DOCSLIB.ORG

324731V1.Full.Pdf

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
324731V1.Full.Pdf bioRxiv preprint doi: https://doi.org/10.1101/324731; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 4-hydroxyphenylpyruvate dioxygenase thermolability is responsible for 2 temperature-dependent melanogenesis in Aeromonas salmonicida subsp. salmonicida 3 4 Yunqian Qiao,a Jiao Wang,a He Wang,a Baozhong Chai,a Chufeng Rao,a Xiangdong 5 Chen,a,b,# Shishen Dua,# 6 7 aState Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 8 China 9 bChina Center for Type Culture Collection, Wuhan, China 10 11 Running Head: Thermosensitive melanization in Aeromonas salmonicida 12 13 #Address correspondence to Xiangdong Chen, [email protected], or Shishen Du, 14 [email protected] 15 16 Keywords: Aeromonas salmonicida subsp. salmonicida, melanin, temperature-dependent, 17 4-hydroxyphenylpyruvate dioxygenase, thermolability, evolution 18 19 20 21 1 bioRxiv preprint doi: https://doi.org/10.1101/324731; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 22 Abstract 23 Aeromonas salmonicida subsp. salmonicida (A.s.s) is a major pathogen affecting fisheries 24 worldwide. It is a well-known member of the pigmented Aeromonas species, which 25 produces melanin at 22 °C. However, melanogenesis decreases as the culture 26 temperature increases and is completely suppressed at 30–35 °C while bacterial growth is 27 not affected. The mechanism and biological significance of this temperature-dependent 28 melanogenesis are not clear. Heterologous expression of an A.s.s. 29 4-hydroxyphenylpyruvate dioxygenase (HppD), the most crucial enzyme in the 30 HGA-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia 31 coli, suggesting that HppD plays a key role in this process. In the current study, we 32 demonstrated that the extreme thermolability of HppD is responsible for the 33 temperature-dependent melanization of A.s.s. Substitutions in three residues, Ser18, 34 Pro103, or Leu119 of HppD from A.s.s increases the thermolability of this enzyme and 35 results in temperature-independent melanogenesis. Moreover, replacing the 36 corresponding residues of HppD from Aeromonas media strain WS, which forms pigment 37 independent of temperature, with those of A.s.s HppD leads to thermosensitive 38 melanogenesis. Structural analysis suggested that mutations at these sites, especially at 39 position P103, can strengthen the secondary structure of HppD and greatly improve its 40 thermal stability. In addition, we found that HppD sequences of all A.s.s isolates are 41 identical and that two of the three residues are completely conserved within A.s.s isolates, 42 which clearly distinguishes these from other Aeromonas strains. We suggest that this 2 bioRxiv preprint doi: https://doi.org/10.1101/324731; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 43 property represents an adaptive strategy to the psychrophilic lifestyle of A.s.s. 44 Importance 45 Aeromonas salmonicida subsp. salmonicida (A.s.s) is the causative agent of furunculosis, 46 a bacterial septicemia of cold water fish of the Salmonidae family. As it has a 47 well-defined host range, A.s.s has become an ideal model to investigate the co-evolution 48 of host and pathogen. For many pathogens, melanin production is associated with 49 virulence. Although other species of Aeromonas can produce melanin, A.s.s is the only 50 member of this genus that has been reported to exhibit temperature-dependent 51 melanization. Here we demonstrate that thermosensitive melanogenesis in A.s.s strains is 52 due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). The strictly 53 conserved hppD sequences among A.s.s and the exclusive thermosensitive pigmentation 54 of these strains might provide insight into the role of melanin in the adaptation to a 55 particular host, and offer a novel molecular marker to readily differentiate A.s.s strains 56 from other A. salmonicida subspecies and Aeromonas species. 57 58 Introduction 59 Melanins are structurally complex high molecular weight pigments formed by the 60 oxidative polymerization of phenolic and/or indolic compounds (1). They represent the 61 most ubiquitous natural pigment and are produced by organisms from every kingdom (2). 62 Melanin has various functions in diverse organisms, and it is believed that it confers a 63 survival advantage for microorganisms, especially under stressful environmental 3 bioRxiv preprint doi: https://doi.org/10.1101/324731; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 64 conditions such as radiation, oxidative stress, the presence of digestive enzymes, and 65 heavy metal toxicity (2). These molecules have also been associated with the virulence 66 and pathogenicity of a variety of microbes such as Cryptococcus neoformans, Wangiella 67 dermatitidis, and Vibrio cholerae (3-5), by protecting pathogens against host defenses and 68 interfering with the host immune response (2). Melanins are classified into several 69 categories based on their synthesis pathway, and bacteria usually synthesize two types, 70 namely eumelanin or pyomelanin. Eumelanin is catalyzed by tyrosinase or other 71 polyphenol oxidases to hydroxylate tyrosine, via the intermediate 72 L-3,4-dihydroxyphenylalanine (L-DOPA), into dopaquinone, which then auto-oxidizes 73 and polymerizes to form melanin (6). Pyomelanin is synthesized by the homogentisic 74 acid (HGA) pathway, in which 4-hydroxyphenylpyruvate is converted to HGA by 75 4-hydroxyphenylpyruvate dioxygenase (HppD), which is followed by the oxidative 76 polymerization of HGA to produce the pigment (6). Our previous study showed that 77 Aeromonas media WS, a strain that produces melanin at high levels, mainly synthesizes 78 pyomelanin through the HGA pathway (7). 79 The Aeromonas genus is a collection of gram-negative, rod-shaped, facultatively 80 anaerobic bacteria that include motile, non-motile, mesophilic, and psychrophilic species. 81 They are widely distributed in aquatic environments and have been frequently isolated 82 from aquatic animal species. Some Aeromonas strains are principal or opportunistic 83 pathogens of fish and other cold-blooded animals, as well as the causative agent of 84 infectious complications in humans (8). Melanogenesis has been described in some 4 bioRxiv preprint doi: https://doi.org/10.1101/324731; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 85 species of this genus, such as A. media, Aeromonas salmonicida, Aeromonas bestiarum, 86 and Aeromonas eucrenophila (9). However, a number of Aeromonas species are not 87 considered to produce melanin, including Aeromonas hydrophila, Aeromonas 88 allosaccharophila, and Aeromonas encheleia (10). In fact, melanin synthesis is one of the 89 key criteria for the taxonomy of this genus. 90 Among melanogenic Aeromonas species, A. salmonicida subsp. salmonicida (A.s.s), 91 an important salmonid fish pathogen and the etiologic agent of furunculosis, is the only 92 member that has been described to exhibit temperature-dependent melanogenesis. It has 93 been demonstrated that A.s.s can initiate pigmentation at lower temperatures such as 94 22 °C. However, this process is thermosensitive. The level of melanin production reduces 95 as the incubation temperature increases, and this process is completely suppressed at 96 temperatures > 30 °C, at which the bacteria still grow normally (11-13). Nevertheless, the 97 reason for this phenomenon is not yet clear. Increasing the temperature can also alter 98 some other characteristics of this bacterium, including the loss of virulence (> 25 °C) 99 (14-16) and the auto-agglutination trait (>25 °C) (17). Since A.s.s is psychrophilic 100 (optimal growth at 22–25 °C) , these temperature-dependent phenotypes probably 101 represent adaptations to psychrophilic environments. 102 In the recent study, we suggested that Aeromonas species mainly produce 103 pyomelanin through the HGA pathway (7) and showed that heterologous expression of 104 hppD from an A.s.s strain in Escherichia coli BL21 leads to similar 105 temperature-dependent pigmentation. This result suggests that the abolished pigmentation 5 bioRxiv preprint doi: https://doi.org/10.1101/324731; this version posted May 17, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 106 of A.s.s at elevated temperatures is likely due to the thermolabile activity of HppD. In this 107 study, we confirmed this hypothesis and identified the critical residues of HppD, 108 specifically, S18, P103, and L119, that are responsible for its thermolability. Substitutions 109 at these residues resulted in temperature-independent melanogenesis in A.s.s. Moreover, 110 replacing the corresponding residues of HppD from other Aeromonas species with those 111 from A.s.s resulted in thermal sensitive melanization.
Load more

Recommended publications
  • Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents
    International Journal of Molecular Sciences Article Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents 1, 2,3, 1 2, Elena V. Eremeeva y , Tianyu Jiang y , Natalia P. Malikova , Minyong Li * and Eugene S. Vysotski 1,* 1 Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk 660036, Russia; [email protected] (E.V.E.); [email protected] (N.P.M.) 2 Key Laboratory of Chemical Biology (MOE), Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China; [email protected] 3 State Key Laboratory of Microbial Technology, Shandong University–Helmholtz Institute of Biotechnology, Shandong University, Qingdao 266237, China * Correspondence: [email protected] (M.L.); [email protected] (E.S.V.); Tel.: +86-531-8838-2076 (M.L.); +7-(391)-249-44-30 (E.S.V.); Fax: +86-531-8838-2076 (M.L.); +7-(391)-290-54-90 (E.S.V.) These authors contributed equally to this work. y Received: 23 June 2020; Accepted: 27 July 2020; Published: 30 July 2020 Abstract: Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial.
    [Show full text]
  • Discovery and Protein Engineering of Baeyer-Villiger Monooxygenases
    Discovery and Protein Engineering of Baeyer-Villiger monooxygenases Inauguraldissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Andy Beier geboren am 11.10.1988 in Parchim Greifswald, den 02.08.2017 I Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Uwe T. Bornscheuer 2. Gutachter: Prof. Dr. Marko Mihovilovic Tag der Promotion: 24.10.2017 II We need to learn to want what we have, not to have what we want, in order to get stable and steady happiness. - The Dalai Lama - III List of abbreviations % Percent MPS Methyl phenyl sulfide % (v/v) % volume per volume MPSO Methyl phenyl sulfoxide % (w/v) % weight per volume MPSO2 Methyl phenyl sulfone °C Degrees Celsius MTS Methyl p-tolyl sulfide µM µmol/L MTSO Methyl p-tolyl sulfoxide aa Amino acids MTSO2 Methyl p-tolyl sulfone + AGE Agarose gel electrophoresis NAD Nicotinamide adenine dinucleotide, oxidized aq. dest. Distilled water NADH Nicotinamide adenine dinucleotide, reduced + BLAST Basic Local Alignment Search NADP Nicotinamide adenine dinucleotide Tool phosphate, oxidized bp Base pair(s) NADPH Nicotinamide adenine dinucleotide phosphate, reduced BVMO Baeyer-Villiger monooxyge- OD600 Optical density at 600 nm nase CHMO Cyclohexanone monooxyge- PAGE Polyacrylamide gel electrophoresis nase Da Dalton PAMO Phenylacetone monooxygenase DMF Dimethyl formamide PCR Polymerase chain reaction DMSO Dimethyl sulfoxide PDB Protein Data Bank DMSO2 Dimethyl sulfone rpm Revolutions per minute DNA Desoxyribonucleic acid rv Reverse dNTP Desoxynucleoside triphosphate SDS Sodium dodecyl sulfate E. coli Escherichia coli SOC Super Optimal broth with Catabolite repression ee Enantiomeric excess TAE TRIS-Acetate-EDTA FAD Flavin adenine dinucleotide TB Terrific broth Fig.
    [Show full text]
  • Cloning of Firefly Luciferase Cdna and the Expression of Active
    Proc. Natl. Acad. Sci. USA Vol. 82, pp. 7870-7873, December 1985 Biochemistry Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli (bioluminescence/Photinus pyralis/antibody screening/expression vector/recombinant DNA) JEFFREY R. DE WET*, KEITH V. WOODt, DONALD R. HELINSKI*, AND MARLENE DELUCAt Departments of *Biology and tChemistry, University of California, San Diego, La Jolla, CA 92093 Communicated by W. D. McElroy, July 26, 1985 ABSTRACT A cDNA library was constructed from firefly library was screened with anti-P. pyralis luciferase antibody, (Photinuspyralis) lantern poly(A)I RNA, using the Escherichia using a chromogenic detection technique (8), and several coli expression vector Xgtll. The library was screened with cDNA clones were isolated and characterized. These clones anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidore- were found to be homologous to the mRNA that encodes ductase, EC 1.13.12.7) antibody, and several cDNA clones luciferase. The largest luciferase cDNA clone that was expressing luciferase antigens were isolated. One clone, ALucl, isolated was able to direct the synthesis of active luciferase contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- in E. coli. to 2.0-kilobase band on a nitrocellulose blot of electrophoreti- cally fractionated lantern RNA. Hybridization of the cloned MATERIALS AND METHODS cDNA to lantern poly(A)I RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide Enzymes and Strains. Restriction endonucleases and E. coli comigrated with luciferase on NaDodSO4/PAGE and produced DNA polymerase I were purchased from New England bioluminescence upon the addition of luciferin and ATP.
    [Show full text]
  • Bioluminescence Is Produced by a Firefly-Like Luciferase but an Entirely
    www.nature.com/scientificreports OPEN New Zealand glowworm (Arachnocampa luminosa) bioluminescence is produced by a Received: 8 November 2017 Accepted: 1 February 2018 frefy-like luciferase but an entirely Published: xx xx xxxx new luciferin Oliver C. Watkins1,2, Miriam L. Sharpe 1, Nigel B. Perry 2 & Kurt L. Krause 1 The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the frefy. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1H NMR spectroscopy. The purifed luciferase enzyme is in the same protein family as frefy luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely diferent to that of the frefy and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confrmed by chemical synthesis and bioluminescence assays. These fndings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally diferent luciferins. Glowworms are found in New Zealand and Australia, and are a major tourist attraction at sites located across both countries. In contrast to luminescent beetles such as the frefy (Coleoptera), whose bioluminescence has been well characterised (reviewed by ref.1), the molecular details of glowworm bioluminescence have remained elusive. Tese glowworms are the larvae of fungus gnats of the genus Arachnocampa, with eight species endemic to Australia and a single species found only in New Zealand2.
    [Show full text]
  • Muscle Regeneration Controlled by a Designated DNA Dioxygenase
    Wang et al. Cell Death and Disease (2021) 12:535 https://doi.org/10.1038/s41419-021-03817-2 Cell Death & Disease ARTICLE Open Access Muscle regeneration controlled by a designated DNA dioxygenase Hongye Wang1, Yile Huang2,MingYu3,YangYu1, Sheng Li4, Huating Wang2,5,HaoSun2,5,BingLi 3, Guoliang Xu6,7 andPingHu4,8,9 Abstract Tet dioxygenases are responsible for the active DNA demethylation. The functions of Tet proteins in muscle regeneration have not been well characterized. Here we find that Tet2, but not Tet1 and Tet3, is specifically required for muscle regeneration in vivo. Loss of Tet2 leads to severe muscle regeneration defects. Further analysis indicates that Tet2 regulates myoblast differentiation and fusion. Tet2 activates transcription of the key differentiation modulator Myogenin (MyoG) by actively demethylating its enhancer region. Re-expressing of MyoG in Tet2 KO myoblasts rescues the differentiation and fusion defects. Further mechanistic analysis reveals that Tet2 enhances MyoD binding by demethylating the flanking CpG sites of E boxes to facilitate the recruitment of active histone modifications and increase chromatin accessibility and activate its transcription. These findings shed new lights on DNA methylation and pioneer transcription factor activity regulation. Introduction Ten-Eleven Translocation (Tet) family of DNA dioxy- 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Skeletal muscles can regenerate due to the existence of genases catalyze the active DNA demethylation and play muscle stem cells (MuSCs)1,2. The normally quiescent critical roles in embryonic development, neural regen- MuSCs are activated after muscle injury and further dif- eration, oncogenesis, aging, and many other important – ferentiate to support muscle regeneration3,4.
    [Show full text]
  • Relating Metatranscriptomic Profiles to the Micropollutant
    1 Relating Metatranscriptomic Profiles to the 2 Micropollutant Biotransformation Potential of 3 Complex Microbial Communities 4 5 Supporting Information 6 7 Stefan Achermann,1,2 Cresten B. Mansfeldt,1 Marcel Müller,1,3 David R. Johnson,1 Kathrin 8 Fenner*,1,2,4 9 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, 10 Switzerland. 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, 8092 11 Zürich, Switzerland. 3Institute of Atmospheric and Climate Science, ETH Zürich, 8092 12 Zürich, Switzerland. 4Department of Chemistry, University of Zürich, 8057 Zürich, 13 Switzerland. 14 *Corresponding author (email: [email protected] ) 15 S.A and C.B.M contributed equally to this work. 16 17 18 19 20 21 This supporting information (SI) is organized in 4 sections (S1-S4) with a total of 10 pages and 22 comprises 7 figures (Figure S1-S7) and 4 tables (Table S1-S4). 23 24 25 S1 26 S1 Data normalization 27 28 29 30 Figure S1. Relative fractions of gene transcripts originating from eukaryotes and bacteria. 31 32 33 Table S1. Relative standard deviation (RSD) for commonly used reference genes across all 34 samples (n=12). EC number mean fraction bacteria (%) RSD (%) RSD bacteria (%) RSD eukaryotes (%) 2.7.7.6 (RNAP) 80 16 6 nda 5.99.1.2 (DNA topoisomerase) 90 11 9 nda 5.99.1.3 (DNA gyrase) 92 16 10 nda 1.2.1.12 (GAPDH) 37 39 6 32 35 and indicates not determined. 36 37 38 39 S2 40 S2 Nitrile hydration 41 42 43 44 Figure S2: Pearson correlation coefficients r for rate constants of bromoxynil and acetamiprid with 45 gene transcripts of ECs describing nucleophilic reactions of water with nitriles.
    [Show full text]
  • Functions and Applications of Nmos
    Functions and applications of NMOs Willem Dijkman (1606212), June 20 2009 Assisted by drs. Anette Riebel and prof. dr. ir. Marco W. Fraaije Abstract The hydroxylation of amino groups is performed by different types of enzymes. Some of these types are metal dependent whereas others need a flavin cofactor. These flavoproteins (NMOs, for N-hydroxylating monooxygenases) can be assigned to subclass B of the external flavoproteins, an enzyme class consisting of enzymes with a broad range of monooxygenating activity. NMOs are mostly found in the biosynthesis of siderophores of different bacteria. Recently a NMO has been identified in the biosynthesis of a kutzneride. NMOs have a narrow substrate range, making them less applicable in industry. NMOs in siderophore biosynthesis can however be targeted to slow down bacterial growth. Contents enzymes which perform these monooxygenations often contain metal Introduction 1 atoms. Different types of enzymes are Classification of flavoproteins 2 known. P450 enzymes as well as non-heme 2 The catalytic cycle of flavins 3 monooxygenases contain iron and copper- N-hydroxylation in nature 4 dependent enzymes also perform Characteristics of NMOs 4 monooxygenations. Not all enzymes depend - NMOs in siderophore synthesis 5 on a (metallic) cofactor for their activity: - A NMO in kutzneride synthesis 7 flavin-dependent monooxygenases make use - N-hydroxylation in valanimycin synthesis 7 of a flavin group instead of a metal ion and Recombinant NMOs 8 recently enzymes without any cofactor have 3,4 Pharmaceutical applications 8 shown monooxygenase activity . Industrial applications 9 In this article one type of monooxygenation Conclusion 10 will be described, focusing on one group of Literature 10 monooxygenating enzymes.
    [Show full text]
  • Nitric Oxide Prevents a Pathogen-Permissive Granulocytic Inflammation During Tuberculosis
    ARTICLES PUBLISHED: 15 MAY 2017 | VOLUME: 2 | ARTICLE NUMBER: 17072 Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis Bibhuti B. Mishra1, Rustin R. Lovewell1,AndrewJ.Olive1, Guoliang Zhang2, Wenfei Wang2, Eliseo Eugenin3, Clare M. Smith1,JiaYaoPhuah1, Jarukit E. Long1, Michelle L. Dubuke4, Samantha G. Palace1, Jon D. Goguen1,RichardE.Baker1, Subhalaxmi Nambi1, Rabinarayan Mishra5, Matthew G. Booty1,ChristinaE.Baer1, Scott A. Shaffer4, Veronique Dartois3,BethA.McCormick1, Xinchun Chen2,6* and Christopher M. Sassetti1* Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen’s environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis
    [Show full text]
  • Mechanisms Underlying Platelet Function
    Received Date : 09-Dec-2013 Accepted Date : 19-Feb-2014 Article type : Original Article - Platelets Mechanisms underlying platelet function defect in a pedigree with FPD/AML: potential role for candidate RUNX1-targets Glembotsky AC,* Bluteau D,†‡ Espasandin YR,* Goette NP,* Marta RF,* Marin Oyarzun CP,* Korin L,* Lev PR,* Laguens RP, § Molinas FC,* Raslova H,† Heller PG* *Departamento de Hematología Investigación. Instituto de Investigaciones Médicas Article Alfredo Lanari. Universidad de Buenos Aires. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Buenos Aires. Argentina †INSERM, UMR1009. Equipe Labellisée Ligue Contre le Cancer. Gustave Roussy. Université Paris-Sud. Villejuif. France ‡Ecole Pratique des Hautes Etudes (EPHE), Paris, France §Departamento de Patología. Universidad de Favaloro. Buenos Aires. Argentina Corresponding author: Paula G. Heller. Departamento de Hematología Investigación. Instituto de Investigaciones Médicas Alfredo Lanari. Universidad de Buenos Aires. CONICET. Combatientes de Malvinas 3150. Buenos Aires 1427. Argentina This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jth.12550 Accepted This article is protected by copyright. All rights reserved. Phone/Fax: +54 11 4523 89 47 Email address: [email protected] Short title Platelet function defect in FPD/AML Summary Background FPD/AML is an inherited platelet disorder caused by germline RUNX1 mutation and characterized by thrombocytopenia, platelet function defect and leukemia Article predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype.
    [Show full text]
  • Redirecting Primary Metabolism to Boost Production of Tyrosine
    www.nature.com/scientificreports OPEN Redirecting Primary Metabolism to Boost Production of Tyrosine- Derived Specialised Metabolites Received: 28 February 2018 Accepted: 5 October 2018 in Planta Published: xx xx xxxx Alfonso Timoneda1, Hester Sheehan1, Tao Feng1, Samuel Lopez-Nieves2, Hiroshi A. Maeda2 & Samuel Brockington 1 L-Tyrosine-derived specialized metabolites perform many important functions in plants, and have valuable applications in human health and nutrition. A necessary step in the overproduction of specialised tyrosine-derived metabolites in planta is the manipulation of primary metabolism to enhance the availability of tyrosine. Here, we utilise a naturally occurring de-regulated isoform of the key enzyme, arogenate dehydrogenase, to re-engineer the interface of primary and specialised metabolism, to boost the production of tyrosine-derived pigments in a heterologous plant host. Through manipulation of tyrosine availability, we report a 7-fold increase in the production of tyrosine- derived betalain pigments, with an upper range of 855 mg·kg−1·FW, which compare favourably to many in vitro and commercial sources of betalain pigments. Since the most common plant pathway for tyrosine synthesis occurs via arogenate, the de-regulated arogenate dehydrogenase isoform is a promising route for enhanced production of tyrosine-derived pharmaceuticals in diverse plant hosts. L-Tyrosine (Tyr) is an essential aromatic amino acid required for protein biosynthesis in all organisms, and is synthesised de novo in bacteria, fungi and plants, but not in animals. In addition to protein synthesis, plants utilise Tyr to produce a diverse array of specialized metabolites, which also have important applications as phar- maceuticals (e.g. epinephrine, noradrenaline, apomorphine, and morphinans)1.
    [Show full text]
  • Illuminating Insights Into Firefly Luciferase and Other
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Review Illuminating Insights into Firefly Luciferase and Other Bioluminescent Reporters Used in Chemical Biology Natasha Thorne,1 James Inglese,1 and Douglas S. Auld1,* 1NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD 20892-3370, USA *Correspondence: [email protected] DOI 10.1016/j.chembiol.2010.05.012 Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhibit FLuc. In some cell- based assays, these inhibitors can act in a counterintuitive way, leading to a gain in luminescent signal. Although formerly attributed to transcriptional activation, intracellular stabilization of FLuc is the primary mechanism underlying this observation. FLuc inhibition and stabilization can be complex, as illustrated by the compound PTC124, which is converted by FLuc in the presence of ATP to a high affinity multisubstrate adduct inhibitor, PTC124-AMP. The potential influence these findings can have on drug discovery efforts is provided here. Introduction of certain beetles known as glow worms (Phengodidae), and Technical advances in molecular biology, microtiter plate–based various marine organisms such as the sea pansy (Renillidae). spectrophotometry, and automation have enabled the develop- This review provides a summary of commonly used luciferases ment of biologically relevant assays with robust performance and suggests recommendations for their appropriate use in that can be utilized in high-throughput screening (HTS) for chem- common HTS assays.
    [Show full text]
  • Supplemental Material-PDF
    List of supplementary tables and figures. Table S1 – Strains sequenced and assembly information (n=72) Table S2 – Genetic clusters obtained using BAPS Table S3 – Genes commonly found to be under positive selection in the three subspecies (n=14) Table S4 - Recombining genes in X. fastidiosa subsp. pauca strains 9a5c and U24D (Citrus) Table S5 – Recombination genes for X. fastidiosa 32 (Coffee) Table S6 - Genes found to be under selection in each subspecies Table S7 – Recombining genes for each X. fastidiosa subspecies Table S8 – Genes found in the accessory genome of each subspecies Figure S1 - Phylogeny of X. fastidiosa strains (n=72) and pan-genome visualization Figure S2 - Detection of temporal signal in the dataset Figure S3 - Rate comparison amongst subspecies. The boxplot (A) was constructed my merging the rate of evolution of each branch (internal and external) of the X. fastidiosa subsp. fastidiosa, pauca and multiplex clades together (obtained though rate-dating) as illustrated by the colored frames (B) Figure S4 - Comparison between clonalFrameML and FastGEAR. (A) ClonalFrameML output, (B) FastGEAR output, (C) comparison between the genes found to be recombining between the two algorithms that were mapped to the X. fastidiosa reference genome Table S1 – Strains sequenced and assembly information (n=72). Strain names in parentheses represent synonyms. Name N50 L50 Total length Country Host Subspecies Isolation date Accession CO33 176,135 6 2,681,926 Italy Coffea arabica ? 2015 SAMN04100274 MUL0034 2,642,186 1 2,666,577 USA Morus alba morus 2003 SAMN03081485 Mul-MD 134,146 6 2,520,555 USA Morus alba morus 2011 SAMN02630185 Ann-1 44,355 22 2,729,755 USA Nerium oleander sandyi SAMN03081476 Xf_ATCC_35879 500,648 2 2,522,328 USA Vitis vinifera fastidiosa 1987 SAMN02997312 DSM10026 89,916 11 2,431,652 USA Vitis vinifera fastidiosa 1987 SAMN05660380 EB92-1 44,355 22 2,729,755 USA Sambucus sp.
    [Show full text]