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  • 51.
    Chandra, Naresh
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Liu, Yan
    Liu, Jing-Xia
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Frängsmyr, Lars
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Wu, Nian
    Silva, Lisete M
    Lindström, Mona
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Chai, Wengang
    Domellöf, Fatima Pedrosa
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Feizi, Ten
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Sulfated Glycosaminoglycans as Viral Decoy Receptors for Human Adenovirus Type 372019In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 11, no 3, article id E247Article in journal (Refereed)
    Abstract [en]

    Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.

  • 52.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Doudna, Jennifer A.
    Biotechnology: rewriting a genome2013In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 495, no 7439, p. 50-51Article in journal (Other academic)
  • 53.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Richter, Hagen
    van der Oost, John
    White, Malcolm F.
    Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity2015In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, no 3, p. 428-441Article, review/survey (Refereed)
    Abstract [en]

    CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

  • 54. Chen, Zhen
    et al.
    Mori, Wakana
    Deng, Xiaoyun
    Cheng, Ran
    Ogasawara, Daisuke
    Zhang, Genwei
    Schafroth, Michael A.
    Dahl, Kenneth
    Fu, Hualong
    Hatori, Akiko
    Shao, Tuo
    Zhang, Yiding
    Yamasaki, Tomoteru
    Zhang, Xiaofei
    Rong, Jian
    Yu, Qngzhen
    Hu, Kuan
    Fujinaga, Masayuki
    Xie, Lin
    Kumata, Katsushi
    Gou, Yuancheng
    Chen, Jingjin
    Gu, Shuyin
    Bao, Liang
    Wang, Lu
    Collier, Thomas Lee
    Vasdev, Neil
    Shao, Yihan
    Ma, Jun-An
    Cravatt, Benjamin F.
    Fowler, Christopher
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Josephson, Lee
    Zhang, Ming-Rong
    Liang, Steven H.
    Design, Synthesis, and Evaluation of Reversible and Irreversible Monoacylglycerol Lipase Positron Emission Tomography (PET) Tracers Using a "Tail Switching" Strategy on a Piperazinyl Azetidine Skeleton2019In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 62, no 7, p. 3336-3353Article in journal (Refereed)
    Abstract [en]

    Monoacylglycerol lipase (MAGL) is a senile hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the endocannabinoid system (eCB). Selective inhibition of MAGL has emerged as a potential therapeutic approach for the treatment of diverse pathological conditions, including chronic pain, inflammation, cancer, and neurodegeneration. Herein, we disclose a novel array of reversible and irreversible MAGL inhibitors by means of "tail switching" on a piperazinyl azetidine scaffold. We developed a lead irreversible-binding MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with C-11 or F-18. [C-11]8 ([C-11]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible radioligands [C-11]17 ([C-11]PAD) and [F-18]37 ([F-18]MAGL-4-11) also demonstrated excellent in vivo binding specificity toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission tomography tracers with tunability in reversible and irreversible binding mechanisms.

  • 55. Cheng, Ran
    et al.
    Mori, Wakana
    Ma, Longle
    Alhouayek, Mireille
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Hatori, Akiko
    Zhang, Yiding
    Ogasawara, Daisuke
    Yuan, Gengyang
    Chen, Zhen
    Zhang, Xiaofei
    Shi, Hang
    Yamasaki, Tomoteru
    Xie, Lin
    Kumata, Katsushi
    Fujinaga, Masayuki
    Nagai, Yuji
    Minamimoto, Takafumi
    Svensson, Mona
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Wang, Lu
    Du, Yunfei
    Ondrechen, Mary Jo
    Vasdev, Neil
    Cravatt, Benjamin F.
    Fowler, Christopher
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Zhang, Ming-Rong
    Liang, Steven H.
    In Vitro and in Vivo Evaluation of C-11-Labeled Azetidinecarboxylates for Imaging Monoacylglycerol Lipase by PET Imaging Studies2018In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 6, p. 2278-2291Article in journal (Refereed)
    Abstract [en]

    Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing endogenous cannabinoid ligand 2-arachidonoyglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation, and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including C-11-carbonylation and spirocyclic iodonium ylide (SCIDY) radio fluorination. The lead compound [C-11]10 (MAGL-0519) demonstrated high specific binding and selectivity in vitro and in vivo. We also observed unexpected washout kinetics with these irreversible radiotracers, in which in vivo evidence for turnover of the covalent residue was unveiled between MAGL and azetidine carboxylates. This work may lead to new directions for drug discovery and PET tracer development based on azetidine carboxylate inhibitor scaffold.

  • 56.
    Chermenina, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pokrzywa, Malgorzata
    Antti, Henrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Strömberg, Ingrid
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Single injection of small-molecule amyloid accelerator results in cell death of nigral dopamine neurons in mice2015In: Parkinson's Disease, ISSN 2090-8083, E-ISSN 2042-0080, Vol. 1, article id 15024Article in journal (Refereed)
    Abstract [en]

    The assembly process of a-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson´s disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of a-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of a-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and follwed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substansia nigra of normal and a-synuclein knockout mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed singificant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in a-synuclein knock-out mice injected with accelerator intor the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson´s disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice.

  • 57.
    Ching, Rosanna C.
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    The role of exosomes in peripheral nerve regeneration2015In: Neural Regeneration Research, ISSN 1673-5374, E-ISSN 1876-7958, Vol. 10, no 5, p. 743-747Article, review/survey (Refereed)
    Abstract [en]

    Peripheral nerve injuries remain problematic to treat, with poor functional recovery commonly observed. Injuries resulting in a nerve gap create specific difficulties for axonal regeneration. Approaches to address these difficulties include autologous nerve grafts (which are currently the gold standard treatment) and synthetic conduits, with the latter option being able to be impregnated with Schwann cells or stem cells which provide an appropriate micro-environment for neuronal regeneration to occur. Transplanting stem cells, however, infers additional risk of malignant transformation as well as manufacturing difficulties and ethical concerns, and the use of autologous nerve grafts and Schwann cells requires the sacrifice of a functioning nerve. A new approach utilizing exosomes, secreted extracellular vesicles, could avoid these complications. In this review, we summarize the current literature on exosomes, and suggest how they could help to improve axonal regeneration following peripheral nerve injury.

  • 58. Chiruvella, Kishore K
    et al.
    Rajaei, Naghmeh
    Jonna, Venkateswara Rao
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Åstrom, Stefan U
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 21671Article in journal (Refereed)
    Abstract [en]

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  • 59.
    Chylinski, Krzysztof
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max F. Perutz Laboratories, University of Vienna, Austria .
    Makarova, Kira S.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Koonin, Eugene V.
    Classification and evolution of type II CRISPR-Cas systems2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 10, p. 6091-6105Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.

  • 60. Clendenen, Tess V
    et al.
    Koenig, Karen L
    Arslan, Alan A
    Lukanova, Annekatrin
    Berrino, Franco
    Gu, Yian
    Hallmans, Göran
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Nutritional Research.
    Idahl, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Krogh, Vittorio
    Lokshin, Anna E
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Muti, Paola
    Marrangoni, Adele
    Nolen, Brian M
    Ohlson, Nina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Shore, Roy E
    Sieri, Sabina
    Zeleniuch-Jacquotte, Anne
    Factors associated with inflammation markers, a cross-sectional analysis2011In: Cytokine, ISSN 1043-4666, E-ISSN 1096-0023, Vol. 56, no 3, p. 769-778Article in journal (Refereed)
    Abstract [en]

    Epidemiological studies have reported associations between circulating inflammation markers and risk of chronic diseases. It is of interest to examine whether risk factors for these diseases are associated with inflammation. We conducted a cross-sectional analysis to evaluate whether reproductive and lifestyle factors and circulating vitamin D were associated with inflammation markers, including C-reactive protein, cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-13, TNFα), and cytokine modulators (IL-1RA, sIL-1RII, sIL-2Ra, sIL-4R, sIL-6R, sTNF-R1/R2), among 616 healthy women. We confirmed associations of several inflammation markers with age and BMI. We also observed significantly higher levels of certain inflammation markers in postmenopausal vs. premenopausal women (TNFα, sIL-1RII, sIL-2Ra), with increasing parity (IL-12p40), and with higher circulating 25(OH) vitamin D (IL-13) and lower levels among current users of non-steroidal anti-inflammatory drugs (NSAIDs) (IL-1β, IL-2, IL-10, IL-12p70, and IL-12p40), current smokers (IL-4, IL-13, IL-12p40), and women with a family history of breast or ovarian cancer (IL-4, IL-10, IL-13). Our findings suggest that risk factors for chronic diseases (age, BMI, menopausal status, parity, NSAID use, family history of breast and ovarian cancer, and smoking) are associated with inflammation markers in healthy women.

  • 61. Cooper, Helen M.
    et al.
    Yang, Yang
    Ylikallio, Emil
    Khairullin, Rafil
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia.
    Woldegebriel, Rosa
    Lin, Kai-Lan
    Euro, Liliya
    Palin, Eino
    Wolf, Alexander
    Trokovic, Ras
    Isohanni, Pirjo
    Kaakkola, Seppo
    Auranen, Mari
    Lönnqvist, Tuula
    Wanrooij, Sjoerd
    Tyynismaa, Henna
    ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia2017In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 26, no 8, p. 1432-1443Article in journal (Refereed)
    Abstract [en]

    De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

  • 62.
    Costa, Tiago
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College, London, UK.
    Francis, Monika K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Region Västerbotten.
    Farag, Salah
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Measurement of Yersinia translocon pore formation in erythrocytes2019In: Pathogenic Yersinia: methods and protocols / [ed] Viveka Vadyvaloo and Matthew B. Lawrenz, New York, NY, U.S.A.: Humana Press, 2019, p. 211-229Chapter in book (Refereed)
    Abstract [en]

    Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.

  • 63. Daumke, Oliver
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Vallis, Yvonne
    Martens, Sascha
    Butler, P Jonathan G
    McMahon, Harvey T
    Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 449, no 7164, p. 923-927Article in journal (Refereed)
    Abstract [en]

    The ability to actively remodel membranes in response to nucleotide hydrolysis has largely been attributed to GTPases of the dynamin superfamily, and these have been extensively studied. Eps15 homology (EH)-domain-containing proteins (EHDs/RME-1/pincher) comprise a less-well-characterized class of highly conserved eukaryotic ATPases implicated in clathrin-independent endocytosis, and recycling from endosomes. Here we show that EHDs share many common features with the dynamin superfamily, such as a low affinity for nucleotides, the ability to tubulate liposomes in vitro, oligomerization around lipid tubules in ring-like structures and stimulated nucleotide hydrolysis in response to lipid binding. We present the structure of EHD2, bound to a non-hydrolysable ATP analogue, and provide evidence consistent with a role for EHDs in nucleotide-dependent membrane remodelling in vivo. The nucleotide-binding domain is involved in dimerization, which creates a highly curved membrane-binding region in the dimer. Oligomerization of dimers occurs on another interface of the nucleotide-binding domain, and this allows us to model the EHD oligomer. We discuss the functional implications of the EHD2 structure for understanding membrane deformation.

  • 64.
    Desmarais, Samantha M
    et al.
    Department of Bioengineering, Stanford University, Stanford, CA, USA.
    De Pedro, Miguel A
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Huang, Kerwyn Casey
    Department of Bioengineering, Stanford University, Stanford, CA, USA ; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
    Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 89, no 1, p. 1-13Article in journal (Refereed)
    Abstract [en]

    The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

  • 65. Desmarais, Samantha M
    et al.
    Tropini, Carolina
    Miguel, Amanda
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Monds, Russell D
    de Pedro, Miguel A
    Huang, Kerwyn Casey
    High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 52, p. 31090-31100Article in journal (Refereed)
    Abstract [en]

    The bacterial cell wall is a network of glycan strands crosslinked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.

  • 66.
    di Summa, Pietro G
    et al.
    University of Manchester, University Hospital of Lausanne.
    Kalbermatten, Daniel F
    University Hospital of Basel.
    Raffoul, Wassim
    University Hospital of Lausanne.
    Terenghi, Giorgio
    University of Manchester.
    Kingham, Paul J
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. University of Manchester.
    Extracellular matrix molecules enhance the neurotrophic effect of Schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions2013In: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 19, no 3-4, p. 368-379Article in journal (Refereed)
    Abstract [en]

    Since the first reports of induction of adipose-derived stem cells (ASC) into neuronal and glial cell phenotypes, expectations have increased regarding their use in tissue engineering applications for nerve repair. Cell adhesion to extracellular matrix (ECM) is a basic feature of survival, differentiation, and migration of Schwann cells (SC) during nerve regeneration, and fibronectin and laminin are two key molecules of this process. Interaction between ECM and SC-like differentiated ASC (dASC) could potentially improve the neurotrophic potential of the stem cells. We have investigated the effect of ECM molecules on SC-like dASC in terms of proliferation, adhesion, and cell viability. Fibronectin and laminin did not affect the proliferation of dASC when compared with cell adherent tissue culture plastic, but significantly improved viability and cell attachment when dASC were exposed to apoptotic conditions. To assess the influence of the ECM molecules on dASC neurotrophic activity, dASC were seeded onto ECM-coated culture inserts suspended above dorsal root ganglia (DRG) sensory neurons. Neurite outgrowth of DRG neurons was enhanced when dASC were seeded on fibronectin and laminin when compared with controls. When DRG neurons and dASC were in direct contact on the various surfaces there was significantly enhanced neurite outgrowth and coculture with laminin-conditioned dASC produced the longest neurites. Compared with primary SCs, dASC grown on laminin produced similar levels of neurite outgrowth in the culture insert experiments but neurite length was shorter in the direct contact groups. Anti beta 1 integrin blocking antibody could inhibit baseline and dASC evoked neurite elongation but had no effect on outgrowth mediated by laminin-conditioned dASC. ECM molecules had no effect on the levels of nerve growth factor and brain-derived neurotrophic factor secretion from dASC. The results of the study suggest that ECM molecules can significantly improve the potential of dASC for nerve regeneration.

  • 67.
    Do, Lan
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Dahl, Christen P
    Kerje, Susanne
    Hansell, Peter
    Mörner, Stellan
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Cardiology.
    Lindqvist, Ulla
    Engström-Laurent, Anna
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hellman, Urban
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    High Sensitivity Method to Estimate Distribution of Hyaluronan Molecular Sizes in Small Biological Samples Using Gas-Phase Electrophoretic Mobility Molecular Analysis.2015In: International Journal of Cell Biology, ISSN 1687-8876, E-ISSN 1687-8884, article id 938013Article in journal (Refereed)
    Abstract [en]

    Hyaluronan is a negatively charged polydisperse polysaccharide where both its size and tissue concentration play an important role in many physiological and pathological processes. The various functions of hyaluronan depend on its molecular size. Up to now, it has been difficult to study the role of hyaluronan in diseases with pathological changes in the extracellular matrix where availability is low or tissue samples are small. Difficulty to obtain large enough biopsies from human diseased tissue or tissue from animal models has also restricted the study of hyaluronan. In this paper, we demonstrate that gas-phase electrophoretic molecular mobility analyzer (GEMMA) can be used to estimate the distribution of hyaluronan molecular sizes in biological samples with a limited amount of hyaluronan. The low detection level of the GEMMA method allows for estimation of hyaluronan molecular sizes from different parts of small organs. Hence, the GEMMA method opens opportunity to attain a profile over the distribution of hyaluronan molecular sizes and estimate changes caused by disease or experimental conditions that has not been possible to obtain before.

  • 68. Doherty, Gary J
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    GRAF1-dependent endocytosis2009In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 37, no 5, p. 1061-1065Article in journal (Refereed)
    Abstract [en]

    The role of endocytosis in controlling a multitude of cell biological events is well established. Molecular and mechanistic characterization of endocytosis has predominantly focused on CME (clathrin-mediated endocytosis), although many other endocytic pathways have been described. it was recently shown that the BAR (Bin/amphiphysin/Rvs) and Rho GAP (GTPase-activating protein) domain-containing protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) is found on prevalent, pleiomorphic endocytic membranes, and is essential for the major, clathrin-independent endocytic pathway that these membranes mediate. This pathway is characterized by its ability to internalize GPI (glycosylphosphatidylinositol)anchored proteins, bacterial toxins and large amounts of extracellular fluid. These membrane carriers are highly dynamic and associated with the activity of the small G-protein Cdc42 (cell division cycle 42). in the present paper, we review the role of GRAF1 in this CLIC (clathrin-independent carrier)/GEEC (GPI-anchored protein-enriched early endocytic compartment) endocytic pathway and discuss the current understanding regarding how this multidomain protein functions at the interface between membrane sculpting, small G-protein signalling and endocytosis.

  • 69. Dopson, Mark
    et al.
    Ossandon, Francisco J.
    Lövgren, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Holmes, David S.
    Metal resistance or tolerance?: Acidophiles confront high metal loads via both abiotic and biotic mechanisms2014In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 5, p. 157-Article in journal (Refereed)
    Abstract [en]

    All metals are toxic at high concentrations and consequently their intracellular concentrations must be regulated. Extremely acidophilic microorganisms have an optimum growth of pH <3 and proliferate in natural and anthropogenic low pH environments. Some acidophiles are involved in the catalysis of sulfide mineral dissolution, resulting in high concentrations of metals in solution. Acidophiles are often described as highly metal resistant via mechanisms such as multiple and/or more efficient active resistance systems than are present in neutrophiles. However, this is not the case for all acidophiles and we contend that their growth in high metal concentrations is partially due to an intrinsic tolerance as a consequence of the environment in which they live. In this perspective, we highlight metal tolerance via complexation of free metals by sulfate ions and passive tolerance to metal influx via an internal positive cytoplasmic transmembrane potential. These tolerance mechanisms have been largely ignored in past studies of acidophile growth in the presence of metals and should be taken into account.

  • 70.
    Dubbaka Venu, Pradeep Reddy
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Molecular studies of intra-oocyte phosphatidylinositol 3 kinase (PI3K) signaling pathway in controlling female fertility2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The primordial follicle pool is the main source of developing follicles in the ovary. The length of reproductive life and the onset of menopause are governed by the amount of primordial follicles in the ovary. The genetic factors and molecular mechanisms that maintain the primordial follicles in a dormant and surviving state for the whole of reproductive life are not well understood. The phosphatidylinositol 3 kinase (PI3K) signaling pathways in the oocyte that control oocyte growth and early follicular development are largely unknown. The major aim of this thesis was to investigate the functional role of the intra-oocyte PI3K pathway in the regulation of primordial follicle activation and survival.

     Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a major negative regulator of PI3K. The conditional deletion of Pten in the oocytes of primordial follicles led to the overgrowth of oocytes and activation of the entire pool of primordial follicles. There were higher numbers of activated primordial follicles at postnatal day 8 (PD8) in ovaries lacking PTEN in oocytes; by PD35 all the primordial follicles were activated and all the follicles were depleted by 12 weeks, causing premature ovarian failure (POF). In addition, the rate of follicular death that occurs during sexual maturity is reduced in ovaries that lack PTEN in oocytes. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling and upregulation of both expression and activation of ribosomal protein S6 (rpS6). The overactivation of primordial follicles in ovaries that lack PTEN in oocytes is believed to be due to elevated expression and activation of rpS6. PTEN in oocytes is indispensable for the maintenance of primordial follicles in dormancy.

     To study the role of the intra-oocyte PI3K signaling pathway in controlling the survival and maintenance of primordial follicles, 3-phosphoinositide-dependent protein kinase-1 (PDK1) was deleted in oocytes of primordial follicle. The loss of Pdk1 in oocytes led to the depletion of most primordial follicles around the onset of sexual maturity, causing POF during early adulthood. Furthermore, the activation of Akt, p70 S6 kinase 1 (S6K1), and rpS6 was impaired in oocytes that lacked PDK1. The suppressed PDK1–Akt–S6K1–rpS6 signaling in oocytes appears to be responsible for the loss of primordial follicles. The excessive activation of primordial follicles seen in the absence of Pten in oocytes could be reversed by concurrent deletion of Pdk1. In addition, the elevated activation of Akt and S6K1 in the absence of PTEN in oocytes was not observed in PTEN and PDK1 double mutant mice. Similarly, the hyperphosphorylation of rpS6 in oocytes that lack PTEN was prevented in double mutant mice, which was most likely due to downregulation of S6K1 activation. Thus, inactivation of rpS6 in double mutant mice might be the reason for the prevention of excessive primordial follicular activation and survival.

     PTEN and PDK1 in oocytes are essential for the maintenance of quiescence and survival of primordial follicles. The molecular network involving PI3K/PTEN–PDK1 signaling in oocyte controls the survival, loss, and activation of primordial follicles, which together govern reproductive aging and determine the length of reproductive life in females. The results of the above studies indicate that the mammalian oocyte serves as the seat of programming of follicular activation and survival.

  • 71. Duffy, Margaret R.
    et al.
    Alonso-Padilla, Julio
    John, Lijo
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Chandra, Naresh
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Khan, Selina
    Ballmann, Monika Z.
    Lipiec, Agnieszka
    Heemskerk, Evert
    Custers, Jerome
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Havenga, Menzo
    Baker, Andrew H.
    Lemckert, Angelique
    Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 562018In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 99, p. 135-147Article in journal (Refereed)
    Abstract [en]

    The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.

  • 72.
    Dwibedi, Chinmay Kumar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Swedish Defence Research Agency.
    Francisella tularensis: persistence, dissemination and source attribution: a theoretical and computational approach2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The bacterium Francisella tularensis causing tularemia in humans and other mammals displays little genetic diversity among genomes across temporal and spatial scales. F. tularensis infects humans with an extremely low infectious dose and causes natural seasonal tularemia outbreaks. During the Cold War, this bacterium was developed as a biological weapon.

    In paper I, we aimed at investigating the genetic diversity of F. tularensis over space and time and were especially interested in the influence of spatial dispersal on the genetic diversity. By analyses of single-nucleotide polymorphisms (SNPs) among 205 F. tularensis genomes, we found that tularemia had moved from East to West over the European continent by dispersal patterns characterized by multiple long-range dispersal events. Evolutionary rate estimates based on the year of bacterial isolation from 1947 to 2012 indicated non-measurable rates. In outbreak areas with multiple recent outbreaks, however, there was a measurable rate of 0.4 SNPs/genome/year indicating that in areas with more intense disease activity, there is a detectable evolutionary rate. The findings suggest that long-range geographical dispersal events and mostly very low evolutionary rates are important factors contributing to a very low genetic diversity of F. tularensis populations.

    In paper II, we focused on a geographically restricted area with a history of frequent tularemia outbreaks to study F. tularensis persistence. By analyzing F. tularensis genomes from 138 individuals infected from 1994 to 2010 in Örebro County in Sweden and performing a long-term laboratory storage experiment, we explored the microbial population concept of a pathogen seed-bank. We found that eight indistinguishable genomes – each of them defined by no SNPs across 1.65 million whole-genome nucleotides – locally persisted over 2-9 years. We found unmeasurable SNP accumulation rates and overlapping bacterial generations among the outbreak genomes and that F. tularensis survived in saline for four years without nutrients. By these findings, and analyses of nucleotide substitution patterns, we suggest that a pathogen seed-bank effect is an important feature of F. tularensis ecology influencing genetic diversity.

    In paper III, we developed a new concept for source attribution of a F. tularensis sample. We aimed to identify genetic variation that is characteristic to laboratory culturing and we used culture amplification to identify genetic variation present at exceedingly low frequencies in a sample. Based on a biological enrichment scheme followed by high-throughput sequencing, we could track genetic variation back to a source sample. These results suggest that the concept has potential for linking a F. tularensis sample to its laboratory source sample.

    Taken together, the results presented in this thesis provide new understanding of the dissemination patterns and local persistence of tularemia. This is important for the interpretation of molecular epidemiology investigations of the disease. In a wider context, the results demonstrate how spatial dispersal and a microbial seed-bank effect may contribute to the diversity of a disease-causing agent. Finally, we have described a promising concept for source attribution of F. tularensis samples

  • 73.
    Dwibedi, Chinmay Kumar
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Larsson, P.
    Lindgren, P.
    Myrtennäs, K.
    Granberg, M.
    Larsson, E.
    Öhrman, C.
    Sjödin, A.
    Stenberg, P.
    Ahlinder, J.
    Forsman, M.
    Johansson, A.
    Biological amplification of low frequency mutations for source attribution of Francisella tularensisManuscript (preprint) (Other academic)
  • 74.
    Dwibedi, Chinmay Kumar
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Myrtennäs, K.
    Thelaus, J.
    Öhrman, C.
    Lärkeryd, A.
    Birdsell, D.
    Keim, P.
    Bäckman, S.
    Noppa, L.
    Näslund, J.
    Lundmark, E.
    Vidman, L.
    Lindgren, P.
    Eliasson, H.
    Fredlund, H.
    Ryden, P.
    Larsson, P.
    Wagner, D.
    Forsman, M.
    Johansson, A.
    Tularemia outbreaks are caused by infective pathogen seedbanksManuscript (preprint) (Other academic)
  • 75.
    Dörr, Tobias
    et al.
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). 3 Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.
    Lam, Hubert
    Discovery Research, Sanofi Pasteur, Cambridge, MA, USA.
    Davis, Brigid M
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Waldor, Matthew K
    Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, Massachusetts, USA ; Department of Microbiology and Immunobiology, Harvard Medical School and HHMI, Boston, MA, USA .
    Substrate specificity of an elongation-specific peptidoglycan endopeptidase and its implications for cell wall architecture and growth of Vibrio cholerae2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 89, no 5, p. 949-962Article in journal (Refereed)
    Abstract [en]

    The bacterial cell wall consists of peptidoglycan (PG), a sturdy mesh of glycan strands cross-linked by short peptides. This rigid structure constrains cell shape and size, yet is sufficiently dynamic to accommodate insertion of newly synthesized PG, which was long hypothesized, and recently demonstrated, to require cleavage of the covalent peptide cross-links that couple previously inserted material. Here, we identify several genes in Vibrio cholerae that collectively are required for growth - particularly elongation - of this pathogen. V. cholerae encodes three putative periplasmic proteins, here denoted ShyA, ShyB, and ShyC, that contain both PG binding and M23 family peptidase domains. While none is essential individually, the absence of both ShyA and ShyC results in synthetic lethality, while the absence of ShyA and ShyB causes a significant growth deficiency. ShyA is a D,d-endopeptidase able to cleave most peptide chain cross-links in V. cholerae's PG. PG from a ∆shyA mutant has decreased average chain length, suggesting that ShyA may promote removal of short PG strands. Unexpectedly, ShyA has little activity against muropeptides containing pentapeptides, which typically characterize newly synthesized material. ShyA's substrate-dependent activity may contribute to selection of cleavage sites in PG, whose implications for the process of side-wall growth are discussed.

  • 76. Earp, Caroline
    et al.
    Rowbotham, Samuel
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cha, Rita S
    Acute sensitivity of DNA replication to reduction in dNTP pools following Mec1ATR inactivation2015In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, no Suppl. 1, p. S56-S56Article in journal (Other academic)
    Abstract [en]

    Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by a fatal mitotic catastrophe. The mec1 S phase arrest was proposed to stem from a defect in the Mec1-Rad53-Dun1 dependent removal of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), at the onset of S phase: According to this view, Sml1 removal and the ensuing RNR activation would promote the dNTP production necessary for genome duplication. In support for this view, dNTP levels in hypomorphic mec1 or rad53 mutants and a dun1∆ strain were shown to be reduced by as much as 46% compared to a MEC1 control strain. Notably however, nearly all analyses on a lethal mec1 allele (e.g. mec1∆ or mec1-kd [kinase dead]) have been performed in a strain background that was either deleted for SML1 or over-expressing RNR1, a requirement for maintaining viability of a mutant lacking Mec1's essential function. As a result, while it is clear that absence of Mec1 causes dNTP pool to decrease, the true extent of the reduction and whether it would be sufficient to account for the replication arrest remain elusive. Here, we addressed these questions utilizing a temperature sensitive mutant, mec1-4, which maintains its viability at permissive temperature in an otherwise wild-type background, circumventing the need to exogenously manipulate Sml1 and/or RNR activity. Results show that Mec1 inactivation leads to an S phase arrest and a ~17% reduction in dNTP pool; expression of a novel suppressor, GIS2 (glucose inhibition of gluconeogenic growth suppressor 2), rescues the arrest and partially restores the dNTP pool to ~ 93% of a control. Unexpectedly modest effects of mec1 and GIS2 on dNTP levels demonstrate that the arrest does result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to its availability

  • 77.
    East, Emma
    et al.
    Department of Life Health & Chemical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
    Johns, Noémie
    Department of Life Health & Chemical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
    Georgiou, Melanie
    Department of Life Health & Chemical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
    Golding, Jon P.
    Department of Life Health & Chemical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
    Loughlin, A. Jane
    Department of Life Health & Chemical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Phillips, James B.
    Department of Life Health & Chemical Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK, Department of Biomaterials & Tissue Engineering, UCL Eastman Dental Institute, 256 Gray’s Inn Road, London WC1X 8LD, UK.
    A 3D in vitro model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury2013In: Regenerative Medicine, ISSN 1746-0751, E-ISSN 1746-076X, Vol. 8, no 6, p. 739-746Article in journal (Refereed)
    Abstract [en]

    AIM: This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS repair success.

    MATERIALS & METHODS: The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control.

    RESULTS: NCSCs and differentiated Schwann cell-like adipose-derived stem cells did not increase astrocyte reactivity. Highly reactive responses to bone marrow mesenchymal stem cells and Schwann cells were equivalent.

    CONCLUSION: This approach can screen therapeutic cells prior to in vivo testing, allowing cells likely to trigger a substantial astrocyte response to be identified at an early stage. NCSCs and differentiated Schwann cell-like adipose-derived stem cells may be useful in treating CNS damage without increasing astrogliosis.

  • 78. Egecioglu, Emil
    et al.
    Jerlhag, Elisabet
    Salome, Nicolas
    Skibicka, Karolina P.
    Haage, David
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Physiology.
    Bohlooly-Y, Mohammad
    Andersson, Daniel
    Bjursell, Mikael
    Perrissoud, Daniel
    Engel, Jorgen A.
    Dickson, Suzanne L.
    Ghrelin increases intake of rewarding food in rodents2010In: Addiction Biology, ISSN 1355-6215, E-ISSN 1369-1600, Vol. 15, no 3, p. 304-311Article in journal (Refereed)
    Abstract [en]

    We investigated whether ghrelin action at the level of the ventral tegmental area (VTA), a key node in the mesolimbic reward system, is important for the rewarding and motivational aspects of the consumption of rewarding/palatable food. Mice with a disrupted gene encoding the ghrelin receptor (GHS-R1A) and rats treated peripherally with a GHS-R1A antagonist both show suppressed intake of rewarding food in a free choice (chow/rewarding food) paradigm. Moreover, accumbal dopamine release induced by rewarding food was absent in GHS-R1A knockout mice. Acute bilateral intra-VTA administration of ghrelin increased 1-hour consumption of rewarding food but not standard chow. In comparison with sham rats, VTA-lesioned rats had normal intracerebroventricular ghrelin-induced chow intake, although both intake of and time spent exploring rewarding food was decreased. Finally, the ability of rewarding food to condition a place preference was suppressed by the GHS-R1A antagonist in rats. Our data support the hypothesis that central ghrelin signaling at the level of the VTA is important for the incentive value of rewarding food.

  • 79.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Augusti, Angela
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Betson, Tatiana R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B.
    Marshall, John D.
    Schleucher, Juergen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Detecting long-term metabolic shifts using isotopomers: CO2-driven suppression of photorespiration in C-3 plants over the 20th century2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 51, p. 15585-15590Article in journal (Refereed)
    Abstract [en]

    Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C-3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C-3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C-3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the similar to 100-ppm CO2 increase between similar to 1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.

  • 80. Ekblad, Torun
    et al.
    Lindgren, Anders E. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, C. David
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Caraballo, Remi
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Thorsell, Ann-Gerd
    Karlberg, Tobias
    Spjut, Sara
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schuler, Herwig
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Towards small molecule inhibitors of mono-ADP-ribosyltransferases2015In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 95, p. 546-551Article in journal (Refereed)
    Abstract [en]

    Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50.

  • 81. Ekblad, Torun
    et al.
    Verheugd, Patricia
    Lindgren, Anders E.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nyman, Tomas
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schüler, Herwig
    Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol2018In: SLAS DISCOVERY, ISSN 2472-5552, Vol. 23, no 4, p. 353-362Article in journal (Refereed)
    Abstract [en]

    Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a similar to 1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.

  • 82.
    Ekblom, Kim
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Marklund, Stefan L
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Jansson, Jan-Håkan
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Hallmans, Göran
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Nutritional Research.
    Weinehall, Lars
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Hultdin, Johan
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Response to letter regarding article "Plasma bilirubin and UGT1A1*28 are not protective factors against first-time myocardial infarction in a prospective nested case-referent setting"2011In: Circulation: Cardiovascular Genetics, ISSN 1942-325X, E-ISSN 1942-3268, Vol. 4, no 1, p. e2-Article in journal (Refereed)
  • 83.
    Ekestubbe, Sofie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 175Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.

  • 84.
    Ekström, Fredrik
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Stier, Gunter
    Sauer, Uwe
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Crystallization of the actin-binding domain of human alpha-actinin: analysis of microcrystals of SeMet-labelled protein2003In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, no Pt 4, p. 724-726Article in journal (Refereed)
    Abstract [en]

    Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.

  • 85.
    Ekström, Jens-Ola
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). BioMediTech, FI-33014 University of Tampere, Finland.
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). BioMediTech, FI-33014 University of Tampere, Finland.
    A Novel Strategy for Live Detection of Viral Infection in Drosophila melanogaster2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 26250Article in journal (Refereed)
    Abstract [en]

    We have created a transgenic reporter for virus infection, and used it to study Nora virus infection in Drosophila melanogaster. The transgenic construct, Munin, expresses the yeast transcription factor Gal4, tethered to a transmembrane anchor via a linker that can be cleaved by a viral protease. In infected cells, liberated Gal4 will then transcribe any gene that is linked to a promoter with a UAS motif, the target for Gal4 transcription. For instance, infected cells will glow red in the offspring of a cross between the Munin stock and flies with a UAS-RFPnls transgene (expressing a red fluorescent protein). In such flies we show that after natural infection, via the faecal-oral route, 5-15% of the midgut cells are infected, but there is little if any infection elsewhere. By contrast, we can detect infection in many other tissues after injection of virus into the body cavity. The same principle could be applied for other viruses and it could also be used to express or suppress any gene of interest in infected cells.

  • 86.
    Elfving, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Transcriptional regulation of mouse ribonucleotide reductase2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.

  • 87.
    Elfving, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Characterization of the promoters of the mouse ribonucleotide reductase R1, R2 and p53R2-genesManuscript (preprint) (Other academic)
    Abstract [en]

    Reduction of ribonucleotides into deoxyribonucleotides is catalyzed by the enzyme ribonucleotide reductase. The mouse enzyme is composed of two protein subunits, the R1 protein and the R2/p53R2 protein, and its subunit constellation differs during the cell cycle. We report here how the promoters of each of these subunits are regulated during the cell cycle. Previous DNase footprinting experiments of the R1 and the R2 promoter gave us an idea of how these promoters are structured. The R1 promoter contains four elements; Inr, α (binding YY1), β (binding YY1) and γ, while the R2 promoter contains four different elements; TATA-box (binding TBP), CCAAT-box (binding NFY), E2F element (binding E2F4) and an upstream activating region. The p53R2 promoter is uncharacterized; only the transcription start has been suggested in Genebank.

    We found that activation of both subunits needed for S phase specific activity (R1 and R2) is dependent of release of the repressor E2F4 from each promoter. Previous results showed that the mouse R2 promoter harbors an E2F4 binding element and our result, using transient transfections, indicates that this is also the case for the mouse R1 promoter. Using primer extension experiments on the mouse p53R2 promoter we show that the transcription start colocalizes with an earlier unidentified Inr element similar to the Inr element in the mouse R1 promoter. Our transcription start site is localized 126 bp downstream from the start site indicated in Genebank. We also show that it is possible to partially purify the transcription factor(s) binding to the upstream activating region in the mouse R2 promoter by using phosphocellulose chromatography and oligonucleotides immobilized on magnetic beads.

  • 88. Elfving, Karin
    et al.
    Olsen, Björn
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Waldenström, Jonas
    Lundkvist, Åke
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Mejlon, Hans
    Nilsson, Kenneth
    Dissemination of spotted fever rickettsia agents in Europe by migrating birds2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 1, p. e8572-Article in journal (Refereed)
    Abstract [en]

    Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp.IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.

  • 89.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal-regulated kinase 1/2 signaling2018In: Muscle and Nerve, ISSN 0148-639X, E-ISSN 1097-4598, Vol. 57, no 2, p. 305-311Article in journal (Refereed)
    Abstract [en]

    Introduction: In this study we investigated the interaction between adipose tissue-derived stem cells (ASCs) and myoblasts in co-culture experiments. Methods: Specific inductive media were used to differentiate ASCs in vitro into a Schwann cell-like phenotype (differentiated adipose tissuederived stem cells, or dASCs) and, subsequently, the expression of acetylcholine (ACh)-related machinery was determined. In addition, the expression of muscarinic ACh receptors was examined in denervated rat gastrocnemius muscles. Results: In contrast to undifferentiated ASCs, dASCs expressed more choline acetyltransferase and vesicular acetylcholine transporter. When co-cultured with myoblasts, dASCs enhanced the proliferation rate, as did ACh administration alone. Western blotting and pharmacological inhibitor studies showed that phosphorylated extracellular signal-regulated kinase 1/2 signaling mediated these effects. In addition, denervated muscle showed higher expression of muscarinic ACh receptors than control muscle. Discussion: Our findings suggest that dASCs promote proliferation of myoblasts through paracrine secretion of ACh, which could explain some of their regenerative capacity in vivo.

  • 90.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, L. J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts2018In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 9, article id 352Article in journal (Refereed)
    Abstract [en]

    Background: Adipose tissue is an excellent source for isolation of stem cells for treating various clinical conditions including injuries to the neuromuscular system. Many previous studies have focused on differentiating these adipose stem cells (ASCs) towards a Schwann cell-like phenotype (dASCs), which can enhance axon regeneration and reduce muscle atrophy. However, the stromal vascular fraction (SVF), from which the ASCs are derived, also exerts broad regenerative potential and might provide a faster route to clinical translation of the cell therapies for treatment of neuromuscular disorders.

    Methods: The aim of this study was to establish the effects of SVF cells on the proliferation and differentiation of myoblasts using indirect co-culture experiments. A Growth Factor PCR Array was used to compare the secretomes of SVF and dASCs, and the downstream signaling pathways were investigated.

    Results: SVF cells, unlike culture-expanded dASCs, expressed and secreted hepatocyte growth factor (HGF) at concentrations sufficient to enhance the proliferation of myoblasts. Pharmacological inhibitor studies revealed that the signal is mediated via ERK1/2 phosphorylation and that the effect is significantly reduced by the addition of 100 pM Norleual, a specific HGF inhibitor. When myoblasts were differentiated into multinucleated myotubes, the SVF cells reduced the expression levels of fast-type myosin heavy chain (MyHC2) suggesting an inhibition of the differentiation process.

    Conclusions: In summary, this study shows the importance of HGF as a mediator of the SVF effects on myoblasts and provides further evidence for the importance of the secretome in cell therapy and regenerative medicine applications.

  • 91.
    Elluri, Sridhar
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
    Enow Oben Ayuk, Constance
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Vdovikova, Svitlana
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rompikuntal, Pramod K
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Dongre, Mitesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pal, Amit
    Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 9, article id e106731Article in journal (Refereed)
    Abstract [en]

    Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.

    Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.

    Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.

  • 92.
    Engels, Patricia E.
    et al.
    University Hospital Basel.
    Tremp, Mathias
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    di Summa, Pietro G.
    Largo, Rene D.
    Schaefer, Dirk J.
    Kalbermatten, Daniel F
    Harvest site influences the growth properties of adipose derived stem cells2013In: Cytotechnology (Dordrecht), ISSN 0920-9069, E-ISSN 1573-0778, Vol. 65, no 3, p. 437-445Article in journal (Refereed)
    Abstract [en]

    The therapeutic potential of adult stem cells may become a relevant option in clinical care in the future. In hand and plastic surgery, cell therapy might be used to enhance nerve regeneration and help surgeons and clinicians to repair debilitating nerve injuries. Adipose-derived stem cells (ASCs) are found in abundant quantities and can be harvested with a low morbidity. In order to define the optimal fat harvest location and detect any potential differences in ASC proliferation properties, we compared biopsies from different anatomical sites (inguinal, flank, pericardiac, omentum, neck) in Sprague-Dawley rats. ASCs were expanded from each biopsy and a proliferation assay using different mitogenic factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) was performed. Our results show that when compared with the pericardiac region, cells isolated from the inguinal, flank, omental and neck regions grow significantly better in growth medium alone. bFGF significantly enhanced the growth rate of ASCs isolated from all regions except the omentum. PDGF had minimal effect on ASC proliferation rate but increases the growth of ASCs from the neck region. Analysis of all the data suggests that ASCs from the neck region may be the ideal stem cell sources for tissue engineering approaches for the regeneration of nervous tissue.

  • 93.
    Enow Oben Ayuk, Constance
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Oscarsson, Jan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Zlatkov, Nikola
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Westermark, Marie
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Duperthuy, Marylise
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Elevated recombinant clyA gene expression in the uropathogenic Escherichia coli strain 536, a clue to explain pathoadaptive mutations in a subset of extraintestinal E. coli strains2014In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, p. 216-Article in journal (Refereed)
    Abstract [en]

    There are at least four different variants of ΔclyA, suggesting that such deletions in clyA have arisen at more than one occasion. On the basis of this occurrence of the truncated clyA genes, we considered that there may be a patho-adaptive selection for deletions in clyA in extraintestinal pathogenic E. coli. In E. coli K-12 the clyA gene has been viewed as “cryptic” since it is tightly silenced by the nucleoid structuring protein H-NS. We constructed a restored clyA+ locus in derivatives of the UPEC strain 536 for further investigation of this hypothesis and, in particular, how the gene would be expressed. Our results show that the level of clyA+ expression is highly increased in the UPEC derivatives in comparison with the non-pathogenic E. coli K-12. Transcription of the clyA+ gene was induced to even higher levels when the SfaX regulatory protein was overproduced. The derivative with a restored clyA+ locus displayed a somewhat slower growth than the parental UPEC strain 536 when a sub-inhibitory concentration of the antimicrobial peptide Polymyxin B was added to the growth medium.

  • 94.
    Eriksson, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis2012In: Advances in Yersinia Research, New York: Springer, 2012, Vol. 954, p. 357-363Chapter in book (Refereed)
    Abstract [en]

    Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.

  • 95.
    Escamez, Sacha
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Tuominen, Hannele
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Contribution of cellular autolysis to tissular functions during plant development2017In: Current opinion in plant biology, ISSN 1369-5266, E-ISSN 1879-0356, Vol. 35, p. 124-130Article, review/survey (Refereed)
    Abstract [en]

    Plant development requires specific cells to be eliminated in a predictable and genetically regulated manner referred to as programmed cell death (PCD). However, the target cells do not merely die but they also undergo autolysis to degrade their cellular corpses. Recent progress in understanding developmental cell elimination suggests that distinct proteins execute PCD sensu stricto and autolysis. In addition, cell death alone and cell dismantlement can fulfill different functions. Hence, it appears biologically meaningful to distinguish between the modules of PCD and autolysis during plant development.

  • 96.
    Eschbach, Judith
    et al.
    Neurology, Ulm University, Germany.
    Schwalenstocker, Birgit
    Neurology, Ulm University, Germany.
    Soyal, Selma M.
    Pharmacology, Paracelsus Medical University, Salzburg, Austria.
    Bayer, Hanna
    Neurology, Ulm University, Germany.
    Wiesner, Diana
    Neurology, Ulm University, Germany.
    Akimoto, Chizuru
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Nilsson, Ann-Charloth
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Birve, Anna
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Meyer, Thomas
    Neurology, Charité University Hospital, Berlin, Germany.
    Dupuis, Luc
    INSERM, Strasbourg, France ; Faculté de Médecine, Université de Strasbourg, , France.
    Danzer, Karin M.
    Neurology, Ulm University, Germany.
    Andersen, Peter M.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience. Neurology, Ulm University, Germany.
    Witting, Anke
    Neurology, Ulm University, Germany.
    Ludolph, Albert C.
    Neurology, Ulm University, Germany.
    Patsch, Wolfgang
    Pharmacology, Paracelsus Medical University, Salzburg, Austria.
    Weydt, Patrick
    Neurology, Ulm University, Germany.
    PGC-1 is a male-specific disease modifier of human and experimental amyotrophic lateral sclerosis2013In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 22, no 17, p. 3477-3484Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a devastating, adult-onset neurodegenerative disorder of the upper and lower motor systems. It leads to paresis, muscle wasting and inevitably to death, typically within 35 years. However, disease onset and survival vary considerably ranging in extreme cases from a few months to several decades. The genetic and environmental factors underlying this variability are of great interest as potential therapeutic targets. In ALS, men are affected more often and have an earlier age of onset than women. This gender difference is recapitulated in transgenic rodent models, but no underlying mechanism has been elucidated. Here we report that SNPs in the brain-specific promoter region of the transcriptional co-activator PGC-1, a master regulator of metabolism, modulate age of onset and survival in two large and independent ALS populations and this occurs in a strictly male-specific manner. In complementary animal studies, we show that deficiency of full-length (FL) Pgc-1 leads to a significantly earlier age of onset and a borderline shortened survival in male, but not in female ALS-transgenic mice. In the animal model, FL Pgc-1-loss is associated with reduced mRNA levels of the trophic factor Vegf-A in males, but not in females. In summary, we indentify PGC-1 as a novel and clinically relevant disease modifier of human and experimental ALS and report a sex-dependent effect of PGC-1 in this neurodegenerative disorder.

  • 97.
    Espaillat, Akbar
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Carrasco-Lopez, Cesar
    Bernardo-Garcia, Noelia
    Pietrosemoli, Natalia
    Otero, Lisandro H.
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    de Pedro, Miguel A.
    Pazos, Florencio
    Davis, Brigid M.
    Waldor, Matthew K.
    Hermoso, Juan A.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Structural basis for the broad specificity of a new family of amino-acid racemases2014In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 70, p. 79-90Article in journal (Refereed)
    Abstract [en]

    Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

  • 98. Eter, Wael A.
    et al.
    Parween, Saba
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Joosten, Lieke
    Frielink, Cathelijne
    Eriksson, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Brom, Maarten
    Ahlgren, Ulf
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Gotthardt, Martin
    SPECT-OPT multimodal imaging enables accurate evaluation of radiotracers for beta-cell mass assessments2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 24576Article in journal (Refereed)
    Abstract [en]

    Single Photon Emission Computed Tomography (SPECT) has become a promising experimental approach to monitor changes in beta-cell mass (BCM) during diabetes progression. SPECT imaging of pancreatic islets is most commonly cross-validated by stereological analysis of histological pancreatic sections after insulin staining. Typically, stereological methods do not accurately determine the total beta-cell volume, which is inconvenient when correlating total pancreatic tracer uptake with BCM. Alternative methods are therefore warranted to cross-validate beta-cell imaging using radiotracers. In this study, we introduce multimodal SPECT - optical projection tomography (OPT) imaging as an accurate approach to cross-validate radionuclide-based imaging of beta-cells. Uptake of a promising radiotracer for beta-cell imaging by SPECT, In-111-exendin-3, was measured by ex vivo-SPECT and cross evaluated by 3D quantitative OPT imaging as well as with histology within healthy and alloxan-treated Brown Norway rat pancreata. SPECT signal was in excellent linear correlation with OPT data as compared to histology. While histological determination of islet spatial distribution was challenging, SPECT and OPT revealed similar distribution patterns of In-111-exendin-3 and insulin positive beta-cell volumes between different pancreatic lobes, both visually and quantitatively. We propose ex vivo SPECT-OPT multimodal imaging as a highly accurate strategy for validating the performance of beta-cell radiotracers.

  • 99. Fallath, Thorya
    et al.
    Kidd, Brendan N.
    Stiller, Jiri
    Davoine, Celine
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Manners, John M.
    Kazan, Kemal
    Schenk, Peer M.
    MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 4, article id e0176022Article in journal (Refereed)
    Abstract [en]

    The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways.

  • 100. Fang, Hanwei
    et al.
    Gomes, Ana Rita
    Klages, Natacha
    Pino, Paco
    Maco, Bohumil
    Walker, Eloise M.
    Zenonos, Zenon A.
    Angrisano, Fiona
    Baum, Jake
    Doerig, Christian
    Baker, David A.
    Billker, Oliver
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Brochet, Mathieu
    Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4248Article in journal (Refereed)
    Abstract [en]

    In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.

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