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  • 1.
    Abdalaal, Hind
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pundir, Shreya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Collateral toxicity limits the evolution of bacterial Release Factor 2 towards total omnipotence2020In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 37, no 10, p. 2918-2930Article in journal (Refereed)
    Abstract [en]

    When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.

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  • 2.
    Abdelaziz, Nouha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The role of RNA-binding protein FUBL-1 in epigenetic inheritance in C. elegans2022Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
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  • 3.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Strålin, Kristoffer
    Department of Infectious Diseases, Örebro University Hospital.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Olcén, Per
    Department of Clinical Microbiology, Örebro University Hospital.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction2009In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 64, no 4, p. 366-373Article in journal (Refereed)
    Abstract [en]

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  • 4.
    Abdul Rahman, Zozek
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Expression of FLAG-tagged argonautes in Dictyostelium discoideum2022Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Argonautes are conserved RNA-binding proteins that can regulate gene expression post transcriptionally through a process known as RNA interference (RNAi). This is done through the use of small RNAs, e.g. sRNAs that act as a guide for the argonautes, allowing for sequence-specific binding to the target site. This interaction has been studied in many organisms, one of which is the model organism Dictyostelium discoideum. D. discoideum is an amoeba that has been used extensively in genetic experiments due to its unique lifestyle, and ease of use. Being a eukaryotic, unicellular organism, it proves to be a great tool for the study of regulatory systems in eukaryotes, allowing us to study this argonaute-sRNA interaction in detail. By analysing which RNAs bind to the argonautes, we can better understand which genes these proteins regulate and what role RNAi has in the organisms as a whole. 

    In this study, I investigate three of the five argonautes found in D. discoideum, namely agnA, agnC and agnE. By transforming FLAG-tagged versions of these genes into the amoeba, I successfully express two of these modified proteins in D. discoideum and verified expression by using antibodies designed specifically to recognise the FLAG-tags. This opens up the possibility for the characterisation of the argonaute proteins to better understand their role and function in the regulation of genes. 

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  • 5. Abola, EE
    et al.
    Bairoch, A
    Barker, WC
    Beck, S
    Benson, DA
    Berman, H
    Cantor, C
    Cantor, C
    Doubet, S
    Hubbard, TJP
    Jones, T. A.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kleywegt, G J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kolaskar, AS
    van Kuik, A
    Lesk, A M
    Mewes, H W
    Neuhaus, D
    Pfeiffer, F
    Ten Eyck, LF
    Simpson, RJ
    Stoesser, G
    Sussman, J L
    Tateno, Y
    Tsugita, A
    Ulrich, EL
    Vliegenthart, JFG
    Quality control in databanks for molecular biology2000In: BioEssays, Vol. 22, p. 1024-1034Article, review/survey (Other (popular scientific, debate etc.))
  • 6.
    Abu Sabaa, Amal
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Centre for Research and Development, Gävleborg. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Mörth, Charlott
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Centre for Clinical Research Sörmland. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Berglund, Mattias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Hashemi, Jamileh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Amini, Rose-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Freyhult, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kamali-Moghaddam, Masood
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular Tools and Functional Genomics.
    Robelius, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    T-cell Leukaemia/Lymphoma Protein 1A (TCL1A) In Diffuse Large B-cell lymphoma (DLBCL)Manuscript (preprint) (Other academic)
  • 7.
    Abu Sabaa, Amal
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Centre for Research and Development, Gävleborg. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Mörth, Charlott
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Centre for Clinical Research Sörmland. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Molin, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Freyhult, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kamali-Moghaddam, Masood
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular Tools and Functional Genomics.
    Robelius, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
    Enblad, Gunilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Plasma Protein Profiling using Multiplex Extension Assay in Diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP: A descriptive studyManuscript (preprint) (Other academic)
  • 8.
    Acharya, P
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Chattopadhyaya, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry. Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Electrostatic Cross-modulation of the Pseudoaromatic Character in Single Stranded RNA by Nearest-neighbor interactions2005In: Pure and Applied Chemistry, Vol. 77, no 1, p. 291-311Article in journal (Refereed)
  • 9. Adamczyk, Andrew J.
    et al.
    Cao, Jie
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Warshel, Arieh
    Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 34, p. 14115-14120Article in journal (Refereed)
    Abstract [en]

    The proposal that enzymatic catalysis is due to conformational fluctuations has been previously promoted by means of indirect considerations. However, recent works have focused on cases where the relevant motions have components toward distinct conformational regions, whose population could be manipulated by mutations. In particular, a recent work has claimed to provide direct experimental evidence for a dynamical contribution to catalysis in dihydrofolate reductase, where blocking a relevant conformational coordinate was related to the suppression of the motion toward the occluded conformation. The present work utilizes computer simulations to elucidate the true molecular basis for the experimentally observed effect. We start by reproducing the trend in the measured change in catalysis upon mutations (which was assumed to arise as a result of a "dynamical knockout" caused by the mutations). This analysis is performed by calculating the change in the corresponding activation barriers without the need to invoke dynamical effects. We then generate the catalytic landscape of the enzyme and demonstrate that motions in the conformational space do not help drive catalysis. We also discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. Finally, we point out that the changes in the reaction potential surface modify the reorganization free energy (which includes entropic effects), and such changes in the surface also alter the corresponding motion. However, this motion is never the reason for catalysis, but rather simply a reflection of the shape of the reaction potential surface.

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  • 10.
    Adams, Christopher
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences.
    Kjeldsen, Frank
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
    Patriksson, Alexandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    van Der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Gräslund, Astrid
    Papadopolous, Evangelos
    Zubarev, Roman
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Probing Solution-Phase and Gas-Phase Structures of Trp-cage Cations by Chiral Substitution and Spectroscopic Techniques2006In: International Journal of Mass Spectrometry, ISSN 1387-3806, E-ISSN 1873-2798, Vol. 253, no 3, p. 263-273Article in journal (Refereed)
    Abstract [en]

    The relevance of gas-phase protein structure to its solution structure is of the utmost importance in studying biomolecules by mass spectrometry. D-Amino acid substitutions within a minimal protein. Trp-cage. were used to correlate solution-phase properties as measured by circular dichroism with solution/gas-phase conformational features of protein cations probed via charge state distribution (CSD) in electrospray ionization. and gas-phase features revealed by tandem mass spectrometry (MS/MS). The gas-phase features were additionally supported by force-field molecular dynamics simulations. CD data showed that almost any single-residue D-substitution destroys the most prominent CD feature of the "native" all-L isomer, alpha-helicity. CSD was able to qualitatively assess the degree of compactness of solution-phase molecular structures. CSD results were consistent with the all-L form being the most compact in solution among all studied stereoisomers except for the D-Asn(1) isomer. D-substitutions of the aromatic Y(3), W(6) and Q(5) residues generated the largest deviations in CSD data among single amino acid substitutions. consistent with the critical role of these residues in Trp-cage stability. Electron capture dissociation of the stereoisomer dications gave an indication that some gas-phase structural features of Trp-cage are similar to those in solution. This result is supported by MDS data oil five of the studied stereoisomer dications in the gas-phase. The MDS-derived minimum-energy structures possessed more extensive hydrogen bonding than the solution-phase structure of the native form, deviating from the latter by 3-4 angstrom and were not 'inside-out' compared to native structures. MDS data could be correlated with CD data and even with ECD results. which aided in providing a long-range structural constraint for MDS. The overall conclusion is the general resemblance, despite the difference on the detailed level, of the preferred structures in both phases for the mini protein Trp-cage.

  • 11.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto, G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms2014In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, p. 1526-1535Article in journal (Refereed)
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

  • 12.
    Adler, Sara
    et al.
    Umea Univ, Unit Clin Res Ctr Ostersund, Dept Publ Hlth & Clin Med, Umea, Sweden..
    Widerstrom, Micael
    Umea Univ, Unit Communicable Dis Control & Prevent Ostersund, Dept Clin Microbiol, Umea, Sweden..
    Lindh, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lilja, Mikael
    Umea Univ, Unit Clin Res Ctr Ostersund, Dept Publ Hlth & Clin Med, Umea, Sweden..
    Symptoms and risk factors of Cryptosporidium hominis infection in children: data from a large waterborne outbreak in Sweden2017In: Parasitology Research, ISSN 0932-0113, E-ISSN 1432-1955, Vol. 116, no 10, p. 2613-2618Article in journal (Refereed)
    Abstract [en]

    Cryptosporidium is a major cause of diarrheal disease worldwide. In developing countries, this infection is endemic and in children, associated with growth faltering and cognitive function deficits, with the most severe impact on those aged <2 years. Little has been reported about symptoms and risk factors for children in industrialized countries, although the disease incidence is increasing in such regions. In November 2010, a large waterborne outbreak of C. hominis occurred in the city of Ostersund in Sweden. Approximately 27,000 of the 60,000 inhabitants were symptomatic. We aimed to describe duration of symptoms and the risk factors for infection with C. hominis in children aged <15 years in a Western setting. Within 2 months after a boil water advisory, a questionnaire was sent to randomly selected inhabitants of all ages, including 753 children aged <15 years. Those with >= 3 loose stools/day were defined as cases of diarrhoea. The response rate was 70.3%, and 211 children (39.9%) fulfilled the case definition. Mean duration of diarrhoea was 7.5 days (median 6, range 1-80 days). Recurrence, defined as a new episode of diarrhoea after >= 2 days of normal stools, occurred in 52.5% of the cases. Significant risk factors for infection, besides living within the distribution area of the contaminated water plant, included a high level of water consumption, male sex, and a previous history of loose stools. The outbreak was characterized by high attack and recurrence rates, emphasizing the necessity of water surveillance to prevent future outbreaks.

  • 13.
    Aguileta, Gabriela
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Bielawski, Joseph P.
    Yang, Ziheng
    Proposed standard nomenclature for the alpha- and beta-globin gene families2006In: Genes & Genetic Systems, ISSN 1341-7568, E-ISSN 1880-5779, Vol. 81, no 5, p. 367-371Article in journal (Refereed)
    Abstract [en]

    The globin family of genes and proteins has been a recurrent object of study for many decades. This interest has generated a vast amount of knowledge. However it has also created an inconsistent and confusing nomenclature, due to the lack of a systematic approach to naming genes and failure to reflect the phylogenetic relationships among genes of the gene family. To alleviate the problems with the existing system, here we propose a standardized nomenclature for the alpha and beta globin family of genes, based on a phylogenetic analysis of vertebrate alpha and beta globins, and following the Guidelines for Human Gene Nomenclature.

  • 14.
    Aguirre Rivera, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Tracking single molecules in uncharted territory: A single-molecule method to study kinetics in live bacteria2020Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.

    This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.

    To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.

    List of papers
    1. tRNA tracking for direct measurements of protein synthesis kinetics in live cells
    Open this publication in new window or tab >>tRNA tracking for direct measurements of protein synthesis kinetics in live cells
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    2018 (English)In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed) Published
    Abstract [en]

    Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

    Place, publisher, year, edition, pages
    NATURE PUBLISHING GROUP, 2018
    National Category
    Biochemistry and Molecular Biology Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-359663 (URN)10.1038/s41589-018-0063-y (DOI)000435445100019 ()29769736 (PubMedID)
    Funder
    Swedish Research Council, 2015-04111EU, European Research Council, ERC-2013-CoG 616047 SMILEKnut and Alice Wallenberg FoundationWenner-Gren FoundationsCarl Tryggers foundation , CTS 15:243
    Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2020-08-21Bibliographically approved
    2. Real-time measurements of aminoglycoside effects on protein synthesis in live cells
    Open this publication in new window or tab >>Real-time measurements of aminoglycoside effects on protein synthesis in live cells
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    Single-molecule translation protein synthesis fluorescence microscopy bacteria antibiotics aminoglycoside gentamicin paromomycin apramycin
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-417619 (URN)
    Available from: 2020-08-21 Created: 2020-08-21 Last updated: 2020-09-08
    3. Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells
    Open this publication in new window or tab >>Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells
    2021 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 433, no 10, article id 166942Article in journal (Refereed) Published
    Abstract [en]

    Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA(Phe) on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA(fMet). By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA(Phe), underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.

    Place, publisher, year, edition, pages
    Elsevier, 2021
    Keywords
    Single-molecule translation protein synthesis fluorescence microscopy bacteria antibiotics erythromycin
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-417623 (URN)10.1016/j.jmb.2021.166942 (DOI)000643684400005 ()33744313 (PubMedID)
    Funder
    Swedish Research Council, 2015-04111Swedish Research Council, 2016-06264Swedish Research Council, 2019-03714Wenner-Gren FoundationsCarl Tryggers foundation , 15:243Carl Tryggers foundation , 17:226
    Available from: 2020-08-21 Created: 2020-08-21 Last updated: 2024-01-15Bibliographically approved
    4. An extended C-terminal tail of the ribosomal protein S13 modulates the speed of ribosomal translocation.
    Open this publication in new window or tab >>An extended C-terminal tail of the ribosomal protein S13 modulates the speed of ribosomal translocation.
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    Protein synthesis S13 single-molecule tRNA translocation
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-417624 (URN)
    Available from: 2020-08-21 Created: 2020-08-21 Last updated: 2020-08-21
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  • 15.
    Aguirre Rivera, Javier
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Universitet.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Seefeldt, A. Carolin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real-time measurements of aminoglycoside effects on protein synthesis in live cellsManuscript (preprint) (Other academic)
  • 16.
    Aguirre Rivera, Javier
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Volkov, Ivan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Seefeldt, A. Carolin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real-time measurements of aminoglycoside effects on protein synthesis in live cells2021In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 9, article id e2013315118Article in journal (Refereed)
    Abstract [en]

    The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

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  • 17.
    Agullo, Luis
    et al.
    Univ Vic Cent Univ Catalonia UVIC UCC, Dept Syst Biol, U Sci Tech, Sagrada Familia 7, Vic 08500, Spain..
    Buch, Ignasi
    Hosp Del Mar Med Res Inst IMIM, Computat Biophys Lab, Barcelona 08003, Spain..
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Garcia-Dorado, David
    Vall DHebron Res Inst VHIR, Cardiocirculatory Pathol Grp, Barcelona 08035, Spain..
    Villa-Freixa, Jordi
    Univ Vic Cent Univ Catalonia UVIC UCC, Dept Syst Biol, U Sci Tech, Sagrada Familia 7, Vic 08500, Spain..
    Computational exploration of the binding mode of heme-dependent stimulators into the active catalytic domain of soluble guanylate cyclase2016In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 84, no 10, p. 1534-1548Article in journal (Refereed)
    Abstract [en]

    Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), has been proven to have a significant role in coronary artery disease, pulmonary hypertension, erectile dysfunction, and myocardial infarction. One of its agonists, BAY 41-2272 (Riociguat), has been recently approved for treatment of pulmonary arterial hypertension (PHA), while some others are in clinical phases of development. However, the location of the binding sites for the two known types of agonists, heme-dependent stimulators and heme-independent activators, is a matter of debate, particularly for the first group where both a location on the regulatory (H-NOX) and on the catalytic domain have been suggested by different authors. Here, we address its potential location on the catalytic domain, the unique well characterized at the structural level, by an in silico approach. Homology models of the catalytic domain of sGC in inactive or active conformations were constructed using the structure of previously described crystals of the catalytic domains of inactive sGCs (2WZ1, 3ET6) and of active adenylate cyclase (1CJU). Each model was submitted to six independent molecular dynamics simulations of about 1 s. Docking of YC-1, a classic heme-dependent stimulator, to all frames of representative trajectories of inactive and active conformations, followed by calculation of absolute binding free energies with the linear interaction energy (LIE) method, revealed a potential high-affinity binding site on the active structure. The site, located between the pseudo-symmetric and the catalytic site just over the loop (2)-(3), does not overlap with the forskolin binding site on adenylate cyclases.

  • 18.
    Ahluwalia, Tarunveer S.
    et al.
    Steno Diabet Ctr Copenhagen, DK-2820 Gentofte, Denmark.;Univ Copenhagen, Bioinformat Ctr, Dept Biol, DK-2200 Copenhagen, Denmark..
    Prins, Bram P.
    Univ Groningen, Univ Med Ctr Groningen, Dept Epidemiol, NL-9700 RB Groningen, Netherlands..
    Abdollahi, Mohammadreza
    Univ Groningen, Univ Med Ctr Groningen, Dept Epidemiol, NL-9700 RB Groningen, Netherlands..
    Armstrong, Nicola J.
    Murdoch Univ, Math & Stat, Perth, WA 6150, Australia..
    Aslibekyan, Stella
    Univ Alabama Birmingham, Sch Publ Hlth, Dept Epidemiol, Birmingham, AL 35233 USA..
    Bain, Lisa
    QIMR Berghofer Med Res Inst, Brisbane, Qld 4006, Australia..
    Jefferis, Barbara
    UCL, UCL Inst Epidemiol & Hlth Care, Dept Primary Care & Populat Hlth, London NW3 2PF, England..
    Baumert, Jens
    Helmholtz Zentrum Munchen, Inst Epidemiol, German Res Ctr Environm Hlth, D-85764 Neuherberg, Germany..
    Beekman, Marian
    Leiden Univ Med Ctr, Dept Biomed Data Sci, Sect Mol Epidemiol, NL-2300 RC Leiden, Netherlands..
    Ben-Shlomo, Yoav
    Univ Bristol, Populat Hlth Sci, Bristol BS8 2PS, Avon, England..
    Bis, Joshua C.
    Univ Washington, Dept Med, Cardiovasc Hlth Res Unit, Seattle, WA 98101 USA..
    Mitchell, Braxton D.
    Univ Maryland, Dept Med, Sch Med, Baltimore, MD 21202 USA..
    de Geus, Eco
    Vrije Univ Amsterdam, Dept Biol Psychol Behav & Movement Sci, NL-1081 BT Amsterdam, Netherlands.;Amsterdam Univ Med Ctr, Amsterdam Publ Hlth Res Inst, NL-1105 AZ Amsterdam, Netherlands..
    Delgado, Graciela E.
    Heidelberg Univ, Med Fac Mannheim, Dept Med Nephrol Hypertensiol Rheumatol Endocrino, D-68167 Mannheim, Germany..
    Marek, Diana
    SIB Swiss Inst Bioinformat, CH-1015 Lausanne, Switzerland..
    Eriksson, Joel
    Univ Gothenburg, Sahlgrenska Acad, Ctr Bone & Arthrit Res CBAR, Dept Internal Med & Clin Nutr, S-41345 Gothenburg, Sweden..
    Kajantie, Eero
    Natl Inst Hlth & Welf, Chron Dis Prevent Unit, POB 30, Helsinki 00271, Finland.;Helsinki Univ Cent Hosp, Hosp Children & Adolescents, Helsinki 00014, Finland.;Univ Helsinki, Helsinki 00014, Finland..
    Kanoni, Stavroula
    Queen Mary Univ London, Barts & London Med Sch, William Harvey Res Inst, London EC1M 6BQ, England..
    Kemp, John P.
    Univ Queensland, Univ Queensland Diamantina Inst, Woolloongabba, Qld 4102, Australia.;Univ Bristol, MRC Integrat Epidemiol Unit, Bristol BS8 2BN, Avon, England..
    Lu, Chen
    Boston Univ, Dept Biostat, Sch Publ Hlth, Boston, MA 02118 USA..
    Marioni, Riccardo E.
    Univ Edinburgh, Ctr Genom & Expt Med, Inst Genet & Mol Med, Edinburgh EH4 2XU, Midlothian, Scotland.;Univ Edinburgh, Ctr Cognit Ageing & Cognit Epidemiol, Edinburgh EH8 9JZ, Midlothian, Scotland..
    McLachlan, Stela
    Univ Edinburgh, Usher Inst, Edinburgh EH8 9AG, Midlothian, Scotland..
    Milaneschi, Yuri
    Vrije Univ, Dept Psychiat, Amsterdam UMC, NL-1081 HJ Amsterdam, Netherlands..
    Nolte, Ilja M.
    Univ Groningen, Univ Med Ctr Groningen, Dept Epidemiol, NL-9700 RB Groningen, Netherlands..
    Petrelis, Alexandros M.
    Univ Lorraine, IGE PCV, INSERM, F-54000 Nancy, France..
    Porcu, Eleonora
    CNR, Ist Ric Genet & Biomed, I-09042 Monserrato, CA, Italy..
    Sabater-Lleal, Maria
    Karolinska Inst, Ctr Mol Med, Dept Med Solna, Cardiovasc Med, S-17176 Stockholm, Sweden.;Inst Invest Biomed St Pau IIB St Pau, Unit Genom Complex Dis, Barcelona 08041, Spain..
    Naderi, Elnaz
    Univ Groningen, Univ Med Ctr Groningen, Dept Epidemiol, NL-9700 RB Groningen, Netherlands..
    Seppala, Ilkka
    Tampere Univ, Fac Med & Hlth Technol, Fimlab Labs, Dept Clin Chem, Tampere 33520, Finland.;Tampere Univ, Fac Med & Hlth Technol, Finnish Cardiovasc Res Ctr Tampere, Tampere 33520, Finland..
    Shah, Tina
    UCL, Inst Cardiovasc Sci, London WC1E 6BT, England..
    Singhal, Gaurav
    Univ Adelaide, Adelaide Med Sch, Discipline Psychiat, Adelaide, SA 5005, Australia..
    Standl, Marie
    Helmholtz Zentrum Munchen, Inst Epidemiol, German Res Ctr Environm Hlth, D-85764 Neuherberg, Germany..
    Teumer, Alexander
    Univ Med Greifswald, Inst Community Med, D-17475 Greifswald, Germany..
    Thalamuthu, Anbupalam
    Univ New South Wales, Ctr Hlth Brain Ageing, Sch Psychiat, Sydney, NSW 2052, Australia..
    Thiering, Elisabeth
    Helmholtz Zentrum Munchen, Inst Epidemiol, German Res Ctr Environm Hlth, D-85764 Neuherberg, Germany.;Ludwig Maximilians Univ Munchen, Dr von Hauner Childrens Hosp, Div Metab Dis & Nutr Med, D-80337 Munich, Germany..
    Trompet, Stella
    Leiden Univ Med Ctr, Dept Cardiol, NL-2300 RC Leiden, Netherlands.;Leiden Univ Med Ctr, Dept Internal Med, Sect Gerontol & Geriatr, NL-2333 ZA Leiden, Netherlands..
    Ballantyne, Christie M.
    Baylor Coll Med, Houston, TX 77030 USA..
    Benjamin, Emelia J.
    Natl Heart Lung & Blood Inst, Framingham, MA 01702 USA.;Boston Univ, Framingham Heart Study, Framingham, MA 01702 USA.;Boston Univ, Dept Med, Sect Cardiovasc Med & Prevent Med, Sch Med, Boston, MA 02118 USA..
    Casas, Juan P.
    VA Boston Healthcare Syst, Massachusetts Vet Epidemiol Res & Informat Ctr MA, Boston, MA 02130 USA..
    Toben, Catherine
    Univ Adelaide, Adelaide Med Sch, Discipline Psychiat, Adelaide, SA 5005, Australia..
    Dedoussis, George
    Harokopio Univ, Dept Nutr Dietet, Athens 17671, Greece..
    Deelen, Joris
    Leiden Univ Med Ctr, Dept Biomed Data Sci, Sect Mol Epidemiol, NL-2300 RC Leiden, Netherlands.;Max Planck Inst Biol Ageing, D-50931 Cologne, Germany..
    Durda, Peter
    Univ Vermont, Larner Coll Med, Dept Pathol & Lab Med, Burlington, VT 05405 USA..
    Engmann, Jorgen
    UCL, Inst Cardiovasc Sci, London WC1E 6BT, England..
    Feitosa, Mary F.
    Washington Univ, Dept Genet, Div Stat Genom, Sch Med, St Louis, MO 63110 USA..
    Grallert, Harald
    Helmholtz Zentrum Munchen, Inst Epidemiol, German Res Ctr Environm Hlth, D-85764 Neuherberg, Germany.;German Ctr Diabet Res DZD, D-85764 Neuherberg, Germany..
    Hammarstedt, Ann
    Univ Gothenburg, Dept Mol & Clin Med, Lundberg Lab Diabet Res, Sahlgrenska Acad, SE-41345 Gothenburg, Sweden..
    Harris, Sarah E.
    Univ Edinburgh, Ctr Cognit Ageing & Cognit Epidemiol, Edinburgh EH8 9JZ, Midlothian, Scotland.;Univ Edinburgh, Dept Psychol, Edinburgh EH8 9JZ, Midlothian, Scotland..
    Homuth, Georg
    Univ Med Greifswald, Interfac Inst Genet & Funct Genom, D-17475 Greifswald, Germany..
    Hottenga, Jouke-Jan
    Vrije Univ Amsterdam, Dept Biol Psychol Behav & Movement Sci, NL-1081 BT Amsterdam, Netherlands.;Amsterdam Univ Med Ctr, Amsterdam Publ Hlth Res Inst, NL-1105 AZ Amsterdam, Netherlands..
    Jalkanen, Sirpa
    Univ Turku, MediCity Res Lab, Turku 20520, Finland.;Univ Turku, Dept Med Microbiol & Immunol, Turku 20520, Finland..
    Jamshidi, Yalda
    St Georges Univ London, Mol & Clin Sci Inst, Genet Res Ctr, London SW17 0RE, England..
    Jawahar, Magdalene C.
    Univ Adelaide, Adelaide Med Sch, Discipline Psychiat, Adelaide, SA 5005, Australia..
    Jess, Tine
    Statens Serum Inst, Dept Epidemiol Res, DK-2300 Copenhagen, Denmark..
    Kivimaki, Mika
    UCL, UCL Inst Epidemiol & Hlth Care, Dept Epidemiol & Publ Hlth, London WC1E 7HB, England..
    Kleber, Marcus E.
    Heidelberg Univ, Med Fac Mannheim, Dept Med Nephrol Hypertensiol Rheumatol Endocrino, D-68167 Mannheim, Germany..
    Lahti, Jari
    Univ Turku, Turku Inst Adv Studies, Turku 20014, Finland.;Univ Helsinki, Dept Psychol & Logoped, Helsinki 00014, Finland..
    Liu, Yongmei
    Wake Forest Sch Med, Dept Epidemiol & Prevent, Winston Salem, NC 27157 USA..
    Marques-Vidal, Pedro
    Lausanne Univ Hosp CHUV, Dept Internal Med, CH-1011 Lausanne, Switzerland.;Univ Lausanne, CH-1011 Lausanne, Switzerland..
    Mellstrom, Dan
    Univ Gothenburg, Sahlgrenska Acad, Ctr Bone & Arthrit Res CBAR, Dept Internal Med & Clin Nutr, S-41345 Gothenburg, Sweden..
    Mooijaart, Simon P.
    Leiden Univ Med Ctr, Dept Internal Med, Sect Gerontol & Geriatr, NL-2333 ZA Leiden, Netherlands..
    Muller-Nurasyid, Martina
    Ludwig Maximilians Univ LMU Munich, Fac Med, IBE, D-81377 Munich, Germany.;Johhanes Gutenberg Univ, Univ Med Ctr, Inst Med Biostat Epidemiol & Informat IMBEI, D-55101 Mainz, Germany..
    Penninx, Brenda
    Vrije Univ, Dept Psychiat, Amsterdam UMC, NL-1081 HJ Amsterdam, Netherlands..
    Revez, Joana A.
    QIMR Berghofer Med Res Inst, Brisbane, Qld 4006, Australia..
    Rossing, Peter
    Steno Diabet Ctr Copenhagen, DK-2820 Gentofte, Denmark.;Univ Copenhagen, Dept Clin Med, DK-2200 Copenhagen, Denmark..
    Raikkonen, Katri
    Univ Helsinki, Dept Psychol & Logoped, Helsinki 00014, Finland..
    Sattar, Naveed
    BHF Glasgow Cardiovasc Res Ctr, Fac Med, Glasgow G12 8TA, Lanark, Scotland..
    Scharnagl, Hubert
    Med Univ Graz, Clin Inst Med & Chem Lab Diagnost, A-8036 Graz, Austria..
    Sennblad, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Ctr Mol Med, Dept Med Solna, Cardiovasc Med, S-17176 Stockholm, Sweden.
    Silveira, Angela
    Karolinska Inst, Ctr Mol Med, Dept Med Solna, Cardiovasc Med, S-17176 Stockholm, Sweden..
    St Pourcain, Beate
    Univ Bristol, MRC Integrat Epidemiol Unit, Bristol BS8 2BN, Avon, England.;Max Planck Inst Psycholinguist, NL-6525 XD Nijmegen, Netherlands.;Radboud Univ Nijmegen, Donders Inst Brain Cognit & Behav, NL-6525 AJ Nijmegen, Netherlands..
    Timpson, Nicholas J.
    Univ Bristol, MRC Integrat Epidemiol Unit, Bristol BS8 2BN, Avon, England..
    Trollor, Julian
    Univ New South Wales, Ctr Hlth Brain Ageing, Sch Psychiat, Sydney, NSW 2052, Australia.;Univ New South Wales, Sch Psychiat, Dept Dev Disabil Neuropsychiat, Sydney, NSW 2031, Australia..
    van Dongen, Jenny
    Vrije Univ Amsterdam, Dept Biol Psychol Behav & Movement Sci, NL-1081 BT Amsterdam, Netherlands.;Amsterdam Univ Med Ctr, Amsterdam Publ Hlth Res Inst, NL-1105 AZ Amsterdam, Netherlands..
    Van Heemst, Diana
    Baylor Coll Med, Houston, TX 77030 USA..
    Visvikis-Siest, Sophie
    Univ Lorraine, IGE PCV, INSERM, F-54000 Nancy, France..
    Vollenweider, Peter
    Lausanne Univ Hosp CHUV, Dept Internal Med, CH-1011 Lausanne, Switzerland.;Univ Lausanne, CH-1011 Lausanne, Switzerland..
    Volker, Uwe
    Univ Turku, MediCity Res Lab, Turku 20520, Finland..
    Waldenberger, Melanie
    Helmholtz Zentrum Munchen, Inst Epidemiol, German Res Ctr Environm Hlth, D-85764 Neuherberg, Germany..
    Willemsen, Gonneke
    Vrije Univ Amsterdam, Dept Biol Psychol Behav & Movement Sci, NL-1081 BT Amsterdam, Netherlands.;Amsterdam Univ Med Ctr, Amsterdam Publ Hlth Res Inst, NL-1105 AZ Amsterdam, Netherlands..
    Zabaneh, Delilah
    UCL, Dept Genet, Genet Inst, London WC1E 6BT, England..
    Morris, Richard W.
    Univ Bristol, Bristol Med Sch, Dept Populat Hlth Sci, Bristol BS8 1UD, Avon, England..
    Arnett, Donna K.
    Univ Kentucky, Coll Publ Hlth, Deans Off, Lexington, KY 40536 USA..
    Baune, Bernhard T.
    Univ Melbourne, Melbourne Med Sch, Dept Psychiat, Parkville, Vic 3000, Australia.;Univ Munster, Dept Psychiat & Psychotherapy, D-48149 Munster, Germany.;Univ Melbourne, Florey Inst Neurosci & Mental Hlth, Parkville, Vic 3000, Australia..
    Boomsma, Dorret, I
    Vrije Univ Amsterdam, Dept Biol Psychol Behav & Movement Sci, NL-1081 BT Amsterdam, Netherlands.;Amsterdam Univ Med Ctr, Amsterdam Publ Hlth Res Inst, NL-1105 AZ Amsterdam, Netherlands..
    Chang, Yen-Pei C.
    Univ Maryland, Dept Med, Sch Med, Baltimore, MD 21202 USA..
    Deary, Ian J.
    Univ Edinburgh, Ctr Cognit Ageing & Cognit Epidemiol, Edinburgh EH8 9JZ, Midlothian, Scotland.;Univ Edinburgh, Dept Psychol, Edinburgh EH8 9JZ, Midlothian, Scotland..
    Deloukas, Panos
    Queen Mary Univ London, Barts & London Med Sch, William Harvey Res Inst, London EC1M 6BQ, England.;Queen Mary Univ London, Ctr Genom Hlth, London EC1M 6BQ, England..
    Eriksson, Johan G.
    Univ Helsinki, Natl Inst Hlth & Welf, Helsinki 00014, Finland.;Univ Helsinki, Dept Gen Practice & Primary Hlth Care, Helsinki 00014, Finland..
    Evans, David M.
    Univ Queensland, Univ Queensland Diamantina Inst, Woolloongabba, Qld 4102, Australia.;Univ Bristol, MRC Integrat Epidemiol Unit, Bristol BS8 2BN, Avon, England..
    Ferreira, Manuel A.
    QIMR Berghofer Med Res Inst, Brisbane, Qld 4006, Australia..
    Gaunt, Tom
    Univ Bristol, MRC Integrat Epidemiol Unit, Bristol BS6 2BN, Avon, England.;Univ Bristol, Bristol Med Sch, Populat Hlth Sci, Bristol BS8 2BN, Avon, England..
    Gudnason, Vilmundur
    Iceland Heart Assoc, IS-201 Kopavogur, Iceland.;Univ Iceland, Fac Med, IS-101 Reykjavik, Iceland..
    Hamsten, Anders
    Karolinska Inst, Ctr Mol Med, Dept Med Solna, Cardiovasc Med, S-17176 Stockholm, Sweden..
    Heinrich, Joachim
    Helmholtz Zentrum Munchen, Inst Epidemiol, German Res Ctr Environm Hlth, D-85764 Neuherberg, Germany.;Ludwig Maximilians Univ Munchen, Inst & Clin Occupat Social & Environm Med, Univ Hosp, D-81377 Munich, Germany.;Univ Melbourne, Melbourne Sch Populat & Global Hlth, Allergy & Lung Hlth Unit, Melbourne, Vic 3010, Australia..
    Hingorani, Aroon
    UCL, Inst Cardiovasc Sci, London WC1E 6BT, England..
    Humphries, Steve E.
    UCL, Inst Cardiovasc Sci, London WC1E 6BT, England..
    Jukema, J. Wouter
    Leiden Univ Med Ctr, Dept Internal Med, Sect Gerontol & Geriatr, NL-2333 ZA Leiden, Netherlands.;Durrer Ctr Cardiogenet Res, NL-1105 AZ Amsterdam, Netherlands..
    Koenig, Wolfgang
    Tech Univ Munich, Deutsch Herzzentrum Munchen, D-80636 Munich, Germany.;DZHK German Ctr Cardiovasc Res, Partner Site Munich Heart Alliance, D-80336 Munich, Germany.;Univ Ulm, Inst Epidemiol & Med Biometry, D-89081 Ulm, Germany..
    Kumari, Meena
    UCL, UCL Inst Epidemiol & Hlth Care, Dept Epidemiol & Publ Hlth, London WC1E 7HB, England.;Univ Essex, Inst Social & Econ Res, Colchester CO4 3SQ, Essex, England..
    Kutalik, Zoltan
    SIB Swiss Inst Bioinformat, CH-1015 Lausanne, Switzerland.;Univ Lausanne, Univ Ctr Primary Care & Publ Hlth, CH-1010 Lausanne, Switzerland..
    Lawlor, Deborah A.
    Univ Bristol, MRC Integrat Epidemiol Unit, Bristol BS6 2BN, Avon, England.;Univ Bristol, Bristol Med Sch, Populat Hlth Sci, Bristol BS8 2BN, Avon, England..
    Lehtimaki, Terho
    Tampere Univ, Fac Med & Hlth Technol, Fimlab Labs, Dept Clin Chem, Tampere 33520, Finland.;Tampere Univ, Fac Med & Hlth Technol, Finnish Cardiovasc Res Ctr Tampere, Tampere 33520, Finland..
    Marz, Winfried
    Heidelberg Univ, Med Fac Mannheim, Dept Med Nephrol Hypertensiol Rheumatol Endocrino, D-68167 Mannheim, Germany.;Med Univ Graz, Clin Inst Med & Chem Lab Diagnost, A-8036 Graz, Austria.;SYNALB Holding Deutschland GmbH, SYNLAB Acad, D-68163 Mannheim, Germany..
    Mather, Karen A.
    Univ New South Wales, Ctr Hlth Brain Ageing, Sch Psychiat, Sydney, NSW 2052, Australia.;Neurosci Res Australia, Sydney, NSW 2031, Australia..
    Naitza, Silvia
    CNR, Ist Ric Genet & Biomed, I-09042 Monserrato, CA, Italy..
    Nauck, Matthias
    Univ Med Greifswald, Inst Clin Chem & Lab Med, D-17475 Greifswald, Germany.;DZHK German Ctr Cardiovasc Res, Partner Site Greifswald, D-17475 Greifswald, Germany..
    Ohlsson, Claes
    Univ Gothenburg, Sahlgrenska Acad, Ctr Bone & Arthrit Res CBAR, Dept Internal Med & Clin Nutr, S-41345 Gothenburg, Sweden..
    Price, Jackie F.
    Univ Edinburgh, Usher Inst, Edinburgh EH8 9AG, Midlothian, Scotland..
    Raitakari, Olli
    Univ Turku, Turku Univ Hosp, Ctr Populat Hlth Res, Turku 20520, Finland.;Univ Turku, Res Ctr Appl & Prevent Cardiovasc Med, Turku 20520, Finland.;Turku Univ Hosp, Dept Clin Physiol & Nucl Med, Turku 20014, Finland..
    Rice, Ken
    Univ Washington, Dept Biostat, Seattle, WA 98195 USA..
    Sachdev, Perminder S.
    Univ New South Wales, Ctr Hlth Brain Ageing, Sch Psychiat, Sydney, NSW 2052, Australia.;Prince Wales Hosp, Neuropsychiat Inst, Sydney, NSW 2031, Australia..
    Slagboom, Eline
    Leiden Univ Med Ctr, Dept Biomed Data Sci, Sect Mol Epidemiol, NL-2300 RC Leiden, Netherlands.;Max Planck Inst Biol Ageing, D-50931 Cologne, Germany..
    Sorensen, Thorkild I. A.
    Univ Copenhagen, Novo Nordisk Fdn Ctr Basic Metab Res, Fac Hlth & Med Sci, Sect Metab Genet, DK-2200 Copenhagen, Denmark.;Univ Copenhagen, Dept Publ Hlth, Sect Epidemiol, DK-1014 Copenhagen, Denmark..
    Spector, Tim
    Kings Coll London, Dept Twin Res & Genet Epidemiol, London SE1 7EH, England..
    Stacey, David
    Univ Cambridge, Dept Publ Hlth & Primary Care, MRC BHF Cardiovasc Epidemiol Unit, Cambridge CB1 8RN, England..
    Stathopoulou, Maria G.
    Univ Lorraine, IGE PCV, INSERM, F-54000 Nancy, France..
    Tanaka, Toshiko
    NIA, Translat Gerontol Branch, Longitudinal Study Sect, Baltimore, MD 21224 USA..
    Wannamethee, S. Goya
    UCL, UCL Inst Epidemiol & Hlth Care, Dept Primary Care & Populat Hlth, London NW3 2PF, England..
    Whincup, Peter
    St Georges Univ London, Populat Hlth Res Inst, London SW17 0RE, England..
    Rotter, Jerome, I
    Harbor UCLA Med Ctr, Inst Translat Genom & Populat Sci, Dept Pediat, Lundquist Inst, Torrance, CA 90502 USA..
    Dehghan, Abbas
    Erasmus MC, Dept Epidemiol, NL-3000 CA Rotterdam, Netherlands..
    Boerwinkle, Eric
    Univ Texas Hlth Sci Ctr Houston, Human Genet Ctr, Sch Publ Hlth, Houston, TX 77030 USA.;Baylor Coll Med, Human Genome Sequencing Ctr, Houston, TX 77030 USA..
    Psaty, Bruce M.
    Univ Washington, Dept Med, Cardiovasc Hlth Res Unit, Seattle, WA 98101 USA.;Univ Washington, Dept Epidemiol, Seattle, WA 98101 USA.;Univ Washington, Dept Hlth Serv, Seattle, WA 98101 USA..
    Snieder, Harold
    Univ Groningen, Univ Med Ctr Groningen, Dept Epidemiol, NL-9700 RB Groningen, Netherlands..
    Alizadeh, Behrooz Z.
    Univ Groningen, Univ Med Ctr Groningen, Dept Epidemiol, NL-9700 RB Groningen, Netherlands..
    Genome-wide association study of circulating interleukin 6 levels identifies novel loci2021In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 30, no 5, p. 393-409Article in journal (Refereed)
    Abstract [en]

    Interleukin 6 (IL-6) is a multifunctional cytokine with both pro- and anti-inflammatory properties with a heritability estimate of up to 61%. The circulating levels of IL-6 in blood have been associated with an increased risk of complex disease pathogenesis. We conducted a two-staged, discovery and replication meta genome-wide association study (GWAS) of circulating serum IL-6 levels comprising up to 67428 (n(discovery)=52654 and n(replication)=14774) individuals of European ancestry. The inverse variance fixed effects based discovery meta-analysis, followed by replication led to the identification of two independent loci, IL1F10/IL1RN rs6734238 on chromosome (Chr) 2q14, (P-combined=1.8x10(-11)), HLA-DRB1/DRB5 rs660895 on Chr6p21 (P-combined=1.5x10(-10)) in the combined meta-analyses of all samples. We also replicated the IL6R rs4537545 locus on Chr1q21 (P-combined=1.2x10(-122)). Our study identifies novel loci for circulating IL-6 levels uncovering new immunological and inflammatory pathways that may influence IL-6 pathobiology.

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  • 19.
    Ahmed, Engy
    et al.
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden.;Sci Life Lab, Tomtebodavagen 23A, SE-17165 Solna, Sweden..
    Parducci, Laura
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Plant Ecology and Evolution.
    Unneberg, Per
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Ågren, Rasmus
    Chalmers Univ Technol, Dept Chem & Biol Engn, Sci Life Lab, SE-41296 Gothenburg, Sweden..
    Schenk, Frederik
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden..
    Rattray, Jayne E.
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden.;Univ Calgary, Biol Sci, 2500 Univ Dr NW, Calgary, AB, Canada..
    Han, Lu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics. Jilin Univ, Coll Life Sci, Ancient DNA Lab, Changchun, Jilin, Peoples R China..
    Muschitiello, Francesco
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden.;Columbia Univ, Lamont Doherty Earth Observ, 61 Route 9NW, Palisades, NY USA..
    Pedersen, Mikkel W.
    Univ Cambridge, Dept Zool, Downing St, Cambridge CB2 3EJ, England..
    Smittenberg, Rienk H.
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden..
    Yamoah, Kweku Afrifa
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden..
    Slotte, Tanja
    Stockholm Univ, Dept Ecol Environm & Plant Sci, SE-10691 Stockholm, Sweden.;Sci Life Lab, Tomtebodavagen 23A, SE-17165 Solna, Sweden..
    Wohlfarth, Barbara
    Stockholm Univ, Dept Geol Sci, SE-10691 Stockholm, Sweden.;Stockholm Univ, Bolin Ctr Climate Res, SE-10691 Stockholm, Sweden..
    Archaeal community changes in Lateglacial lake sediments: Evidence from ancient DNA2018In: Quaternary Science Reviews, ISSN 0277-3791, E-ISSN 1873-457X, Vol. 181, p. 19-29Article in journal (Refereed)
    Abstract [en]

    The Lateglacial/early Holocene sediments from the ancient lake at Hasseldala Port, southern Sweden provide an important archive for the environmental and climatic shifts at the end of the last ice age and the transition into the present Interglacial. The existing multi-proxy data set highlights the complex interplay of physical and ecological changes in response to climatic shifts and lake status changes. Yet, it remains unclear how microorganisms, such as Archaea, which do not leave microscopic features in the sedimentary record, were affected by these climatic shifts. Here we present the metagenomic data set of Hasseldala Port with a special focus on the abundance and biodiversity of Archaea. This allows reconstructing for the first time the temporal succession of major Archaea groups between 13.9 and 10.8 ka BP by using ancient environmental DNA metagenomics and fossil archaeal cell membrane lipids. We then evaluate to which extent these findings reflect physical changes of the lake system, due to changes in lake-water summer temperature and seasonal lake-ice cover. We show that variations in archaeal composition and diversity were related to a variety of factors (e.g., changes in lake water temperature, duration of lake ice cover, rapid sediment infilling), which influenced bottom water conditions and the sediment-water interface. Methanogenic Archaea dominated during the Allerod and Younger Dryas pollen zones, when the ancient lake was likely stratified and anoxic for large parts of the year. The increase in archaeal diversity at the Younger Dryas/Holocene transition is explained by sediment infilling and formation of a mire/peatbog. (C) 2017 Elsevier Ltd. All rights reserved.

  • 20.
    Ahmed, Laeeq
    et al.
    Royal Inst Technol KTH, Dept Elect Engn & Computat Sci, Lindstedtsvagen 5, S-10044 Stockholm, Sweden..
    Alogheli, Hiba
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Arvidsson Mc Shane, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Alvarsson, Jonathan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Berg, Arvid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Schaal, Wesley
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Laure, Erwin
    Royal Inst Technol KTH, Dept Elect Engn & Computat Sci, Lindstedtsvagen 5, S-10044 Stockholm, Sweden..
    Spjuth, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Predicting target profiles with confidence as a service using docking scores2020In: Journal of Cheminformatics, E-ISSN 1758-2946, Vol. 12, article id 62Article in journal (Refereed)
    Abstract [en]

    Background: Identifying and assessing ligand-target binding is a core component in early drug discovery as one or more unwanted interactions may be associated with safety issues.

    Contributions: We present an open-source, extendable web service for predicting target profiles with confidence using machine learning for a panel of 7 targets, where models are trained on molecular docking scores from a large virtual library. The method uses conformal prediction to produce valid measures of prediction efficiency for a particular confidence level. The service also offers the possibility to dock chemical structures to the panel of targets with QuickVina on individual compound basis.

    Results: The docking procedure and resulting models were validated by docking well-known inhibitors for each of the 7 targets using QuickVina. The model predictions showed comparable performance to molecular docking scores against an external validation set. The implementation as publicly available microservices on Kubernetes ensures resilience, scalability, and extensibility.

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  • 21.
    Aken, Bronwen L.
    et al.
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Ayling, Sarah
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;Genome Anal Ctr, Norwich Res Pk, Norwich NR4 7UH, Norfolk, England..
    Barrell, Daniel
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;Eagle Genom Ltd, Babraham Res Campus, Cambridge CB22 3AT, England..
    Clarke, Laura
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;European Bioinformat Inst, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Curwen, Valery
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Fairley, Susan
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;European Bioinformat Inst, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Banet, Julio Fernandez
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;Pfizer Inc, 10646 Sci Ctr Dr, San Diego, CA 92121 USA..
    Billis, Konstantinos
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Giron, Carlos Garcia
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Hourlier, Thibaut
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Howe, Kevin
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;European Bioinformat Inst, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Kähäri, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.
    Kokocinski, Felix
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Martin, Fergal J.
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Murphy, Daniel N.
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Nag, Rishi
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Ruffier, Magali
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;European Bioinformat Inst, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Schuster, Michael
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Austrian Acad Sci, CeMM Res Ctr Mol Med, A-1090 Vienna, Austria..
    Tang, Y. Amy
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;European Bioinformat Inst, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Vogel, Jan-Hinnerk
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;Genentech Inc, 1 DNAWay, San Francisco, CA 94080 USA..
    White, Simon
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;Baylor Coll Med, Human Genome Sequencing Ctr, Houston, TX 77030 USA..
    Zadissa, Amonida
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England.;European Bioinformat Inst, European Mol Biol Lab, Wellcome Genome Campus, Cambridge CB10 1SD, England..
    Flicek, Paul
    European Bioinformat Inst Wellcome Genome Campus, European Mol Biol Lab, Cambridge CB10 1SD, England.;Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    Searle, Stephen M. J.
    Wellcome Trust Sanger Inst Wellcome Genome Campus, Cambridge CB10 1SA, England..
    The Ensembl gene annotation system2016In: Database: The Journal of Biological Databases and Curation, E-ISSN 1758-0463, article id baw093Article in journal (Refereed)
    Abstract [en]

    The Ensembl gene annotation system has been used to annotate over 70 different vertebrate species across a wide range of genome projects. Furthermore, it generates the automatic alignment-based annotation for the human and mouse GENCODE gene sets. The system is based on the alignment of biological sequences, including cDNAs, proteins and RNA-seq reads, to the target genome in order to construct candidate transcript models. Careful assessment and filtering of these candidate transcripts ultimately leads to the final gene set, which is made available on the Ensembl website. Here, we describe the annotation process in detail.

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  • 22.
    Akula, Srinivas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Analysis of the isotype specificity of three platypus immunoglobulin Fc receptors2012Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    The host’s defense against diseases called immunity acts either via innate or adaptive defense mechanisms. Immunoglobulins (Ig’s) are important players in adaptive immunity. They have evolved both structurally and functionally during vertebrate evolution. The Fc region of Igs can interact with specific receptors on the surface of various immune cells; crosslinking of these Fc receptors can trigger a wide array of immune reactions. To trigger such reactions, higher mammals have five different classes of Igs (IgM, IgG, IgA, IgE and IgD) while amphibians, reptiles and birds have four (IgM, IgD, IgA and IgY).  Our recent studies have revealed that the early mammals (Platypus) have eight Ig isotypes (IgM, IgD IgO, IgG1, IgG2, IgA1, IgA2 and IgE) and at least four Fc receptors: FcRA, FcRB, FcRC and FcRD. In this study we investigated the specificity of three of these platypus Fc receptors to get a better picture of their isotype specificity.   

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    Report-srinivasakula.pdf
  • 23.
    Akula, Srinivas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University.
    The mast cell transcriptome and the evolution of granule proteins and Fc receptors2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Protection against disease-causing pathogens, known as immunity, involves numerous cells organs, tissues and their products. To able to understand the biology of immune cells (hematopoietic cells) and their role in an immune system, we have used several different methods, including transcriptome analyses, bioinformatics, production of recombinant proteins and analyses of some of them, focusing on the granule proteases by substrate phage display.

    Hematopoietic cells express surface receptors interacting with the constant region of immunoglobulins (Igs) known as Fc receptors (FcRs). These receptors play major roles in the immune system, including enhancing phagocytosis, activating antibody dependent cellular cytotoxicity and cell activation. A detailed bioinformatics analysis of FcRs reveals that the poly-Ig receptors (PIGR), FcR-like molecules and common signalling γ chain all appeared very early with the appearance of the bony fishes, and thereby represent the first major evolutionary step in FcR evolution. The FcμR, FcαμR, FcγR and FcεR receptors most likely appeared in reptiles or early mammals, representing the second major step in FcR evolution.

    Cells of several of the hematopoietic cell lineages contain large numbers of cytoplasmic granules, and serine proteases constitute the major protein content of these granules. In mammals, these proteases are encoded from four different loci: the chymase, the met-ase, the granzyme (A/K) and the mast cell tryptase loci. The granzyme (A/K) locus was the first to appear and came with the cartilaginous fishes. This locus is also the most conserved of the three. The second most conserved locus is the met-ase locus, which is found in bony fishes. The chymase locus appeared relatively late, and we find the first traces in frogs, indicating it appeared in early tetrapods.

    To study the early events in the diversification of these hematopoietic serine proteases we have analyzed key characteristics of a protease expressed by an NK-like cell in the channel catfish, catfish granzyme–like I. We have used phage display and further validated the results using a panel of recombinant substrates. This protease showed a strict preference for Met at the P1 (cleavage) position, which indicates met-ase specificity. From the screening of potential in vivo substrates, we found an interesting potential target caspase 6, which indicates that caspase-dependent apoptosis mechanisms have been conserved from fishes to mammals.

    A larger quantitative transcriptome analysis of purified mouse peritoneal mast cells, cultured mast cells (BMMCs), and mast cells isolated from mouse ear and lung tissue identified the major tissue specific transcripts in these mast cells as the granule proteases. Mast cell specific receptors and processing enzymes were expressed at approximately 2 orders of magnitude lower levels. The levels of a few proteases were quite different at various anatomical sites between in vivo and cultured BMMCs. These studies have given us a new insights into mast cells in different tissues, as well as key evolutionary aspects concerning the origins of a number of granule proteases and FcRs.

    List of papers
    1. Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution
    Open this publication in new window or tab >>Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution
    2014 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 9, no 5, p. e96903-Article in journal (Refereed) Published
    Abstract [en]

    Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR gamma chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR.

    National Category
    Biomedical Laboratory Science/Technology
    Identifiers
    urn:nbn:se:uu:diva-228481 (URN)10.1371/journal.pone.0096903 (DOI)000336838000078 ()
    Available from: 2014-07-15 Created: 2014-07-15 Last updated: 2021-06-14Bibliographically approved
    2. Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution
    Open this publication in new window or tab >>Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution
    2015 (English)In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 11, article id e0143091Article in journal (Refereed) Published
    Abstract [en]

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-271027 (URN)10.1371/journal.pone.0143091 (DOI)000365070700134 ()26569620 (PubMedID)
    Funder
    Swedish Research Council, 621-2011-5007
    Available from: 2016-01-05 Created: 2016-01-05 Last updated: 2022-01-29Bibliographically approved
    3. Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells
    Open this publication in new window or tab >>Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells
    2016 (English)In: Developmental And Comparative Immunology, ISSN 0145-305X, Vol. 63, p. 84-95Article in journal (Refereed) Published
    Abstract [en]

    Here we present the extended cleavage specificity of catfish granzyme-like I, previously identified in fish NK-like cells. This protease has been characterised using substrate phage display and further validated by using a panel of recombinant substrates. A strict preference for Met in the P1 (cleavage) position, indicating metase specificity was observed. A screening of potential in vivo substrates was performed based on the derived P5-P3' consensus: Arg-Val-Thr-Gly-Met(down arrow)Ser-Leu-Val. Channel catfish caspase 6 was one very interesting potential target identified. This site was present in an adjacent position to the classic caspase activation site (Asp179 in human caspase 6). Cleavage of this site (hence potential activation) by the catfish granzyme-like I could reveal a novel mechanism of caspase 6 activation. This poses an interesting idea that the role of granzyme-like proteases in the activation of caspase dependent apoptosis mechanisms has been conserved for over 400 million years.

    Keywords
    Fish; Serine protease; Cleavage specificity; Metase; NK cell; Caspase; Evolution
    National Category
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-221554 (URN)10.1016/j.dci.2016.05.013 (DOI)000380623300010 ()27216028 (PubMedID)
    Funder
    Swedish Research Council, 621-2011-5007
    Available from: 2014-04-09 Created: 2014-04-01 Last updated: 2019-04-12Bibliographically approved
    4. The mouse mast cell transcriptome
    Open this publication in new window or tab >>The mouse mast cell transcriptome
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Mast cells (MCs) are highly specialized tissue resident cells that are often found at the interphase between body and environment such as the skin, lung and intestinal mucosa. To obtain a more detailed picture of the biology of MCs we have analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR based transcriptomics.  The results show that MCs at different tissue locations can differ quite substantially in transcript levels of several of the most abundant granule proteins even if they belong to the same basic MC type, i.e connective tissue or mucosal MCs. We can also see that transcript levels for the major granule proteins, like the various proteases and the heparin core protein can be several orders of magnitude higher than the surface receptors.  This also applies for the processing enzymes involved in activation of the proteases and in the synthesis of heparin and histamine. Interestingly also is the almost complete absence of transcripts for cytokines in the MC populations of the various organs, indicating that cytokines only are produced by activated MCs. Bone marrow derived MCs are often used as equivalents of tissue MCs.  We here show that these cells differ substantially in their transcriptome from tissue MCs. They show a transcriptome of relatively immature cells both with respect to the granule components and to the processing enzymes indicating that care should be taken when transferring findings from these cells to the in vivo function of tissue resident MCs.  This latter finding also give clear indication for that additional cytokines are needed, in addition to the stem cell factor (SCF), for the development into fully mature tissue MCs.

    National Category
    Cell and Molecular Biology
    Research subject
    Immunology
    Identifiers
    urn:nbn:se:uu:diva-381501 (URN)
    Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-04-15Bibliographically approved
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  • 24.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Wernersson, Sara
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases2021In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 22, no 20, article id 10975Article, review/survey (Refereed)
    Abstract [en]

    Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.

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    FULLTEXT01
  • 25.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The Appearance and Diversification of Receptors for IgM During Vertebrate Evolution2017In: IGM AND ITS RECEPTORS AND BINDING PROTEINS / [ed] Kubagawa, H Burrows, PD, SPRINGER-VERLAG BERLIN , 2017, p. 1-23Chapter in book (Refereed)
    Abstract [en]

    Three different receptors that interact with the constant domains of IgM have been identified: the polymeric immunoglobulin (Ig) receptor (PIGR), the dual receptor for IgA/IgM (Fc alpha mu R) and the IgM receptor (Fc mu R). All of them are related in structure and located in the same chromosomal region in mammals. The functions of the PIGRs are to transport IgM and IgA into the intestinal lumen and to saliva and tears, whereas the Fc alpha mu Rs enhance uptake of immune complexes and antibody coated bacteria and viruses by B220+ B cells and phagocytes, as well as dampening the Ig response to thymus-independent antigens. The Fc mu Rs have broad-spectrum effects on B-cell development including effects on IgM homeostasis, B-cell survival, humoral immune responses and also in autoantibody formation. The PIGR is the first of these receptors to appear during vertebrate evolution and is found in bony fish and all tetrapods but not in cartilaginous fish. The Fc mu R is present in all extant mammalian lineages and also in the Chinese and American alligators, suggesting its appearance with early reptiles. Currently the Fc alpha mu R has only been found in mammals and is most likely the evolutionary youngest of the three receptors. In bony fish, the PIGR has either 2, 3, 4, 5 or 6 extracellular Ig-like domains, whereas in amphibians, reptiles and birds it has 4 domains, and 5 in all mammals. The increase in domain number from 4 to 5 in mammals has been proposed to enhance the interaction with IgA. Both the Fc alpha mu Rs and the Fc mu Rs contain only one Ig domain; the domain that confers Ig binding. In both of these receptors this domain shows the highest degree of sequence similarity to domain 1 of the PIGR. All Ig domains of these three receptors are V type domains, indicating they all have the same origin although they have diversified extensively in function during vertebrate evolution by changing expression patterns and cytoplasmic signaling motifs.

  • 26.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, BOX 7011, SE-75007 Uppsala, Sweden..
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Aviles, Francesc Xavier
    Univ Autonoma Barcelona, Inst Biotecnol & Biomed IBB, Barcelona, Spain.;Univ Autonoma Barcelona, Dept Bioquim & Biol Mol, Barcelona, Spain..
    Wernersson, Sara
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, BOX 7011, SE-75007 Uppsala, Sweden..
    Analysis of the mast cell expressed carboxypeptidase A3 and its structural and evolutionary relationship to other vertebrate carboxypeptidases2022In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 127, article id 104273Article in journal (Refereed)
    Abstract [en]

    Metallo-carboxypeptidases are exopeptidases with diverse expression and function, found in all kingdoms of life from bacteria to mammals. One of them, the carboxypeptidase A3 (CPA3), has become an important component of the mammalian immune system by its expression in mast cells. Mast cells (MCs) are highly specialized sentinel cells, which store large amounts of bioactive mediators, including CPA3, in very abundant cytoplasmic granules. Clinical studies have found an increased CPA3 expression in asthma but the physiological role as well as the evolutionary origin of CPA3 remains largely unexplored. CPA3 belongs to the M14A subfamily of metallocarboxypeptidases, which among others also includes the digestive enzymes CPA1, CPA2, CPB1 and CPO. To study the appearance of CPA3 during vertebrate evolution, we here performed bioinformatic analyses of homologous genes and gene loci from a broad panel of metazoan animals from invertebrates to mammals. The phylogenetic analysis indicated that CPA3 appeared at the base of tetrapod evolution in a branch closer to CPB1 than to other CPAs. Indeed, CPA3 and CPB1 are also located in the same locus, on chromosome 3 in humans. The presence of CPA3 only in tetrapods and not in fishes, suggested that CPA3 could have appeared by a gene duplication from CPB1 during early tetrapod evolution. However, the apparent loss of CPA3 in several tetrapod lineages, e.g. in birds and monotremes, indicates a complex evolution of the CPA3 gene. Interestingly, in the lack of CPA3 in fishes, zebrafish MCs express instead CPA5 for which the most closely related human carboxypeptidase is CPA1, which has a similar cleavage specificity as CPA3. Collectively, these findings clarify and add to our understanding of the evolution of hematopoietic proteases expressed by mast cells.

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  • 27.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Lara, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines-THP-1 and Mono Mac 6-Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 10, article id 5818Article in journal (Refereed)
    Abstract [en]

    Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

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  • 28.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mohammadamin, Sayran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Fc Receptors for Immunoglobulins and Their Appearance during Vertebrate Evolution2014In: PLOS ONE, E-ISSN 1932-6203, Vol. 9, no 5, p. e96903-Article in journal (Refereed)
    Abstract [en]

    Receptors interacting with the constant domain of immunoglobulins (Igs) have a number of important functions in vertebrates. They facilitate phagocytosis by opsonization, are key components in antibody-dependent cellular cytotoxicity as well as activating cells to release granules. In mammals, four major types of classical Fc receptors (FcRs) for IgG have been identified, one high-affinity receptor for IgE, one for both IgM and IgA, one for IgM and one for IgA. All of these receptors are related in structure and all of them, except the IgA receptor, are found in primates on chromosome 1, indicating that they originate from a common ancestor by successive gene duplications. The number of Ig isotypes has increased gradually during vertebrate evolution and this increase has likely been accompanied by a similar increase in isotype-specific receptors. To test this hypothesis we have performed a detailed bioinformatics analysis of a panel of vertebrate genomes. The first components to appear are the poly-Ig receptors (PIGRs), receptors similar to the classic FcRs in mammals, so called FcRL receptors, and the FcR gamma chain. These molecules are not found in cartilagous fish and may first appear within bony fishes, indicating a major step in Fc receptor evolution at the appearance of bony fish. In contrast, the receptor for IgA is only found in placental mammals, indicating a relatively late appearance. The IgM and IgA/M receptors are first observed in the monotremes, exemplified by the platypus, indicating an appearance during early mammalian evolution. Clearly identifiable classical receptors for IgG and IgE are found only in marsupials and placental mammals, but closely related receptors are found in the platypus, indicating a second major step in Fc receptor evolution during early mammalian evolution, involving the appearance of classical IgG and IgE receptors from FcRL molecules and IgM and IgA/M receptors from PIGR.

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  • 29.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Paivandy, Aida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    How Relevant Are Bone Marrow-Derived Mast Cells (BMMCs) as Models for Tissue Mast Cells?: A Comparative Transcriptome Analysis of BMMCs and Peritoneal Mast Cells2020In: Cells, E-ISSN 2073-4409, Vol. 9, no 9, article id 2118Article in journal (Refereed)
    Abstract [en]

    Bone marrow-derived mast cells (BMMCs) are often used as a model system for studies of the role of MCs in health and disease. These cells are relatively easy to obtain from total bone marrow cells by culturing under the influence of IL-3 or stem cell factor (SCF). After 3 to 4 weeks in culture, a nearly homogenous cell population of toluidine blue-positive cells are often obtained. However, the question is how relevant equivalents these cells are to normal tissue MCs. By comparing the total transcriptome of purified peritoneal MCs with BMMCs, here we obtained a comparative view of these cells. We found several important transcripts that were expressed at very high levels in peritoneal MCs, but were almost totally absent from the BMMCs, including the major chymotryptic granule protease Mcpt4, the neurotrophin receptor Gfra2, the substance P receptor Mrgprb2, the metalloprotease Adamts9 and the complement factor 2 (C2). In addition, there were a number of other molecules that were expressed at much higher levels in peritoneal MCs than in BMMCs, including the transcription factors Myb and Meis2, the MilR1 (Allergin), Hdc (Histidine decarboxylase), Tarm1 and the IL-3 receptor alpha chain. We also found many transcripts that were highly expressed in BMMCs but were absent or expressed at low levels in the peritoneal MCs. However, there were also numerous MC-related transcripts that were expressed at similar levels in the two populations of cells, but almost absent in peritoneal macrophages and B cells. These results reveal that the transcriptome of BMMCs shows many similarities, but also many differences to that of tissue MCs. BMMCs can thereby serve as suitable models in many settings concerning the biology of MCs, but our findings also emphasize that great care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs.

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  • 30.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Paivandy, Aida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, SE-75007 Uppsala, Sweden.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Quantitative In-Depth Analysis of the Mouse Mast Cell Transcriptome Reveals Organ-Specific Mast Cell Heterogeneity2020In: CELLS, E-ISSN 2073-4409, Vol. 9, no 1, article id 211Article in journal (Refereed)
    Abstract [en]

    Mast cells (MCs) are primarily resident hematopoietic tissue cells that are localized at external and internal surfaces of the body where they act in the first line of defense. MCs are found in all studied vertebrates and have also been identified in tunicates, an early chordate. To obtain a detailed insight into the biology of MCs, here we analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR-based transcriptomics. We show that MCs at different tissue locations differ substantially in their levels of transcripts coding for the most abundant MC granule proteins, even within the connective tissue type, or mucosal MC niches. We also demonstrate that transcript levels for the major granule proteins, including the various MC-restricted proteases and the heparin core protein, can be several orders of magnitude higher than those coding for various surface receptors and enzymes involved in protease activation, as well as enzymes involved in the synthesis of heparin, histamine, leukotrienes, and prostaglandins. Interestingly, our analyses revealed an almost complete absence in MCs of transcripts coding for cytokines at baseline conditions, indicating that cytokines are primarily produced by activated MCs. Bone marrow-derived MCs (BMMCs) are often used as equivalents of tissue MCs. Here, we show that these cells differ substantially from tissue MCs with regard to their transcriptome. Notably, they showed a transcriptome indicative of relatively immature cells, both with respect to the expression of granule proteases and of various enzymes involved in the processing/synthesis of granule compounds, indicating that care should be taken when extrapolating findings from BMMCs to the in vivo function of tissue-resident MCs. Furthermore, the latter finding indicates that the development of fully mature tissue-resident MCs requires a cytokine milieu beyond what is needed for in vitro differentiation of BMMCs. Altogether, this study provides a comprehensive quantitative view of the transcriptome profile of MCs resident at different tissue locations that builds nicely on previous studies of both the mouse and human transcriptome, and form a solid base for future evolutionary studies of the role of MCs in vertebrate immunity.

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  • 31.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Paivandy, Aida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pjeler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The mouse mast cell transcriptomeManuscript (preprint) (Other academic)
    Abstract [en]

    Mast cells (MCs) are highly specialized tissue resident cells that are often found at the interphase between body and environment such as the skin, lung and intestinal mucosa. To obtain a more detailed picture of the biology of MCs we have analyzed the transcriptome of MCs from different mouse organs by RNA-seq and PCR based transcriptomics.  The results show that MCs at different tissue locations can differ quite substantially in transcript levels of several of the most abundant granule proteins even if they belong to the same basic MC type, i.e connective tissue or mucosal MCs. We can also see that transcript levels for the major granule proteins, like the various proteases and the heparin core protein can be several orders of magnitude higher than the surface receptors.  This also applies for the processing enzymes involved in activation of the proteases and in the synthesis of heparin and histamine. Interestingly also is the almost complete absence of transcripts for cytokines in the MC populations of the various organs, indicating that cytokines only are produced by activated MCs. Bone marrow derived MCs are often used as equivalents of tissue MCs.  We here show that these cells differ substantially in their transcriptome from tissue MCs. They show a transcriptome of relatively immature cells both with respect to the granule components and to the processing enzymes indicating that care should be taken when transferring findings from these cells to the in vivo function of tissue resident MCs.  This latter finding also give clear indication for that additional cytokines are needed, in addition to the stem cell factor (SCF), for the development into fully mature tissue MCs.

  • 32.
    Akula, Srinivas
    et al.
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, POB 7011, SE-75007 Uppsala, Sweden..
    Riihimaki, Miia
    Swedish Univ Agr Sci, Fac Vet Med & Anim Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    Waern, Ida
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, POB 7011, SE-75007 Uppsala, Sweden..
    Abrink, Magnus
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, SE-75007 Uppsala, Sweden..
    Raine, Amanda
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wernersson, Sara
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, POB 7011, SE-75007 Uppsala, Sweden..
    Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 22, article id 13976Article in journal (Refereed)
    Abstract [en]

    Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.

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  • 33.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Boinapally, Vamsi
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution2015In: PLOS ONE, E-ISSN 1932-6203, Vol. 10, no 11, article id e0143091Article in journal (Refereed)
    Abstract [en]

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

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  • 34.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Tripathi, Shiva Raj
    Charite Univ Med Berlin, Inst Allergol, Hindenburgdamm 30, D-12203 Berlin, Germany.;Freie Univ Berlinand, Hindenburgdamm 30, D-12203 Berlin, Germany.;Humboldt Univ, Hindenburgdamm 30, D-12203 Berlin, Germany.;Fraunhofer Inst Translat Med & Pharmacol ITMP, Immunol & Allergol IA, Hindenburgdamm 30, D-12203 Berlin, Germany..
    Franke, Kristin
    Charite Univ Med Berlin, Inst Allergol, Hindenburgdamm 30, D-12203 Berlin, Germany.;Freie Univ Berlinand, Hindenburgdamm 30, D-12203 Berlin, Germany.;Humboldt Univ, Hindenburgdamm 30, D-12203 Berlin, Germany.;Fraunhofer Inst Translat Med & Pharmacol ITMP, Immunol & Allergol IA, Hindenburgdamm 30, D-12203 Berlin, Germany..
    Wernersson, Sara
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Babina, Magda
    Charite Univ Med Berlin, Inst Allergol, Hindenburgdamm 30, D-12203 Berlin, Germany.;Fraunhofer Inst Translat Med & Pharmacol ITMP, Immunol & Allergol IA, Hindenburgdamm 30, D-12203 Berlin, Germany..
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Cultures of Human Skin Mast Cells, an Attractive In Vitro Model for Studies of Human Mast Cell Biology2024In: Cells, E-ISSN 2073-4409, Vol. 13, no 1, article id 98Article in journal (Refereed)
    Abstract [en]

    Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

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  • 35.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Welinder, Charlotte
    Lund Univ, Dept Clin Sci Lund, Div Mass Spectrometry, SE-22100 Lund, Sweden..
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Identification of the Major Protein Components of Human and Cow Saliva2023In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 24, no 23, article id 16838Article in journal (Refereed)
    Abstract [en]

    Cows produce saliva in very large quantities to lubricate and facilitate food processing. Estimates indicate an amount of 50-150 L per day. Human saliva has previously been found to contain numerous antibacterial components, such as lysozyme, histatins, members of the S-100 family and lactoferrin, to limit pathogen colonization. Cows depend on a complex microbial community in their digestive system for food digestion. Our aim here was to analyze how this would influence the content of their saliva. We therefore sampled saliva from five humans and both nose secretions and saliva from six cows and separated the saliva on SDS-PAGE gradient gels and analyzed the major protein bands with LC-MS/MS. The cow saliva was found to be dominated by a few major proteins only, carbonic anhydrase 6, a pH-stabilizing enzyme and the short palate, lung and nasal epithelium carcinoma-associated protein 2A (SPLUNC2A), also named bovine salivary protein 30 kDa (BSP30) or BPIFA2B. This latter protein has been proposed to play a role in local antibacterial response by binding bacterial lipopolysaccharides (LPSs) and inhibiting bacterial growth but may instead, according to more recent data, primarily have surfactant activity. Numerous peptide fragments of mucin-5B were also detected in different regions of the gel in the MS analysis. Interestingly, no major band on gel was detected representing any of the antibacterial proteins, indicating that cows may produce them at very low levels that do not harm the microbial flora of their digestive system. The nose secretions of the cows primarily contained the odorant protein, a protein thought to be involved in enhancing the sense of smell of the olfactory receptors and the possibility of quickly sensing potential poisonous food components. High levels of secretory IgA were also found in one sample of cow mouth drippings, indicating a strong upregulation during an infection. The human saliva was more complex, containing secretory IgA, amylase, carbonic anhydrase 6, lysozyme, histatins and a number of other less abundant proteins, indicating a major difference to the saliva of cows that show very low levels of antibacterial components, most likely to not harm the microbial flora of the rumen.

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  • 36.
    Akusjärvi, Göran
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kreivi, Jan-Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Petersen-Mahrt, Svend
    Messenger RNA in Eukaryotes2007In: Encyclopedia of Life Sciences, Chichester: John Wiley , 2007, p. 1-8Chapter in book (Other academic)
    Abstract [en]

    Posttranscriptional regulation of gene expression represents an important level at which eukaryotes can expand the coding capacity of their genomes. The concept that one gene makes one protein does not apply to higher eukaryotes. Thus, a eukaryotic cell can use alternative ribonucleic acid (RNA) splicing, alternative polyadenylation and RNA editing to produce hundreds or even several thousands of protein isoforms from a single gene.

  • 37.
    Albers, Suki
    et al.
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Beckert, Bertrand
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Matthies, Marco C.
    Univ Hamburg, Ctr Bioinformat, Hamburg, Germany..
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Schuster, Raphael
    Univ Hamburg, Inst Organ Chem, Hamburg, Germany..
    Seuring, Carolin
    Ctr Struct & Syst Biol, Hamburg, Germany..
    Riedner, Maria
    Univ Hamburg, Inst Organ Chem, Hamburg, Germany..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Torda, Andrew E.
    Univ Hamburg, Ctr Bioinformat, Hamburg, Germany..
    Wilson, Daniel N.
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Ignatova, Zoya
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Repurposing tRNAs for nonsense suppression2021In: Nature Communications, E-ISSN 2041-1723, Vol. 12, article id 3850Article in journal (Refereed)
    Abstract [en]

    Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the T Psi C-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 angstrom resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon. Here, the authors report de novo design, optimization and characterization of tRNAs that decode UGA stop codons in E. coli. The structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon suggests that distinct A-site ligands (tRNAs versus release factors) induce distinct conformation of the stop codon within the mRNA in the decoding center.

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  • 38.
    Ali, Arwa
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer Immunotherapy.
    Gao, Menghan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Iskantar, Alexandros
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wang, Hai
    Chinese Acad Sci, CAS Ctr Excellence Nanosci, Natl Ctr Nanosci & Technol, Key Lab Biomed Effects Nanomat & Nanosafety, Beijing, Peoples R China.;Univ Chinese Acad Sci, Beijing, Peoples R China..
    Karlsson-Parra, Alex
    Mendus AB, Stockholm, Sweden..
    Yu, Di
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Jin, Chuan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Proinflammatory allogeneic dendritic cells enhance the therapeutic efficacy of systemic anti-4-1BB treatment2023In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1146413Article in journal (Refereed)
    Abstract [en]

    As an immune adjuvant, proinflammatory allogeneic dendritic cells (AlloDCs) have demonstrated promising immune-priming effects in several preclinical and clinical studies. The effector cells, including NK cells and T cells are widely acknowledged as pivotal factors in the effectiveness of cancer immunotherapy due to their ability to selectively identify and eradicate malignant cells. 4-1BB, as a costimulatory receptor, plays a significant role in the stimulation of effector cell activation. This study evaluated the anti-tumor effects when combining intratumoral administration of the immune-adjuvant AlloDCs with systemic a4-1BB treatment directly acting on effector cells. In both the CT-26 murine colon carcinoma model and B16 murine melanoma model, AlloDCs demonstrated a significant enhancement in the therapeutic efficacy of a4-1BB antibody. This enhancement was observed through the delayed growth of tumors and prolonged survival. Analysis of the tumor microenvironment (TME) in the combined-treatment group revealed an immune-inflamed TME characterized by increased infiltration of activated endogenous DCs and IFN?(+) CD8(+) T cells, showing reduced signs of exhaustion. Furthermore, there was an augmented presence of tissue-resident memory (T-RM) CD8(+) T cells (CD103(+)CD49a(+)CD69(+)). The combination treatment also led to increased infiltration of CD39(+)CD103(+) tumor-specific CD8(+) T cells and neoantigen-specific T cells into the tumor. Additionally, the combined treatment resulted in a less immunosuppressive TME, indicated by decreased infiltration of myeloid-derived suppressor cells and Tregs. These findings suggest that the combination of intratumoral AlloDCs administration with systemic agonistic a4-1BB treatment can generate a synergistic anti-tumor response, thereby warranting further investigation through clinical studies.

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  • 39. Alizadehheidari, M.
    et al.
    Frykholm, K.
    Fritzsche, J.
    Wigenius, J.
    Modesti, M.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, F.
    Probing the Physical Properties of a DNA-Protein Complex Using Nanofluidic Channels2013In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 42, p. S134-S134Article in journal (Other academic)
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    fulltext
  • 40. Alizadehheidari, Mohammadreza
    et al.
    Werner, Erik
    Noble, Charleston
    Nyberg, Lena
    Fritzsche, Joachim
    Mehlig, Bernhard
    Tegenfeldt, Jonas
    Ambjoernsson, Tobias
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, Fredrik
    Nanoconfined Circular DNA2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 274A-274AArticle in journal (Other academic)
    Abstract [en]

    Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.

  • 41.
    Alizadehheidari, Mohammadreza
    et al.
    Chalmers, Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Werner, Erik
    Gothenburg Univ, Phys, Gothenburg, Sweden..
    Noble, Charleston
    Lund Univ, Phys, Lund, Sweden..
    Nyberg, Lena
    Chalmers, Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Fritzsche, Joachim
    Chalmers, Appl Phys, S-41296 Gothenburg, Sweden..
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mehlig, Bernhard
    Gothenburg Univ, Phys, Gothenburg, Sweden..
    Tegenfeldt, Jonas
    Lund Univ, Solid State Phys, Gothenburg, Sweden..
    Ambjornsson, Tobias
    Lund Univ, Phys, Lund, Sweden..
    Westerlund, Fredrik
    Chalmers, Biol & Biol Engn, S-41296 Gothenburg, Sweden..
    Unfolding of Nanoconfined Circular DNA2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 231A-231AArticle in journal (Other academic)
  • 42. Alizadehheidari, Mohammadreza
    et al.
    Werner, Erik
    Noble, Charleston
    Reiter-Schad, Michaela
    Nyberg, Lena K.
    Fritzsche, Joachim
    Mehlig, Bernhard
    Tegenfeldt, Jonas O.
    Ambjornsson, Tobias
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Westerlund, Fredrik
    Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics2015In: Macromolecules, ISSN 0024-9297, E-ISSN 1520-5835, Vol. 48, no 3, p. 871-878Article in journal (Refereed)
    Abstract [en]

    Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of confined DNA from the circular to linear configuration as a light-induced double-strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to what extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations.

  • 43.
    Allam, Venkata
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Biomed Ctr, Dept Med Biochem & Microbiol, Uppsala, Sweden..
    Chellappan, Dinesh Kumar
    Int Med Univ, Sch Pharm, Dept Life Sci, Kuala Lumpur, Malaysia..
    Jha, Niraj Kumar
    Sharda Univ, Sch Engn & Technol, Dept Biotechnol, Greater Noida, Uttar Pradesh, India..
    Shastri, Madhur D.
    Univ Tasmania, Coll Hlth & Med, Sch Hlth Sci, Launceston, Tas, Australia..
    Gupta, Gaurav
    Suresh Gyan Vihar Univ, Sch Pharm, Jaipur, India..
    Shukla, Shakti D.
    Univ Newcastle, Hunter Med Res Inst, Prior Res Ctr Hlth Lungs, New Lambton Hts, Newcastle, NSW, Australia..
    Singh, Sachin K.
    Lovely Profess Univ, Sch Pharmaceut Sci, Phagwara, Punjab, India..
    Sunkara, Krishna
    John Hunter Hosp, Intens Care Unit, Emergency Clin Management, Newcastle, NSW, Australia..
    Chitranshi, Nitin
    Macquarie Univ, Fac Med Hlth & Human Sci, N Ryde, NSW, Australia..
    Gupta, Vivek
    Macquarie Univ, Fac Med Hlth & Human Sci, N Ryde, NSW, Australia..
    Wich, Peter R.
    Univ New South Wales, Sch Chem Engn, Sydney, NSW, Australia.;Univ New South Wales, Ctr Nanomed, Sydney, NSW, Australia..
    MacLoughlin, Ronan
    Aerogen, IDA Business Pk, Dangan, Galway, Ireland.;Royal Coll Surgeons Ireland, Sch Pharm & Biomol Sci, Dublin, Ireland.;Trinity Coll, Sch Pharm & Pharmaceut Sci, Sydney, NSW, Australia..
    Oliver, Brian Gregory George
    Univ Technol Sydney, Fac Sci, Sch Life Sci, Sydney, NSW, Australia.;Univ Sydney, Woolcock Inst Med Res, Sydney, NSW, Australia..
    Wernersson, Sara
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden..
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Biomed Ctr, Dept Med Biochem & Microbiol, Uppsala, Sweden.;Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden..
    Dua, Kamal
    Univ Technol Sydney, Grad Sch Hlth, Discipline Pharm, Sydney, NSW, Australia..
    Treatment of chronic airway diseases using nutraceuticals: Mechanistic insight2022In: Critical reviews in food science and nutrition, ISSN 1040-8398, E-ISSN 1549-7852, Vol. 62, no 27, p. 7576-7590Article, review/survey (Refereed)
    Abstract [en]

    Respiratory diseases, both acute and chronic, are reported to be the leading cause of morbidity and mortality, affecting millions of people globally, leading to high socio-economic burden for the society in the recent decades. Chronic inflammation and decline in lung function are the common symptoms of respiratory diseases. The current treatment strategies revolve around using appropriate anti-inflammatory agents and bronchodilators. A range of anti-inflammatory agents and bronchodilators are currently available in the market; however, the usage of such medications is limited due to the potential for various adverse effects. To cope with this issue, researchers have been exploring various novel, alternative therapeutic strategies that are safe and effective to treat respiratory diseases. Several studies have been reported on the possible links between food and food-derived products in combating various chronic inflammatory diseases. Nutraceuticals are examples of such food-derived products which are gaining much interest in terms of its usage for the well-being and better human health. As a consequence, intensive research is currently aimed at identifying novel nutraceuticals, and there is an emerging notion that nutraceuticals can have a positive impact in various respiratory diseases. In this review, we discuss the efficacy of nutraceuticals in altering the various cellular and molecular mechanisms involved in mitigating the symptoms of respiratory diseases.

  • 44. Allen, Andrew J.
    et al.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Kaysser-Pyzalla, Anke R.
    Beyond the International Year of Crystallography2015In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 48, no P1, p. 1-2Article in journal (Other academic)
  • 45.
    Allen, Andrew J.
    et al.
    NIST, Mat Measurement Sci Div Gaithersburg, MD USA.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. AS CR, European Extreme Light Infrastruct, Inst Phys, Prague, Czech Republic..
    McIntyre, Garry J.
    Australian Nucl Sci & Technol Org, New Illawarra Rd, Lucas Heights, NSW, Australia.
    Journal of Applied Crystallography: the first 50 years and beyond2018In: Journal of applied crystallography, ISSN 0021-8898, E-ISSN 1600-5767, Vol. 51, no Part: 2, p. 233-234Article in journal (Other academic)
    Abstract [en]

    The Editors of Journal of Applied Crystallography mark the journal's 50th anniversary.

  • 46. Allen, Gregory S
    et al.
    Zavialov, Andrey
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Gursky, Richard
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Frank, Joachim
    The cryo-EM structure of a translation initiation complex from Escherichia coli.2005In: Cell, ISSN 0092-8674, Vol. 121, no 5, p. 703-12Article in journal (Other scientific)
  • 47.
    Allen, Marie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Divne, Anna-Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Universal tag arrays in forensic SNP analysis.2005In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 297, p. 141-154Article in journal (Refereed)
    Abstract [en]

    Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.

  • 48.
    Alm, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Scholz, Birger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kultima, Kim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nilsson, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Andrén, Per E
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Savitski, Mikhail M
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bergman, Åke
    Stigson, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fex-Svenningsen, Åsa
    Institute of Medical Biology, Anatomy and Neurobiology, University of Southern Denmark, Denmark.
    Dencker, Lennart
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    In Vitro Neurotoxicity of PBDE-99: Immediate and Concentration-Dependent Effects on Protein Expression in Cerebral Cortex Cells2010In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 9, no 3, p. 1226-1235Article in journal (Refereed)
    Abstract [en]

    Polybrominated diphenyl ethers (PBDEs) are commonly used flame retardants in various consumer products. Pre- and postnatal exposure to congeners of PBDEs disrupts normal brain development in rodents. Two-dimensional difference gel electrophoresis (2D-DIGE) was used to analyze concentration-dependent differences in protein expression in cultured cortical cells isolated from rat fetuses (GD 21) after 24 h exposure to PBDE-99 (3, 10, or 30 muM). Changes on a post-translational level were studied using a 1 h exposure to 30 muM PBDE-99. The effects of 24 h exposure to 3 and 30 muM PBDE-99 on mRNA levels were measured using oligonucleotide microarrays. A total of 62, 46, and 443 proteins were differentially expressed compared to controls after 24 h of exposure to 3, 10, and 30 muM PDBE-99, respectively. Of these, 48, 43, and 238 proteins were successfully identified, respectively. We propose that the biological effects of low-concentration PBDE-99 exposure are fundamentally different than effects of high-concentration exposure. Low-dose PBDE-99 exposure induced marked effects on cytoskeletal proteins, which was not correlated to cytotoxicity or major morphological effects, suggesting that other more regulatory aspects of cytoskeletal functions may be affected. Interestingly, 0.3 and 3 muM, but not 10 or 30 muM increased the expression of phosphorylated (active) Gap43, perhaps reflecting effects on neurite extension processes.

  • 49.
    Alm Rosenblad, Magnus
    et al.
    Univ Gothenburg, Dept Chem & Mol Biol, Gothenburg, Sweden..
    Abramova, Anna
    Univ Gothenburg, Dept Chem & Mol Biol, Gothenburg, Sweden..
    Lind, Ulrika
    Univ Gothenburg, Dept Chem & Mol Biol, Gothenburg, Sweden..
    Ólason, Páll I.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Giacomello, Stefania
    Stockholm Univ, Dept Biochem & Biophys, Natl Bioinformat Infrastruct Sweden, Sci Life Lab, Box 1031, S-17121 Solna, Sweden..
    Nystedt, Björn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Blomberg, Anders
    Univ Gothenburg, Dept Chem & Mol Biol, Gothenburg, Sweden..
    Genomic Characterization of the Barnacle Balanus improvisus Reveals Extreme Nucleotide Diversity in Coding Regions2021In: Marine Biotechnology, ISSN 1436-2228, E-ISSN 1436-2236, Vol. 23, p. 402-416Article in journal (Refereed)
    Abstract [en]

    Barnacles are key marine crustaceans in several habitats, and they constitute a common practical problem by causing biofouling on man-made marine constructions and ships. Despite causing considerable ecological and economic impacts, there is a surprising void of basic genomic knowledge, and a barnacle reference genome is lacking. We here set out to characterize the genome of the bay barnacle Balanus improvisus (= Amphibalanus improvisus) based on short-read whole-genome sequencing and experimental genome size estimation. We show both experimentally (DNA staining and flow cytometry) and computationally (k-mer analysis) that B. improvisus has a haploid genome size of similar to 740 Mbp. A pilot genome assembly rendered a total assembly size of similar to 600 Mbp and was highly fragmented with an N50 of only 2.2 kbp. Further assembly-based and assembly-free analyses revealed that the very limited assembly contiguity is due to the B. improvisus genome having an extremely high nucleotide diversity (pi) in coding regions (average pi approximate to 5% and average pi in fourfold degenerate sites approximate to 20%), and an overall high repeat content (at least 40%). We also report on high variation in the alpha-octopamine receptor OctA (average pi = 3.6%), which might increase the risk that barnacle populations evolve resistance toward antifouling agents. The genomic features described here can help in planning for a future high-quality reference genome, which is urgently needed to properly explore and understand proteins of interest in barnacle biology and marine biotechnology and for developing better antifouling strategies.

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  • 50.
    Almeida, Pedro
    et al.
    UCL, Dept Genet Evolut & Environm, London, England.
    Proux-Wera, Estelle
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Stockholm, Sweden.
    Churcher, Allison
    Umeå Univ, Dept Mol Biol, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Umeå, Sweden.
    Soler, Lucile
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dainat, Jacques
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Pucholt, Pascal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Rheumatology. Swedish Univ Agr Sci, Uppsala BioCtr, Linnean Ctr Plant Biol, Dept Plant Biol, Uppsala, Sweden.
    Nordlund, Jessica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Martin, Tom
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Rönnberg-Wästljung, Ann-Christin
    Swedish Univ Agr Sci, Uppsala BioCtr, Linnean Ctr Plant Biol, Dept Plant Biol, Uppsala, Sweden.
    Nystedt, Björn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Berlin, Sofia
    Swedish Univ Agr Sci, Uppsala BioCtr, Linnean Ctr Plant Biol, Dept Plant Biol, Uppsala, Sweden.
    Mank, Judith E.
    UCL, Dept Genet Evolut & Environm, London, England; Univ British Columbia, Dept Zool, Vancouver, BC, Canada; Univ British Columbia, Biodivers Res Ctr, Vancouver, BC, Canada.
    Genome assembly of the basket willow, Salix viminalis, reveals earliest stages of sex chromosome expansion2020In: BMC Biology, E-ISSN 1741-7007, Vol. 18, no 1, article id 78Article in journal (Refereed)
    Abstract [en]

    Background

    Sex chromosomes have evolved independently multiple times in eukaryotes and are therefore considered a prime example of convergent genome evolution. Sex chromosomes are known to emerge after recombination is halted between a homologous pair of chromosomes, and this leads to a range of non-adaptive modifications causing gradual degeneration and gene loss on the sex-limited chromosome. However, the proximal causes of recombination suppression and the pace at which degeneration subsequently occurs remain unclear.

    Results

    Here, we use long- and short-read single-molecule sequencing approaches to assemble and annotate a draft genome of the basket willow, Salix viminalis, a species with a female heterogametic system at the earliest stages of sex chromosome emergence. Our single-molecule approach allowed us to phase the emerging Z and W haplotypes in a female, and we detected very low levels of Z/W single-nucleotide divergence in the non-recombining region. Linked-read sequencing of the same female and an additional male (ZZ) revealed the presence of two evolutionary strata supported by both divergence between the Z and W haplotypes and by haplotype phylogenetic trees. Gene order is still largely conserved between the Z and W homologs, although the W-linked region contains genes involved in cytokinin signaling regulation that are not syntenic with the Z homolog. Furthermore, we find no support across multiple lines of evidence for inversions, which have long been assumed to halt recombination between the sex chromosomes.

    Conclusions

    Our data suggest that selection against recombination is a more gradual process at the earliest stages of sex chromosome formation than would be expected from an inversion and may result instead from the accumulation of transposable elements. Our results present a cohesive understanding of the earliest genomic consequences of recombination suppression as well as valuable insights into the initial stages of sex chromosome formation and regulation of sex differentiation.

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