Displaying publications 1 - 20 of 9012 in total

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  1. Fulltext Origin and evolution of European community-acquired methicillin-resistant Staphylococcus aureus
    Stegger M, Wirth T, Andersen PS, Skov RL, De Grassi A, Simões PM, et al.
    mBio, 2014 Aug 26;5(5):e01044-14.
    PMID: 25161186 DOI: 10.1128/mBio.01044-14
    Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations.

    IMPORTANCE: With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection of strains of the S. aureus CC80 lineage. Our study determined that a single descendant of a PVL-positive methicillin-sensitive ancestor circulating in sub-Saharan Africa rose to become the dominant CA-MRSA clone in Europe, the Middle East, and North Africa. In the transition from a methicillin-susceptible lineage to a successful CA-MRSA clone, it simultaneously became resistant to fusidic acid, a widely used antibiotic for skin and soft tissue infections, thus demonstrating the importance of antibiotic selection in the success of this clone. This finding furthermore highlights the significance of horizontal gene acquisitions and underscores the combined importance of these factors for the success of CA-MRSA.

    Matched MeSH terms: Bacterial Proteins/metabolism; Protein Kinases/metabolism
  2. Fulltext Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone
    Forde BM, Phan MD, Gawthorne JA, Ashcroft MM, Stanton-Cook M, Sarkar S, et al.
    mBio, 2015 Nov 17;6(6):e01602-15.
    PMID: 26578678 DOI: 10.1128/mBio.01602-15
    Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.

    IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.

    Matched MeSH terms: DNA, Bacterial/metabolism*; Methyltransferases/metabolism*
  3. Fulltext Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage
    Nhu NTK, Phan MD, Peters KM, Lo AW, Forde BM, Min Chong T, et al.
    mBio, 2018 08 21;9(4).
    PMID: 30131362 DOI: 10.1128/mBio.01462-18
    Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis.
    Matched MeSH terms: Escherichia coli Proteins/metabolism
  4. Fulltext Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli
    Goh KGK, Phan MD, Forde BM, Chong TM, Yin WF, Chan KG, et al.
    mBio, 2017 10 24;8(5).
    PMID: 29066548 DOI: 10.1128/mBio.01558-17
    Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
    Matched MeSH terms: Bacterial Capsules/metabolism; Escherichia coli Proteins/metabolism; Uropathogenic Escherichia coli/metabolism
  5. Fulltext Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions
    Loh SY, Jahans-Price T, Greenwood MP, Greenwood M, Hoe SZ, Konopacka A, et al.
    eNeuro, 2017 12 21;4(6).
    PMID: 29279858 DOI: 10.1523/ENEURO.0243-17.2017
    The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2. Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON.
    Matched MeSH terms: RNA, Messenger/metabolism; Supraoptic Nucleus/metabolism*; RNA-Binding Proteins/metabolism*
  6. Fulltext Regulation of nerve growth and patterning by cell surface protein disulphide isomerase
    Cook GM, Sousa C, Schaeffer J, Wiles K, Jareonsettasin P, Kalyanasundaram A, et al.
    Elife, 2020 05 28;9.
    PMID: 32452761 DOI: 10.7554/eLife.54612
    Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in chick half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth.
    Matched MeSH terms: Membrane Proteins/metabolism*; Nitric Oxide/metabolism*; Procollagen-Proline Dioxygenase/metabolism; Protein Disulfide-Isomerases/metabolism*
  7. Fulltext The Sec61 translocon limits IRE1α signaling during the unfolded protein response
    Sundaram A, Plumb R, Appathurai S, Mariappan M
    Elife, 2017 05 15;6.
    PMID: 28504640 DOI: 10.7554/eLife.27187
    IRE1α is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1α forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1α complexes in cells with blue native PAGE immunoblotting. We find that IRE1α forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1α oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1α-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1α to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1α complex defines the extent of IRE1α activity and may determine cell fate decisions during ER stress conditions.
    Matched MeSH terms: Endoribonucleases/metabolism*; Protein-Serine-Threonine Kinases/metabolism*; SEC Translocation Channels/metabolism*
  8. Fulltext Convergence between the microcosms of Southeast Asian and North American pitcher plants
    Bittleston LS, Wolock CJ, Yahya BE, Chan XY, Chan KG, Pierce NE, et al.
    Elife, 2018 08 28;7.
    PMID: 30152327 DOI: 10.7554/eLife.36741
    The 'pitchers' of carnivorous pitcher plants are exquisite examples of convergent evolution. An open question is whether the living communities housed in pitchers also converge in structure or function. Using samples from more than 330 field-collected pitchers of eight species of Southeast Asian Nepenthes and six species of North American Sarracenia, we demonstrate that the pitcher microcosms, or miniature ecosystems with complex communities, are strikingly similar. Compared to communities from surrounding habitats, pitcher communities house fewer species. While communities associated with the two genera contain different microbial organisms and arthropods, the species are predominantly from the same phylogenetic clades. Microbiomes from both genera are enriched in degradation pathways and have high abundances of key degradation enzymes. Moreover, in a manipulative field experiment, Nepenthes pitchers placed in a North American bog assembled Sarracenia-like communities. An understanding of the convergent interactions in pitcher microcosms facilitates identification of selective pressures shaping the communities.
    Matched MeSH terms: Chitinase/metabolism; Nitrogen/metabolism
  9. Fulltext Single-cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes
    Yang P, Chen Y, Huang Z, Xia H, Cheng L, Wu H, et al.
    Elife, 2022 Oct 06;11.
    PMID: 36200862 DOI: 10.7554/eLife.80127
    Despite the importance of innate immunity in invertebrates, the diversity and function of innate immune cells in invertebrates are largely unknown. Using single-cell RNA-seq, we identified prohemocytes, monocytic hemocytes, and granulocytes as the three major cell-types in the white shrimp hemolymph. Our results identified a novel macrophage-like subset called monocytic hemocytes 2 (MH2) defined by the expression of certain marker genes, including Nlrp3 and Casp1. This subtype of shrimp hemocytes is phagocytic and expresses markers that indicate some conservation with mammalian macrophages. Combined, our work resolves the heterogenicity of hemocytes in a very economically important aquatic species and identifies a novel innate immune cell subset that is likely a critical player in the immune responses of shrimp to threatening infectious diseases affecting this industry.
    Matched MeSH terms: Macrophages/metabolism; Phagocytes/metabolism; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism
  10. Fulltext RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response
    Konopacka A, Greenwood M, Loh SY, Paton J, Murphy D
    Elife, 2015 Nov 12;4.
    PMID: 26559902 DOI: 10.7554/eLife.09656
    In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3' poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.
    Matched MeSH terms: RNA, Messenger/metabolism; RNA-Binding Proteins/metabolism*
  11. Fulltext Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells
    Bhattacharya P, Ellegård R, Khalid M, Svanberg C, Govender M, Keita ÅV, et al.
    Elife, 2020 Sep 02;9.
    PMID: 32876566 DOI: 10.7554/eLife.57869
    HIV transmission via genital and colorectal mucosa are the most common routes of dissemination. Here, we explored the effects of free and complement-opsonized HIV on colorectal tissue. Initially, there was higher antiviral responses in the free HIV compared to complement-opsonized virus. The mucosal transcriptional response at 24 hr revealed the involvement of activated T cells, which was mirrored in cellular responses observed at 96 hr in isolated mucosal T cells. Further, HIV exposure led to skewing of T cell phenotypes predominantly to inflammatory CD4+ T cells, that is Th17 and Th1Th17 subsets. Of note, HIV exposure created an environment that altered the CD8+ T cell phenotype, for example expression of regulatory factors, especially when the virions were opsonized with complement factors. Our findings suggest that HIV-opsonization alters the activation and signaling pathways in the colorectal mucosa, which promotes viral establishment by creating an environment that stimulates mucosal T cell activation and inflammatory Th cells.
    Matched MeSH terms: Complement System Proteins/metabolism; Opsonin Proteins/metabolism
  12. Fulltext Plasmodium-infected erythrocytes induce secretion of IGFBP7 to form type II rosettes and escape phagocytosis
    Lee WC, Russell B, Sobota RM, Ghaffar K, Howland SW, Wong ZX, et al.
    Elife, 2020 Feb 18;9.
    PMID: 32066522 DOI: 10.7554/eLife.51546
    In malaria, rosetting is described as a phenomenon where an infected erythrocyte (IRBC) is attached to uninfected erythrocytes (URBC). In some studies, rosetting has been associated with malaria pathogenesis. Here, we have identified a new type of rosetting. Using a step-by-step approach, we identified IGFBP7, a protein secreted by monocytes in response to parasite stimulation, as a rosette-stimulator for Plasmodium falciparum- and P. vivax-IRBC. IGFBP7-mediated rosette-stimulation was rapid yet reversible. Unlike type I rosetting that involves direct interaction of rosetting ligands on IRBC and receptors on URBC, the IGFBP7-mediated, type II rosetting requires two additional serum factors, namely von Willebrand factor and thrombospondin-1. These two factors interact with IGFBP7 to mediate rosette formation by the IRBC. Importantly, the IGFBP7-induced type II rosetting hampers phagocytosis of IRBC by host phagocytes.
    Matched MeSH terms: Insulin-Like Growth Factor Binding Proteins/metabolism*
  13. Fulltext ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development
    Zhou L, Wang P, Zhang J, Heng BC, Tong GQ
    Zygote, 2016 Feb;24(1):89-97.
    PMID: 25672483 DOI: 10.1017/S0967199414000768
    ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.
    Matched MeSH terms: Chromatin/metabolism; Cyclin-Dependent Kinase Inhibitor p21/metabolism; Histone Deacetylase 1/metabolism
  14. Diurnal Rhythm of trek2a Expression is Associated with Diurnal Rhythm of gnrh3 Expression in Zebrafish
    Loganathan K, Moriya S, Parhar IS
    Zoolog Sci, 2019 04 01;36(2):167-171.
    PMID: 31120653 DOI: 10.2108/zs180111
    The two-pore domain potassium ion (K + ) channel-related K + (TREK) channel and melatonin receptors play roles in the regulation of reproduction in zebrafish. Since reproduction is regulated by diurnal rhythms, the TREK family and melatonin receptors may exhibit diurnal rhythms in expression. In this study, we aimed to investigate diurnal variations of the gene expressions of TREK family and melatonin receptors and their associations with kisspeptin and gonadotrophin-releasing hormone (GnRH). Diurnal variations of trek1b, trek2a, trek2b, mt1, mt2, mel1a, kiss2 and gnrh3 expressions were examined by real-time PCR. For reproduction-related genes, kiss2 and gnrh3 exhibited diurnal rhythms. trek2a revealed a diurnal rhythm in the TREK family. mt2 and mel1c exhibited diurnal rhythms in the melatonin receptors. Since Trek2a regulates gnrh3 expression, the diurnal rhythm of gnrh3 expression suggests to be regulated by the diurnal rhythm of trek2a expression.
    Matched MeSH terms: Gonadotropin-Releasing Hormone/metabolism*; Pyrrolidonecarboxylic Acid/metabolism; Zebrafish/metabolism*; Potassium Channels, Tandem Pore Domain/metabolism*; Receptors, Melatonin/metabolism; Kisspeptins/metabolism
  15. [Prevalence of CYP2C19 polymorphisms involved in clopidogrel metabolism in Fujian Han population]
    Wei W, Fang L, Wang N, Zhang T, Zeng JB, Lin MT
    Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2012 Aug;29(4):420-5.
    PMID: 22875498 DOI: 10.3760/cma.j.issn.1003-9406.2012.04.009
    To investigate the frequency of CYP2C19 polymorphisms involved in clopidogrel metabolism in Fujian Han population.
    Matched MeSH terms: Ticlopidine/metabolism
  16. High Melatonin Conditions by Constant Darkness and High Temperature Differently Affect Melatonin Receptor mt1 and TREK Channel trek2a in the Brain of Zebrafish
    Loganathan K, Moriya S, Parhar IS
    Zebrafish, 2018 10;15(5):473-483.
    PMID: 30102584 DOI: 10.1089/zeb.2018.1594
    Ambient light and temperature affect reproductive function by regulating kisspeptin and gonadotrophin-releasing hormone (GnRH) in vertebrates. Melatonin and melatonin receptors, as well as the two-pore domain K+ channel-related K+ (TREK) channels, are affected by light and/or temperature; therefore, these molecules could modulate kisspeptin and GnRH against ambient light and temperature. In this study, we investigated the effect of light and temperature, which affect melatonin levels in gene expression levels of TREK channels, kisspeptin, and GnRH. We first investigated the effects of different light and temperature conditions on brain melatonin concentrations by ELISA. Fish were exposed to either constant darkness, constant light, high temperature (35°C), or low temperature (20°C) for 72 h. Brain melatonin levels were significantly high under constant darkness and high temperature. We further investigated the effects of high brain melatonin levels by constant darkness and high temperature on gene expression levels of melatonin receptors (mt1, mt2, and mel1c), TREK channels (trek1b, trek2a, and trek2b), gnrh3, and kiss2 in the adult zebrafish brain by real-time polymerase chain reaction. Fish were exposed to constant darkness or elevated temperatures (35°C) for 72 h. trek2a, kiss2, and gnrh3 levels were increased under constant darkness. High temperature decreased gene expression levels of mt1, mt2, mel1c, and gnrh3 in the preoptic area, whereas other genes remained unchanged. Melatonin receptors, TREK channels, gnrh3, and kiss2 responded differently under high melatonin conditions. The melatonin receptors and the TREK channels could play roles in the regulation of reproduction by environmental cues, especially ambient light and temperature.
    Matched MeSH terms: Brain/metabolism*; Gonadotropin-Releasing Hormone/metabolism; Zebrafish/metabolism*; Potassium Channels/metabolism*; Zebrafish Proteins/metabolism*; Receptor, Melatonin, MT1/metabolism*; Kisspeptins/metabolism
  17. Proteomics Identification of Potential Candidates Involved in Cell Proliferation for Early Stage of Brain Regeneration in the Adult Zebrafish
    Lim FT, Ogawa S, Smith AI, Parhar IS
    Zebrafish, 2017 Feb;14(1):10-22.
    PMID: 27797681 DOI: 10.1089/zeb.2016.1319
    The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.
    Matched MeSH terms: Biomarkers/metabolism
  18. Fulltext Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition
    Ho SY, Goh CW, Gan JY, Lee YS, Lam MK, Hong N, et al.
    Zebrafish, 2014 Oct;11(5):407-20.
    PMID: 24967707 DOI: 10.1089/zeb.2013.0879
    Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.
    Matched MeSH terms: Blastomeres/metabolism; Embryonic Stem Cells/metabolism*
  19. Fulltext Saccharomyces cerevisiae: a potential stereospecific reduction tool for biotransformation of mono- and sesquiterpenoids
    Khor GK, Uzir MH
    Yeast, 2011 Feb;28(2):93-107.
    PMID: 20939023 DOI: 10.1002/yea.1827
    Terpenes and terpenoids are among the key impact substances in the food and fragrance industries. Equipped with pharmacological properties and applications as ideal precursors for the biotechnological production of natural aroma chemicals, interests in these compounds have been escalating. Hence, the syntheses of new derivatives that can show improved properties are often called for. Stereoselective biotransformation offers several benefits to increase the rate of production, in terms of both the percentage yield and its enantiomeric excesses. Baker's yeast (Saccharomyces cerevisiae) is broadly used as a whole cell stereospecific reduction biocatalyst, due to its capability in reducing carbonyls and carbon-carbon double bonds, which also extends its functionality as a versatile biocatalyst in terpenoid biotransformation. This review provides some insights on the development and prospects in the reductive biotransformation of monoterpenoids and sesquiterpenoids using S. cerevisiae, with an overview of strategies to overcome the common challenges in large-scale implementation.
    Matched MeSH terms: Saccharomyces cerevisiae/metabolism*; Sesquiterpenes/metabolism*; Monoterpenes/metabolism*
  20. Fulltext Induction of apoptosis in yeast by L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma
    Ande SR, Fussi H, Knauer H, Murkovic M, Ghisla S, Fröhlich KU, et al.
    Yeast, 2008 May;25(5):349-57.
    PMID: 18437704 DOI: 10.1002/yea.1592
    Here we report for the first time that L-amino acid oxidase (LAAO), a major component of snake venom, induces apoptosis in yeast. The causative agent for induction of apoptosis has been shown to be hydrogen peroxide, produced by the enzymatic activity of LAAO. However, the addition of catalase, a specific hydrogen peroxide scavenger, does not prevent cell demise completely. Intriguingly, depletion of leucine from the medium by LAAO and the interaction of LAAO with yeast cells are shown to be the major factors responsible for cell demise in the presence of catalase.
    Matched MeSH terms: Catalase/metabolism; Leucine/metabolism*
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