2738Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The impact of human mobility on the northern European urban populations during the Viking and Early Middle Ages and its repercussions in Scandinavia itself are still largely unexplored. Our study of the demographics in the final phase of the Viking era is the first comprehensive multidisciplinary investigation that includes genetics, isotopes, archaeology, and osteology on a larger scale. This early Christian dataset is particularly important as the earlier common pagan burial tradition during the Iron Age was cremation, hindering large-scale DNA analyses. We present genome-wide sequence data from 23 individuals from the 10th to 12th century Swedish town of Sigtuna. The data revealed high genetic diversity among the early urban residents. The observed variation exceeds the genetic diversity in distinct modern-day and Iron Age groups of central and northern Europe. Strontium isotope data suggest mixed local and non-local origin of the townspeople. Our results uncover the social system underlying the urbanization process of the Viking World of which mobility was an intricate part and was comparable between males and females. The inhabitants of Sigtuna were heterogeneous in their genetic affinities, probably reflecting both close and distant connections through an established network, confirming that early urbanization processes in northern Europe were driven by migration.

  • 238.
    Krzywkowski, Tomasz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ciftci, Sibel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Assadian, Farzaneh
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Punga, Tanel
    Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection2017Ingår i: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 91, nr 11, artikel-id e00166-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

  • 239.
    Krzywkowski, Tomasz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection2019Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 25, nr 1, s. 82-89Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide poly-morphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.

  • 240.
    Krzywkowski, Tomasz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Detection of miRNAs using chimeric DNA/RNA iLock probes utilizing novel activity of PBCV-1 DNA ligase: RNA-templated ligation of ssRNAManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Accurate detection of miRNAs with complementary probes is challenging due to the short target size, and often high sequence similarity between isoforms belonging to the same miRNA family. Ligation based methods can provide powerful discrimination of subtle sequence variation among target sequences, but they have been difficult to implement for direct RNA analysis due to the sloppiness and inefficiency of most DNA ligases on RNA substrates. In this work, we have studied if RNA substitutions in padlock probes can provide higher catalytic efficiencies for PBCV-1 DNA ligase on RNA substrates. We also characterise end-joining fidelity for Chlorella virus DNA ligase (PBCV DNA ligase 1) and T4RNA ligase 2 (T4Rnl2) in RNA-templated 3'-OH RNA/5’-pDNA chimeric probe ligation. Although we observed considerable ligation efficiency improvement towards short miRNA targets for PBCV-1 ligated chimeric probes, it showed no sequence specificity towards mismatches at the ligation junction. T4Rnl2 showed some base discrimination, but not satisfactory for robust RNA sequence analysis. To increase end-joining fidelity in PBCV-1 DNA ligase catalysed direct RNA detection assays (iLock probes), we have recently introduced an alternative ligation assay design in which ligation probes first undergo sequence- specific 5’ FLAP removal in order to create ligatable substrates. We have tested various chimeric iLock probe designs where RNA substitutions were introduced at different positions in the FLAP and at the ligation junction. We defined two particular nucleotide positions in the iLock probe sequence that when substituted with RNA, significantly increased iLock probe activation and ligation. We further characterized the end-joining fidelity of PBCV-1 and T4Rnl2 catalysed iLock reactions. Both enzymes showed high ligation fidelities for single nucleotide polymorphisms on RNA and miRNA. Finally, we demonstrate a multiplexed chimeric iLock probe miRNA profiling assay using sequencing-by-ligation as readout. 

  • 241.
    Krzywkowski, Tomasz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Reverse-transcriptase activity of Phi29 DNA polymeraseManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    F29 (Phi29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real-time (SMRT) sequencing. Here we report the ability of F29 DNA polymerase to amplify partially RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions support a similar amplification rate as pure DNA substrates. We observed that increasing the number of consecutive RNA substitutions in the circular templates suppress replication, and cannot be recovered by addition of M-MuLV reverse-transcriptase. In summary, this novel ability of F29 to accept RNA-containing substrates broadens the spectrum of applications for F29 mediated RCA. Applications include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes. 

  • 242.
    Krzywkowski, Tomasz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kühnemund, Malte
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Wu, Di
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Limited reverse transcriptase activity of phi29 DNA polymerase2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 7, s. 3625-3632Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Phi29 (Phi 29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

  • 243.
    Krzywkowski, Tomasz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Fidelity of RNA templated end-joining by chlorella virus DNA ligase and a novel iLock assay with improved direct RNA detection accuracy2017Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 18, artikel-id e161Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ligation-based nucleic acid detection methods are primarily limited to DNA, since they exhibit poor performance on RNA. This is attributed to reduced end-joining efficiency and/or fidelity of ligases. Interestingly, chlorella virus DNA ligase (PBCV-1 DNA ligase) has recently been shown to possess high RNA-templated DNA end-joining activity; however, its fidelity has not yet been systematically evaluated. Herein, we characterized PBCV-1 ligase for its RNA-templated end-joining fidelity at single base mismatches in 3′ and 5′ DNA probe termini and found an overall limited end-joining fidelity. To improve the specificity in PBCV-1 ligase-driven RNA detection assays, we utilized structure-specific 5′ exonucleolytic activity of Thermus aquaticus DNA polymerase, used in the invader assay. In the iLock (invader padLock) probe assay, padlock probe molecules are activated prior ligation thus the base at the probe ligation junction is read twice in order to aid successful DNA ligation: first, during structure-specific invader cleavage and then during sequence-specific DNA ligation. We report two distinct iLock probe activation mechanisms and systematically evaluate the assay specificity, including single nucleotide polymorphisms on RNA, mRNA and miRNA. We show significant increase in PBCV-1 ligation fidelity in the iLock probe assay configuration for RNA detection.

  • 244.
    Kudva, Renuka
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tian, Pengfei
    Pardo-Avila, Fátima
    Carroni, Marta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Best, Robert B.
    Bernstein, Harris D.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding2018Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 7, artikel-id e36326Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Delta loop ribosomes, while two similar to 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Delta loop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

  • 245. Kuhnemund, Malte
    et al.
    Witters, Daan
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Lammertyn, Jeroen
    Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection2014Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 14, nr 16, s. 2983-2992Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 246.
    Kurland, Sara
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Wheat, Christopher W.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Celorio Mancera, Maria de la Paz
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Kutschera, Verena E.
    Stockholms universitet, Science for Life Laboratory (SciLifeLab). Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hill, Jason
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Andersson, Anastasia
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Rubin, Carl-Johan
    Andersson, Leif
    Ryman, Nils
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Laikre, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen.
    Exploring a Pool-seq-only approach for gaining population genomic insights in nonmodel species2019Ingår i: Ecology and Evolution, ISSN 2045-7758, E-ISSN 2045-7758, Vol. 9, s. 11448-11463Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Developing genomic insights is challenging in nonmodel species for which resources are often scarce and prohibitively costly. Here, we explore the potential of a recently established approach using Pool-seq data to generate a de novo genome assembly for mining exons, upon which Pool-seq data are used to estimate population divergence and diversity. We do this for two pairs of sympatric populations of brown trout (Salmo trutta): one naturally sympatric set of populations and another pair of populations introduced to a common environment. We validate our approach by comparing the results to those from markers previously used to describe the populations (allozymes and individual-based single nucleotide polymorphisms [SNPs]) and from mapping the Pool-seq data to a reference genome of the closely related Atlantic salmon (Salmo salar). We find that genomic differentiation (F-ST) between the two introduced populations exceeds that of the naturally sympatric populations (F-ST = 0.13 and 0.03 between the introduced and the naturally sympatric populations, respectively), in concordance with estimates from the previously used SNPs. The same level of population divergence is found for the two genome assemblies, but estimates of average nucleotide diversity differ (pi over bar approximate to 0.002 and pi over bar approximate to 0.001 when mapping to S. trutta and S. salar, respectively), although the relationships between population values are largely consistent. This discrepancy might be attributed to biases when mapping to a haploid condensed assembly made of highly fragmented read data compared to using a high-quality reference assembly from a divergent species. We conclude that the Pool-seq-only approach can be suitable for detecting and quantifying genome-wide population differentiation, and for comparing genomic diversity in populations of nonmodel species where reference genomes are lacking.

  • 247.
    Kutsenko, Alexey
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Svensson, Thomas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nystedt, Björn
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Lundeberg, Joakim
    Björk, Petra
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Sonnhammer, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Giacomello, Stefania
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Wieslander, Lars
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    The Chironomus tentans genome sequence and the organization of the Balbiani ring genes2014Ingår i: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 15, s. 819-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes. Results: The genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150-250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations. Conclusions: We show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.

  • 248.
    Kühnemund, Malte
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Hernández-Neuta, Iván
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Sharif, Mohd Istiaq
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Cornaglia, Matteo
    Gijs, Martin A. M.
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules2017Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 8, artikel-id e59Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.

  • 249.
    Kühnemund, Malte
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Wei, Qingshan
    Darai, Evangelia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Wang, Yingjie
    Hernández-Neuta, Iván
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Yang, Zhao
    Tseng, Derek
    Ahlford, Annika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Devyser AB, Sweden.
    Mathot, Lucy
    Sjoblom, Tobias
    Ozcan, Aydogan
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy2017Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, artikel-id 13913Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 250.
    Laenen, Benjamin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för ekologi, miljö och botanik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). University of Liège, Belgium.
    Machac, Antonin
    Gradstein, S. Robbert
    Shaw, Blanka
    Patino, Jairo
    Desamore, Aurelie
    Goffinet, Bernard
    Cox, Cymon J.
    Shaw, A. Jonathan
    Vanderpoorten, Alain
    Increased diversification rates follow shifts to bisexuality in liverworts2016Ingår i: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 210, nr 3, s. 1121-1129Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shifts in sexual systems are one of the key drivers of species diversification. In contrast to angiosperms, unisexuality prevails in bryophytes. Here, we test the hypotheses that bisexuality evolved from an ancestral unisexual condition and is a key innovation in liverworts. We investigate whether shifts in sexual systems influence diversification using hidden state speciation and extinction analysis (HiSSE). This new method compares the effects of the variable of interest to the best-fitting latent variable, yielding robust and conservative tests. We find that the transitions in sexual systems are significantly biased toward unisexuality, even though bisexuality is coupled with increased diversification. Sexual systems are strongly conserved deep within the liverwort tree but become much more labile toward the present. Bisexuality appears to be a key innovation in liverworts. Its effects on diversification are presumably mediated by the interplay of high fertilization rates, massive spore production and long-distance dispersal, which may separately or together have facilitated liverwort speciation, suppressed their extinction, or both. Importantly, shifts in liverwort sexual systems have the opposite effect when compared to angiosperms, leading to contrasting diversification patterns between the two groups. The high prevalence of unisexuality among liverworts suggests, however, a strong selection for sexual dimorphism.

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