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Sample records for alters bacterial gene

  1. Spaceflight Alters Bacterial Gene Expression and Virulence and Reveals Role for Global Regulator Hfq

    Science.gov (United States)

    Wilson, J. W.; Ott, C. M.; zuBentrup, K. Honer; Ramamurthy R.; Quick, L.; Porwollik, S.; Cheng, P.; McClellan, M.; Tsaprailis, G.; Radabaugh, T.; Hunt, A.; Fernandez, D.; Richter, E.; Shah, M.; Kilcoyne, M.; Joshi, L.; Nelman-Gonzalez, M.; Hing, S.; Parra, M.; Dumaras, P.; Norwood, K.; Nickerson, C. A.; Bober, R.; Devich, J.; Ruggles, A.

    2007-01-01

    A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the spaceflight environment has never been accomplished due to significant technological and logistical hurdles. Moreover, the effects of spaceflight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared to identical ground control cultures. Global microarray and proteomic analyses revealed 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground based microgravity culture model. Spaceflight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during spaceflight missions and provide novel therapeutic options on Earth.

  2. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

    Science.gov (United States)

    Mayfield, Jeffrey A; Liang, Zhong; Agrahari, Garima; Lee, Shaun W; Donahue, Deborah L; Ploplis, Victoria A; Castellino, Francis J

    2014-01-01

    The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

  3. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Mayfield

    Full Text Available The cluster of virulence sensor (CovS/responder (CovR two-component operon (CovRS regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448, containing wild-type (WT CovRS (5448/CovR+S+, or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

  4. Artificially inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes.

    Science.gov (United States)

    Kim, Taejoong; Mays, Jody; Fadly, Aly; Silva, Robert F

    2011-06-01

    Researchers reported that co-cultivating the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in an REV long terminal repeat (LTR) being inserted into the internal repeat short (IRS) region of JM/102W. When the resulting recombinant virus was serially passed in cell culture, the initial LTR was duplicated and a second LTR spontaneously appeared in the terminal repeat short (TRS) region of the MDV genome. The virus, designated RM1, was significantly attenuated but still induced severe bursal and thymic atrophy (Isfort et al. PNAS 89:991-995). To determine whether the altered phenotype was due solely to the LTR, we cloned the LTR from the RM1 IRS region and inserted it into the IRS region of a very virulent bacterial artificial clone (BAC) of the Md5 strain of MDV, which we designated rMd5-RM1-LTR. During blind passage in duck embryo fibroblast cultures, the initial LTR in the rMd5-RM1-LTR was also duplicated, with LTRs appearing in both IRS and TRS regions of the MDV genome. The inserted LTR sequences and transcripts associated with the MDV open reading frames MDV085, MDV086, SORF2, US1, and US10 were molecularly characterized. The parental Md5 BAC contains a family of transcripts of 3, 2, and 1 kb that all terminate at the end of the US10 gene. The rMd5-RM1-LTR and RM1 viruses both express an additional 4 kb transcript that originates in the LTR and also terminates after US10. Collectively, the data suggest that our engineered rMd5-RM1-LTR virus very closely resembles the RM1 virus in its structure and transcription patterns.

  5. Canopy soil bacterial communities altered by severing host tree limbs

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    Cody R. Dangerfield

    2017-09-01

    Full Text Available Trees of temperate rainforests host a large biomass of epiphytic plants, which are associated with soils formed in the forest canopy. Falling of epiphytic material results in the transfer of carbon and nutrients from the canopy to the forest floor. This study provides the first characterization of bacterial communities in canopy soils enabled by high-depth environmental sequencing of 16S rRNA genes. Canopy soil included many of the same major taxonomic groups of Bacteria that are also found in ground soil, but canopy bacterial communities were lower in diversity and contained different operational taxonomic units. A field experiment was conducted with epiphytic material from six Acer macrophyllum trees in Olympic National Park, Washington, USA to document changes in the bacterial communities of soils associated with epiphytic material that falls to the forest floor. Bacterial diversity and composition of canopy soil was highly similar, but not identical, to adjacent ground soil two years after transfer to the forest floor, indicating that canopy bacteria are almost, but not completely, replaced by ground soil bacteria. Furthermore, soil associated with epiphytic material on branches that were severed from the host tree and suspended in the canopy contained altered bacterial communities that were distinct from those in canopy material moved to the forest floor. Therefore, the unique nature of canopy soil bacteria is determined in part by the host tree and not only by the physical environmental conditions associated with the canopy. Connection to the living tree appears to be a key feature of the canopy habitat. These results represent an initial survey of bacterial diversity of the canopy and provide a foundation upon which future studies can more fully investigate the ecological and evolutionary dynamics of these communities.

  6. Dynamics of bacterial gene regulation

    Science.gov (United States)

    Narang, Atul

    2009-03-01

    The phenomenon of diauxic growth is a classical problem of bacterial gene regulation. The most well studied example of this phenomenon is the glucose-lactose diauxie, which occurs because the expression of the lac operon is strongly repressed in the presence of glucose. This repression is often explained by appealing to molecular mechanisms such as cAMP activation and inducer exclusion. I will begin by analyzing data showing that these molecular mechanisms cannot explain the strong lac repression because they exert a relatively weak effect. I will then present a minimal model accounting only for enzyme induction and dilution, which yields strong repression despite the absence of catabolite repression and inducer exclusion. The model also explains the growth patterns observed in batch and continuous cultures of various bacterial strains and substrate mixtures. The talk will conclude with a discussion of the experimental evidence regarding positive feedback, the key component of the minimal model.

  7. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes

    Science.gov (United States)

    The long terminal repeat (LTR) sequence of reticuloendotheliosis virus (REV) was inserted into the very virulent Marek’s disease virus (MDV) Md5 bacterial artificial chromosome clone. The insertion site was nearly identical to the REV LTR that was naturally inserted into the JM/102W strain of MDV fo...

  8. Mutations in the EDR1 Gene Alter the Response of Arabidopsis thaliana to Phytophthora infestans and the Bacterial PAMPs flg22 and elf18.

    Science.gov (United States)

    Geissler, Katrin; Eschen-Lippold, Lennart; Naumann, Kai; Schneeberger, Korbinian; Weigel, Detlef; Scheel, Dierk; Rosahl, Sabine; Westphal, Lore

    2015-02-01

    Mechanistically, nonhost resistance of Arabidopsis thaliana against the oomycete Phytophthora infestans is not well understood. Besides PEN2 and PEN3, which contribute to penetration resistance, no further components have been identified so far. In an ethylmethane sulphonate-mutant screen, we mutagenized pen2-1 and screened for mutants with an altered response to infection by P. infestans. One of the mutants obtained, enhanced response to Phytophthora infestans6 (erp6), was analyzed. Whole-genome sequencing of erp6 revealed a single nucleotide polymorphism in the coding region of the kinase domain of At1g08720, which encodes the putative MAPKKK ENHANCED DISEASE RESISTANCE1 (EDR1). We demonstrate that three independent lines with knock-out alleles of edr1 mount an enhanced response to P. infestans inoculation, mediated by increased salicylic acid signaling and callose deposition. Moreover, we show that the single amino acid substitution in erp6 causes the loss of in vitro autophosphorylation activity of EDR1. Furthermore, growth inhibition experiments suggest a so-far-unknown involvement of EDR1 in the response to the pathogen-associated molecular patterns flg22 and elf18. We conclude that EDR1 contributes to the defense response of A. thaliana against P. infestans. Our data position EDR1 as a negative regulator in postinvasive nonhost resistance.

  9. Does circumcision alter the periurethral uropathogenic bacterial flora

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    Mushtaq Ahmad Laway

    2012-01-01

    Full Text Available Background: The aim of this study was to assess the pattern of periurethral bacterial flora in uncircumcised boys and to evaluate the effect of circumcision on alteration of periurethral uropathogenic bacterial flora. Materials and Methods: Pattern of periurethral bacterial flora before and after circumcision was studied prospectively in 124 boys. The results were analysed to compare change in bacterial colonisation before and after circumcision. Results: The age range was 6 weeks to 96 months. Most (94.3% of the boys had religious indication and 5.7% had medical indication for circumcision. E. coli, Proteus and Klebsiella were most common periurethral bacterial flora in uncircumcised subjects. Coagulase-negative staphylococcus and Staphylococcus aureus was most common periurethral bacterial flora in circumcised subjects. In 66.1% of circumcised subjects, no bacteria were grown from periurethral region. Conclusion: We conclude that presence of prepuce is associated with great quantity of periurethral bacteria, greater likelihood of the presence of high concentration of uropathogens and high incidence of urinary tract infection (UTI. This study provides circumstantial evidence supporting the idea that early circumcision may be beneficial for prevention of UTI.

  10. Root bacterial endophytes alter plant phenotype, but not physiology

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    Jeremiah A. Henning

    2016-11-01

    Full Text Available Plant traits, such as root and leaf area, influence how plants interact with their environment and the diverse microbiota living within plants can influence plant morphology and physiology. Here, we explored how three bacterial strains isolated from the Populus root microbiome, influenced plant phenotype. We chose three bacterial strains that differed in predicted metabolic capabilities, plant hormone production and metabolism, and secondary metabolite synthesis. We inoculated each bacterial strain on a single genotype of Populus trichocarpa and measured the response of plant growth related traits (root:shoot, biomass production, root and leaf growth rates and physiological traits (chlorophyll content, net photosynthesis, net photosynthesis at saturating light–Asat, and saturating CO2–Amax. Overall, we found that bacterial root endophyte infection increased root growth rate up to 184% and leaf growth rate up to 137% relative to non-inoculated control plants, evidence that plants respond to bacteria by modifying morphology. However, endophyte inoculation had no influence on total plant biomass and photosynthetic traits (net photosynthesis, chlorophyll content. In sum, bacterial inoculation did not significantly increase plant carbon fixation and biomass, but their presence altered where and how carbon was being allocated in the plant host.

  11. A study of bacterial gene regulatory mechanisms

    DEFF Research Database (Denmark)

    Hansen, Sabine

    the different regulatory mechanisms affect system dynamics. We have designed a synthetic gene regulatory network (GRN) in bacterial cells that enables us to study the dynamics of GRNs. The results presented in this PhD thesis show that model equations based on the established mechanisms of action of each...... of a particular type of regulatory mechanism. The synthetic system presented in this thesis is, to our knowledge, the first of its kind to allow a direct comparison of the dynamic behaviors of gene regulatory networks that employ different mechanisms of regulation. In addition to studying the dynamic behavior...... of GRNs this thesis also provided the first evidence of the sensor histidine kinase VC1831 being an additional player in the Vibrio cholerae quorum sensing (QS) GRN. Bacteria use a process of cell-cell communication called QS which enable the bacterial cells to collectively control their gene expression...

  12. Horizontal gene transfer and bacterial diversity

    Indian Academy of Sciences (India)

    Chitra Dutta; Archana Pan

    2002-02-01

    Bacterial genomes are extremely dynamic and mosaic in nature. A substantial amount of genetic information is inserted into or deleted from such genomes through the process of horizontal transfer. Through the introduction of novel physiological traits from distantly related organisms, horizontal gene transfer often causes drastic changes in the ecological and pathogenic character of bacterial species and thereby promotes microbial diversification and speciation. This review discusses how the recent influx of complete chromosomal sequences of various microorganisms has allowed for a quantitative assessment of the scope, rate and impact of horizontally transmitted information on microbial evolution.

  13. Bacterial Cellular Engineering by Genome Editing and Gene Silencing

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    Nobutaka Nakashima

    2014-02-01

    Full Text Available Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption, knock-in (insertion, and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering.

  14. Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes.

    Science.gov (United States)

    Abdel-Ghany, Salah E; Day, Irene; Heuberger, Adam L; Broeckling, Corey D; Reddy, Anireddy S N

    2013-11-01

    1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.

  15. Bacterial chitinolytic communities respond to chitin and pH alteration in soil.

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    Kielak, Anna M; Cretoiu, Mariana Silvia; Semenov, Alexander V; Sørensen, Søren J; van Elsas, Jan Dirk

    2013-01-01

    Chitin amendment is a promising soil management strategy that may enhance the suppressiveness of soil toward plant pathogens. However, we understand very little of the effects of added chitin, including the putative successions that take place in the degradative process. We performed an experiment in moderately acid soil in which the level of chitin, next to the pH, was altered. Examination of chitinase activities revealed fast responses to the added crude chitin, with peaks of enzymatic activity occurring on day 7. PCR-denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA and chiA genes showed structural changes of the phylogenetically and functionally based bacterial communities following chitin addition and pH alteration. Pyrosequencing analysis indicated (i) that the diversity of chiA gene types in soil is enormous and (i) that different chiA gene types are selected by the addition of chitin at different prevailing soil pH values. Interestingly, a major role of Gram-negative bacteria versus a minor one of Actinobacteria in the immediate response to the added chitin (based on 16S rRNA gene abundance and chiA gene types) was indicated. The results of this study enhance our understanding of the response of the soil bacterial communities to chitin and are of use for both the understanding of soil suppressiveness and the possible mining of soil for novel enzymes.

  16. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production.

    Science.gov (United States)

    Lemmer, Kimberly C; Zhang, Weiping; Langer, Samantha J; Dohnalkova, Alice C; Hu, Dehong; Lemke, Rachelle A; Piotrowski, Jeff S; Orr, Galya; Noguera, Daniel R; Donohue, Timothy J

    2017-05-23

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in Rhodobacter sphaeroides By screening an R. sphaeroides Tn5 mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.IMPORTANCE This paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase microbial lipid production. We also find that the utility of some of these alterations can be

  17. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    Energy Technology Data Exchange (ETDEWEB)

    Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice C.; Hu, Dehong; Lemke, Rachelle A.; Piotrowski, Jeff S.; Orr, Galya; Noguera, Daniel R.; Donohue, Timothy J.; Ruby, Edward G.

    2017-05-23

    ABSTRACT

    Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL) mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

    IMPORTANCEThis paper reports on experiments to understand how to increase microbial lipid production. Microbial lipids are often cited as one renewable replacement for petroleum-based fuels and chemicals, but strategies to increase the yield of these compounds are needed to achieve this goal. While lipid biosynthesis is often well understood, increasing yields of these compounds to industrially relevant levels is a challenge, especially since genetic, synthetic biology, or engineering approaches are not feasible in many microbes. We show that altering the bacterial cell envelope can be used to increase

  18. Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

    Directory of Open Access Journals (Sweden)

    Kimberly C. Lemmer

    2017-05-01

    Full Text Available Lipids from microbes offer a promising source of renewable alternatives to petroleum-derived compounds. In particular, oleaginous microbes are of interest because they accumulate a large fraction of their biomass as lipids. In this study, we analyzed genetic changes that alter lipid accumulation in Rhodobacter sphaeroides. By screening an R. sphaeroides Tn5 mutant library for insertions that increased fatty acid content, we identified 10 high-lipid (HL mutants for further characterization. These HL mutants exhibited increased sensitivity to drugs that target the bacterial cell envelope and changes in shape, and some had the ability to secrete lipids, with two HL mutants accumulating ~60% of their total lipids extracellularly. When one of the highest-lipid-secreting strains was grown in a fed-batch bioreactor, its lipid content was comparable to that of oleaginous microbes, with the majority of the lipids secreted into the medium. Based on the properties of these HL mutants, we conclude that alterations of the cell envelope are a previously unreported approach to increase microbial lipid production. We also propose that this approach may be combined with knowledge about biosynthetic pathways, in this or other microbes, to increase production of lipids and other chemicals.

  19. A study of bacterial gene regulatory mechanisms

    DEFF Research Database (Denmark)

    Hansen, Sabine

    regulator studied accurately reproduced the experimental data. Simulations of system dynamics reveals that even two step regulatory cascades significantly increase response times compared to direct allosteric regulation of a transcription factor. It is observed that while many system behaviors...... of GRNs this thesis also provided the first evidence of the sensor histidine kinase VC1831 being an additional player in the Vibrio cholerae quorum sensing (QS) GRN. Bacteria use a process of cell-cell communication called QS which enable the bacterial cells to collectively control their gene expression...

  20. Supplemental dietary inulin of variable chain lengths alters intestinal bacterial populations in young pigs.

    Science.gov (United States)

    Patterson, Jannine K; Yasuda, Koji; Welch, Ross M; Miller, Dennis D; Lei, Xin Gen

    2010-12-01

    Previously, we showed that supplementation of diets with short-chain inulin (P95), long-chain inulin (HP), and a 50:50 mixture of both (Synergy 1) improved body iron status and altered expression of the genes involved in iron homeostasis and inflammation in young pigs. However, the effects of these 3 types of inulin on intestinal bacteria remain unknown. Applying terminal restriction fragment length polymorphism analysis, we determined the abundances of luminal and adherent bacterial populations from 6 segments of the small and large intestines of pigs (n = 4 for each group) fed an iron-deficient basal diet (BD) or the BD supplemented with 4% of P95, Synergy 1, or HP for 5 wk. Compared with BD, all 3 types of inulin enhanced (P inulin on bacterial populations in the lumen contents were found. Meanwhile, all 3 types of inulin suppressed the less desirable bacteria Clostridium spp. and members of the Enterobacteriaceae in the lumen and mucosa of various gut segments. Our findings suggest that the ability of dietary inulin to alter intestinal bacterial populations may partially account for its iron bioavailability-promoting effect and possibly other health benefits.

  1. Bacterial nitrate assimilation: gene distribution and regulation.

    Science.gov (United States)

    Luque-Almagro, Víctor M; Gates, Andrew J; Moreno-Vivián, Conrado; Ferguson, Stuart J; Richardson, David J; Roldán, M Dolores

    2011-12-01

    In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.

  2. The constancy of gene conservation across divergent bacterial orders

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    Ackermann Martin

    2009-01-01

    Full Text Available Abstract Background Orthologous genes are frequently presumed to perform similar functions. However, outside of model organisms, this is rarely tested. One means of inferring changes in function is if there are changes in the level of gene conservation and selective constraint. Here we compare levels of gene conservation across three bacterial groups to test for changes in gene functionality. Findings The level of gene conservation for different orthologous genes is highly correlated across clades, even for highly divergent groups of bacteria. These correlations do not arise from broad differences in gene functionality (e.g. informational genes vs. metabolic genes, but instead seem to result from very specific differences in gene function. Furthermore, these functional differences appear to be maintained over very long periods of time. Conclusion These results suggest that even over broad time scales, most bacterial genes are under a nearly constant level of purifying selection, and that bacterial evolution is thus dominated by selective and functional stasis.

  3. Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1

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    Klingeman Dawn M

    2006-04-01

    Full Text Available Abstract Background Shewanella oneidensis MR-1 is a facultative, gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as oxygen, nitrate and metals, and has potential for bioremediation of heavy metal contaminated sites. The complete 5-Mb genome of S. oneidensis MR-1 was sequenced and standard sequence-comparison methods revealed approximately 42% of the MR-1 genome encodes proteins of unknown function. Defining the functions of hypothetical proteins is a great challenge and may need a systems approach. In this study, by using integrated approaches including whole genomic microarray and proteomics, we examined knockout effects of the gene encoding SO1377 (gi24372955, a member of the conserved, hypothetical, bacterial protein family COG2268 (Clusters of Orthologous Group in bacterium Shewanella oneidensis MR-1, under various physiological conditions. Results Compared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin. Conclusion Our results showed that the knock-out of SO1377 gene had pleiotropic effects and suggested that SO1377 may play a role in iron

  4. Altered Bacterial Profiles in Saliva from Adults with Caries Lesions

    DEFF Research Database (Denmark)

    Belstrøm, D; Fiehn, N-E; Nielsen, C H

    2014-01-01

    The aim of this study was to learn whether presence of caries in an adult population was associated with a salivary bacterial profile different from that of individuals without untreated caries. Stimulated saliva samples from 621 participants of the Danish Health Examination Survey were analyzed...... using the Human Oral Microbe Identification Microarray technology. Samples from 174 individuals with dental caries and 447 from a control cohort were compared using frequency and levels of identified bacterial taxa/clusters as endpoints. Differences at taxon/cluster level were analyzed using Mann......-Whitney's test with Benjamini-Hochberg correction for multiple comparisons. Principal component analysis was used to visualize bacterial community profiles. A reduced bacterial diversity was observed in samples from subjects with dental caries. Five bacterial taxa (Veillonella parvula, Veillonella atypica...

  5. Altering plant-microbe interaction through artificially manipulating bacterial quorum sensing.

    Science.gov (United States)

    Fray, Rupert G

    2002-03-01

    Many bacteria regulate diverse physiological processes in concert with their population size. Bacterial cell-to-cell communication utilizes small diffusible signal molecules, which the bacteria both produce and perceive. The bacteria couple gene expression to cell density by eliciting a response only when the signalling molecules reach a critical threshold (a point at which the population is said to be 'quorate'). The population as a whole is thus able to modify its behaviour as a single unit. Amongst Gram-negative bacteria, the quorum sensing signals most commonly used are N-acylhomoserine lactones (AHLs). It is now apparent that AHLs are used for regulating diverse behaviours in epiphytic, rhizosphere-inhabiting and plant pathogenic bacteria and that plants may produce their own metabolites that interfere with this signalling. Transgenic plants that produce high levels of AHLs or which can degrade bacterial-produced AHLs have been made. These plants have dramatically altered susceptibilities to infection by pathogenic Erwinia species. In addition, such plants will prove useful tools in determining the roles of AHL-regulated density-dependent behaviour in growth promoting, biological control and pathogenic plant-associated bacterial species.

  6. Intravenous lipopolysaccharide challenge alters ruminal bacterial microbiota and disrupts ruminal metabolism in dairy cattle.

    Science.gov (United States)

    Jing, Longhui; Zhang, Ruiyang; Liu, Yujie; Zhu, Weiyun; Mao, Shengyong

    2014-07-28

    In the present study, three primiparous lactating Holstein cows (260-285 d in lactation) were used in a 3 × 3 Latin square design to assess the effects of three doses (0.0, 0.4 and 0.8 μg/kg body weight) of lipopolysaccharide (LPS, Escherichia coli 0111:B4) on changes in ruminal microbiota and ruminal fermentation. Ruminal pH was linearly decreased (Pinfusion linearly decreased (Phay and soyabean meal in the rumen, but did not affect (P>0.10) the gene expression of Na⁺/K⁺-ATPase and monocarboxylic acid transporter-1, -2 and -4. A plot of principal coordinate analysis based on unweighted UniFrac values and analysis of molecular variance revealed that the structure of ruminal bacterial communities in the control was distinct from that of the ruminal microbiota in the cattle exposed to LPS. At the phylum level, when compared with the control group, LPS infusion in the tested cows linearly increased (P< 0.05) the abundance of Firmicutes, and linearly decreased (P< 0.05) the percentage of Bacteroidetes, Tenericutes, Spirochaetes, Chlorobi and Lentisphaerae. To our knowledge, this is the first study to report that intravenously LPS challenge altered the ruminal bacterial microbiota and fermentation profiles. The present data suggest that systemic LPS could alter ruminal environment and ruminal microbiota composition, leading to a general decrease in fermentative activity.

  7. Root bacterial endophytes alter plant phenotype, but not physiology

    DEFF Research Database (Denmark)

    Henning, Jeremiah A.; Weston, David J.; Pelletier, Dale A.

    2016-01-01

    Plant traits, such as root and leaf area, influence how plants interact with their environment and the diverse microbiota living within plants can influence plant morphology and physiology. Here, we explored how three bacterial strains isolated from the Populus root microbiome, influenced plant...... phenotype. We chose three bacterial strains that differed in predicted metabolic capabilities, plant hormone production and metabolism, and secondary metabolite synthesis. We inoculated each bacterial strain on a single genotype of Populus trichocarpa and measured the response of plant growth related traits...... (root:shoot, biomass production, root and leaf growth rates) and physiological traits (chlorophyll content, net photosynthesis, net photosynthesis at saturating light-Asat, and saturating CO2-Amax). Overall, we found that bacterial root endophyte infection increased root growth rate up to 184% and leaf...

  8. A destabilized bacterial luciferase for dynamic gene expression studies

    Science.gov (United States)

    Allen, Michael S.; Wilgus, John R.; Chewning, Christopher S.; Sayler, Gary S.

    2006-01-01

    Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression. PMID:19003433

  9. Activation of the jasmonic acid plant defence pathway alters the composition of rhizosphere bacterial communities.

    Science.gov (United States)

    Carvalhais, Lilia C; Dennis, Paul G; Badri, Dayakar V; Tyson, Gene W; Vivanco, Jorge M; Schenk, Peer M

    2013-01-01

    Jasmonic acid (JA) signalling plays a central role in plant defences against necrotrophic pathogens and herbivorous insects, which afflict both roots and shoots. This pathway is also activated following the interaction with beneficial microbes that may lead to induced systemic resistance. Activation of the JA signalling pathway via application of methyl jasmonate (MeJA) alters the composition of carbon containing compounds released by roots, which are implicated as key determinants of rhizosphere microbial community structure. In this study, we investigated the influence of the JA defence signalling pathway activation in Arabidopsis thaliana on the structure of associated rhizosphere bacterial communities using 16S rRNA gene amplicon pyrosequencing. Application of MeJA did not directly influence bulk soil microbial communities but significant changes in rhizosphere community composition were observed upon activation of the jasmonate signalling pathway. Our results suggest that JA signalling may mediate plant-bacteria interactions in the soil upon necrotrophic pathogen and herbivorous insect attacks.

  10. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    Energy Technology Data Exchange (ETDEWEB)

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  11. Root bacterial endophytes alter plant phenotype, but not physiology

    DEFF Research Database (Denmark)

    Henning, Jeremiah A.; Weston, David J.; Pelletier, Dale A.;

    2016-01-01

    (root:shoot, biomass production, root and leaf growth rates) and physiological traits (chlorophyll content, net photosynthesis, net photosynthesis at saturating light-Asat, and saturating CO2-Amax). Overall, we found that bacterial root endophyte infection increased root growth rate up to 184% and leaf...

  12. Genetic and metabolic signals during acute enteric bacterial infection alter the microbiota and drive progression to chronic inflammatory disease

    Energy Technology Data Exchange (ETDEWEB)

    Kamdar, Karishma; Khakpour, Samira; Chen, Jingyu; Leone, Vanessa; Brulc, Jennifer; Mangatu, Thomas; Antonopoulos, Dionysios A.; Chang, Eugene B; Kahn, Stacy A.; Kirschner, Barbara S; Young, Glenn; DePaolo, R. William

    2016-01-13

    Chronic inflammatory disorders are thought to arise due to an interplay between predisposing host genetics and environmental factors. For example, the onset of inflammatory bowel disease is associated with enteric proteobacterial infection, yet the mechanistic basis for this association is unclear. We have shown previously that genetic defiency in TLR1 promotes acute enteric infection by the proteobacteria Yersinia enterocolitica. Examining that model further, we uncovered an altered cellular immune response that promotes the recruitment of neutrophils which in turn increases metabolism of the respiratory electron acceptor tetrathionate by Yersinia. These events drive permanent alterations in anti-commensal immunity, microbiota composition, and chronic inflammation, which persist long after Yersinia clearence. Deletion of the bacterial genes involved in tetrathionate respiration or treatment using targeted probiotics could prevent microbiota alterations and inflammation. Thus, acute infection can drive long term immune and microbiota alterations leading to chronic inflammatory disease in genetically predisposed individuals.

  13. Detecting rare gene transfer events in bacterial populations

    Directory of Open Access Journals (Sweden)

    Kaare Magne Nielsen

    2014-01-01

    Full Text Available Horizontal gene transfer (HGT enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research.

  14. Nitrogen deposition alters soil chemical properties and bacterial communities in the Inner Mongolia grassland

    Institute of Scientific and Technical Information of China (English)

    Ximei Zhang; Xingguo Han

    2012-01-01

    Nitrogen deposition has dramatically altered biodiversity and ecosystem functioning on the earth; however,its effects on soil bacterial community and the underlying mechanisms of these effects have not been thoroughly examined.Changes in ecosystems caused by nitrogen deposition have traditionally been attributed to increased nitrogen content.In fact,nitrogen deposition not only leads to increased soil total N content,but also changes in the NH4+-N content,NO3--N content and pH,as well as changes in the heterogeneity of the four indexes.The soil indexes for these four factors,their heterogeneity and even the plant community might be routes through which nitrogen deposition alters the bacterial community.Here,we describe a 6-year nitrogen addition experiment conducted in a typical steppe ecosystem to investigate the ecological mechanism by which nitrogen deposition alters bacterial abundance,diversity and composition.We found that various characteristics of the bacterial community were explained by different environmental factors.Nitrogen deposition decreased bacterial abundance that is positively related to soil pH value.In addition,nitrogen addition decreased bacterial diversity,which is negatively related to soil total N content and positively related to soil NO3--N heterogeneity.Finally,nitrogen.addition altered bacterial composition that is significantly related to soil NH4+-N content.Although nitrogen deposition significantly altered plant biomass,diversity and composition,these characteristics of plant community did not have a significant impact on processes of nitrogen deposition that led to alterations in bacterial abundance,diversity and composition.Therefore,more sensitive molecular technologies should be adopted to detect the subtle shifts of microbial community structure induced by the changes of plant community upon nitrogen deposition.

  15. Altered Virome and Bacterial Microbiome in Human Immunodeficiency Virus-Associated Acquired Immunodeficiency Syndrome.

    Science.gov (United States)

    Monaco, Cynthia L; Gootenberg, David B; Zhao, Guoyan; Handley, Scott A; Ghebremichael, Musie S; Lim, Efrem S; Lankowski, Alex; Baldridge, Megan T; Wilen, Craig B; Flagg, Meaghan; Norman, Jason M; Keller, Brian C; Luévano, Jesús Mario; Wang, David; Boum, Yap; Martin, Jeffrey N; Hunt, Peter W; Bangsberg, David R; Siedner, Mark J; Kwon, Douglas S; Virgin, Herbert W

    2016-03-09

    Human immunodeficiency virus (HIV) infection is associated with increased intestinal translocation of microbial products and enteropathy as well as alterations in gut bacterial communities. However, whether the enteric virome contributes to this infection and resulting immunodeficiency remains unknown. We characterized the enteric virome and bacterial microbiome in a cohort of Ugandan patients, including HIV-uninfected or HIV-infected subjects and those either treated with anti-retroviral therapy (ART) or untreated. Low peripheral CD4 T cell counts were associated with an expansion of enteric adenovirus sequences and this increase was independent of ART treatment. Additionally, the enteric bacterial microbiome of patients with lower CD4 T counts exhibited reduced phylogenetic diversity and richness with specific bacteria showing differential abundance, including increases in Enterobacteriaceae, which have been associated with inflammation. Thus, immunodeficiency in progressive HIV infection is associated with alterations in the enteric virome and bacterial microbiome, which may contribute to AIDS-associated enteropathy and disease progression.

  16. Lateral Gene Transfer Dynamics in the Ancient Bacterial Genus Streptomyces.

    Science.gov (United States)

    McDonald, Bradon R; Currie, Cameron R

    2017-06-06

    Lateral gene transfer (LGT) profoundly shapes the evolution of bacterial lineages. LGT across disparate phylogenetic groups and genome content diversity between related organisms suggest a model of bacterial evolution that views LGT as rampant and promiscuous. It has even driven the argument that species concepts and tree-based phylogenetics cannot be applied to bacteria. Here, we show that acquisition and retention of genes through LGT are surprisingly rare in the ubiquitous and biomedically important bacterial genus Streptomyces Using a molecular clock, we estimate that the Streptomyces bacteria are ~380 million years old, indicating that this bacterial genus is as ancient as land vertebrates. Calibrating LGT rate to this geologic time span, we find that on average only 10 genes per million years were acquired and subsequently maintained. Over that same time span, Streptomyces accumulated thousands of point mutations. By explicitly incorporating evolutionary timescale into our analyses, we provide a dramatically different view on the dynamics of LGT and its impact on bacterial evolution.IMPORTANCE Tree-based phylogenetics and the use of species as units of diversity lie at the foundation of modern biology. In bacteria, these pillars of evolutionary theory have been called into question due to the observation of thousands of lateral gene transfer (LGT) events within and between lineages. Here, we show that acquisition and retention of genes through LGT are exceedingly rare in the bacterial genus Streptomyces, with merely one gene acquired in Streptomyces lineages every 100,000 years. These findings stand in contrast to the current assumption of rampant genetic exchange, which has become the dominant hypothesis used to explain bacterial diversity. Our results support a more nuanced understanding of genetic exchange, with LGT impacting evolution over short timescales but playing a significant role over long timescales. Deeper understanding of LGT provides new

  17. Bacteriophage-encoded shiga toxin gene in atypical bacterial host

    Directory of Open Access Journals (Sweden)

    Casas Veronica

    2011-07-01

    Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.

  18. Bacterial Toxins for Oncoleaking Suicidal Cancer Gene Therapy.

    Science.gov (United States)

    Pahle, Jessica; Walther, Wolfgang

    For suicide gene therapy, initially prodrug-converting enzymes (gene-directed enzyme-producing therapy, GDEPT) were employed to intracellularly metabolize non-toxic prodrugs into toxic compounds, leading to the effective suicidal killing of the transfected tumor cells. In this regard, the suicide gene therapy has demonstrated its potential for efficient tumor eradication. Numerous suicide genes of viral or bacterial origin were isolated, characterized, and extensively tested in vitro and in vivo, demonstrating their therapeutic potential even in clinical trials to treat cancers of different entities. Apart from this, growing efforts are made to generate more targeted and more effective suicide gene systems for cancer gene therapy. In this regard, bacterial toxins are an alternative to the classical GDEPT strategy, which add to the broad spectrum of different suicide approaches. In this context, lytic bacterial toxins, such as streptolysin O (SLO) or the claudin-targeted Clostridium perfringens enterotoxin (CPE) represent attractive new types of suicide oncoleaking genes. They permit as pore-forming proteins rapid and also selective toxicity toward a broad range of cancers. In this chapter, we describe the generation and use of SLO as well as of CPE-based gene therapies for the effective tumor cell eradication as promising, novel suicide gene approach particularly for treatment of therapy refractory tumors.

  19. Genome engineering and gene expression control for bacterial strain development.

    Science.gov (United States)

    Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

    2015-01-01

    In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen.

  20. Chronic N-amended soils exhibit an altered bacterial community structure in Harvard Forest, MA, USA

    Science.gov (United States)

    Swathi A. Turlapati; Rakesh Minocha; Premsai S. Bhiravarasa; Louise S. Tisa; William K. Thomas; Subhash C. Minocha

    2013-01-01

    At the Harvard Forest, Petersham, MA, the impact of 20 years of annual ammonium nitrate application to the mixed hardwood stand on soil bacterial communities was studied using 16S rRNA genes pyrosequencing. Amplification of 16S rRNA genes was done using DNA extracted from 30 soil samples (three treatments x two horizons x five subplots) collected from untreated (...

  1. Activation of the jasmonic acid plant defence pathway alters the composition of rhizosphere bacterial communities.

    Directory of Open Access Journals (Sweden)

    Lilia C Carvalhais

    Full Text Available Jasmonic acid (JA signalling plays a central role in plant defences against necrotrophic pathogens and herbivorous insects, which afflict both roots and shoots. This pathway is also activated following the interaction with beneficial microbes that may lead to induced systemic resistance. Activation of the JA signalling pathway via application of methyl jasmonate (MeJA alters the composition of carbon containing compounds released by roots, which are implicated as key determinants of rhizosphere microbial community structure. In this study, we investigated the influence of the JA defence signalling pathway activation in Arabidopsis thaliana on the structure of associated rhizosphere bacterial communities using 16S rRNA gene amplicon pyrosequencing. Application of MeJA did not directly influence bulk soil microbial communities but significant changes in rhizosphere community composition were observed upon activation of the jasmonate signalling pathway. Our results suggest that JA signalling may mediate plant-bacteria interactions in the soil upon necrotrophic pathogen and herbivorous insect attacks.

  2. Changes in rhizosphere bacterial gene expression following glyphosate treatment.

    Science.gov (United States)

    Newman, Molli M; Lorenz, Nicola; Hoilett, Nigel; Lee, Nathan R; Dick, Richard P; Liles, Mark R; Ramsier, Cliff; Kloepper, Joseph W

    2016-05-15

    In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria. Copyright © 2016 The Authors. Published by Elsevier B.V. All

  3. Subgingival bacterial colonization profiles correlate with gingival tissue gene expression

    Directory of Open Access Journals (Sweden)

    Handfield Martin

    2009-10-01

    Full Text Available Abstract Background Periodontitis is a chronic inflammatory disease caused by the microbiota of the periodontal pocket. We investigated the association between subgingival bacterial profiles and gene expression patterns in gingival tissues of patients with periodontitis. A total of 120 patients undergoing periodontal surgery contributed with a minimum of two interproximal gingival papillae (range 2-4 from a maxillary posterior region. Prior to tissue harvesting, subgingival plaque samples were collected from the mesial and distal aspects of each tissue sample. Gingival tissue RNA was extracted, reverse-transcribed, labeled, and hybridized with whole-genome microarrays (310 in total. Plaque samples were analyzed using checkerboard DNA-DNA hybridizations with respect to 11 bacterial species. Random effects linear regression models considered bacterial levels as exposure and expression profiles as outcome variables. Gene Ontology analyses summarized the expression patterns into biologically relevant categories. Results Wide inter-species variation was noted in the number of differentially expressed gingival tissue genes according to subgingival bacterial levels: Using a Bonferroni correction (p -7, 9,392 probe sets were differentially associated with levels of Tannerella forsythia, 8,537 with Porphyromonas gingivalis, 6,460 with Aggregatibacter actinomycetemcomitans, 506 with Eikenella corrodens and only 8 with Actinomyces naeslundii. Cluster analysis identified commonalities and differences among tissue gene expression patterns differentially regulated according to bacterial levels. Conclusion Our findings suggest that the microbial content of the periodontal pocket is a determinant of gene expression in the gingival tissues and provide new insights into the differential ability of periodontal species to elicit a local host response.

  4. Subgingival bacterial colonization profiles correlate with gingival tissue gene expression.

    Science.gov (United States)

    Papapanou, Panos N; Behle, Jan H; Kebschull, Moritz; Celenti, Romanita; Wolf, Dana L; Handfield, Martin; Pavlidis, Paul; Demmer, Ryan T

    2009-10-18

    Periodontitis is a chronic inflammatory disease caused by the microbiota of the periodontal pocket. We investigated the association between subgingival bacterial profiles and gene expression patterns in gingival tissues of patients with periodontitis. A total of 120 patients undergoing periodontal surgery contributed with a minimum of two interproximal gingival papillae (range 2-4) from a maxillary posterior region. Prior to tissue harvesting, subgingival plaque samples were collected from the mesial and distal aspects of each tissue sample. Gingival tissue RNA was extracted, reverse-transcribed, labeled, and hybridized with whole-genome microarrays (310 in total). Plaque samples were analyzed using checkerboard DNA-DNA hybridizations with respect to 11 bacterial species. Random effects linear regression models considered bacterial levels as exposure and expression profiles as outcome variables. Gene Ontology analyses summarized the expression patterns into biologically relevant categories. Wide inter-species variation was noted in the number of differentially expressed gingival tissue genes according to subgingival bacterial levels: Using a Bonferroni correction (p < 9.15 x 10(-7)), 9,392 probe sets were differentially associated with levels of Tannerella forsythia, 8,537 with Porphyromonas gingivalis, 6,460 with Aggregatibacter actinomycetemcomitans, 506 with Eikenella corrodens and only 8 with Actinomyces naeslundii. Cluster analysis identified commonalities and differences among tissue gene expression patterns differentially regulated according to bacterial levels. Our findings suggest that the microbial content of the periodontal pocket is a determinant of gene expression in the gingival tissues and provide new insights into the differential ability of periodontal species to elicit a local host response.

  5. CRISPR/Cas systems: new players in gene regulation and bacterial physiology

    Directory of Open Access Journals (Sweden)

    David eWeiss

    2014-04-01

    Full Text Available CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP. Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2, CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes.

  6. Gene calling and bacterial genome annotation with BG7.

    Science.gov (United States)

    Tobes, Raquel; Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Kovach, Evdokim; Alekhin, Alexey; Pareja, Eduardo

    2015-01-01

    New massive sequencing technologies are providing many bacterial genome sequences from diverse taxa but a refined annotation of these genomes is crucial for obtaining scientific findings and new knowledge. Thus, bacterial genome annotation has emerged as a key point to investigate in bacteria. Any efficient tool designed specifically to annotate bacterial genomes sequenced with massively parallel technologies has to consider the specific features of bacterial genomes (absence of introns and scarcity of nonprotein-coding sequence) and of next-generation sequencing (NGS) technologies (presence of errors and not perfectly assembled genomes). These features make it convenient to focus on coding regions and, hence, on protein sequences that are the elements directly related with biological functions. In this chapter we describe how to annotate bacterial genomes with BG7, an open-source tool based on a protein-centered gene calling/annotation paradigm. BG7 is specifically designed for the annotation of bacterial genomes sequenced with NGS. This tool is sequence error tolerant maintaining their capabilities for the annotation of highly fragmented genomes or for annotating mixed sequences coming from several genomes (as those obtained through metagenomics samples). BG7 has been designed with scalability as a requirement, with a computing infrastructure completely based on cloud computing (Amazon Web Services).

  7. Identification of genes and gene products necessary for bacterial bioluminescence.

    OpenAIRE

    Engebrecht, J; Silverman, M.

    1984-01-01

    Expression of luminescence in Escherichia coli was recently achieved by cloning genes from the marine bacterium Vibrio fischeri. One DNA fragment on a hybrid plasmid encoded regulatory functions and enzymatic activities necessary for light production. We report the results of a genetic analysis to identify the luminescence genes (lux) that reside on this recombinant plasmid. lux gene mutations were generated by hydroxylamine treatment, and these mutations were ordered on a linear map by compl...

  8. Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

    NARCIS (Netherlands)

    Andreote, Fernando D.; Mendes, Rodrigo; Dini-Andreote, Francisco; Rossetto, Priscilla B.; Labate, Carlos A.; Pizzirani-Kleiner, Aline A.; van Elsas, Jan Dirck; Azevedo, Joao L.; Araujo, Welington L.

    2008-01-01

    The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities ass

  9. Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

    NARCIS (Netherlands)

    Andreote, Fernando D.; Mendes, Rodrigo; Dini-Andreote, Francisco; Rossetto, Priscilla B.; Labate, Carlos A.; Pizzirani-Kleiner, Aline A.; van Elsas, Jan Dirck; Azevedo, Joao L.; Araujo, Welington L.

    The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities

  10. Soil bacterial community and functional shifts in response to altered snowpack in moist acidic tundra of northern Alaska

    Science.gov (United States)

    Ricketts, Michael P.; Poretsky, Rachel S.; Welker, Jeffrey M.; Gonzalez-Meler, Miquel A.

    2016-09-01

    functional potential was inferred using ancestral state reconstruction to approximate functional gene abundance, revealing a decreased abundance of genes required for soil organic matter (SOM) decomposition in the organic layers of the deep snow accumulation zones. These results suggest that predicted climate change scenarios may result in altered soil bacterial community structure and function, and indicate a reduction in decomposition potential, alleviated temperature limitations on extracellular enzymatic efficiency, or both. The fate of stored C in Arctic soils ultimately depends on the balance between these mechanisms.

  11. State-related alterations of gene expression in bipolar disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Vinberg, Maj; Berk, Michael

    2012-01-01

    Munkholm K, Vinberg M, Berk M, Kessing LV. State-related alterations of gene expression in bipolar disorder: a systematic review. Bipolar Disord 2012: 14: 684-696. © 2012 The Authors. Journal compilation © 2012 John Wiley & Sons A/S. Objective:  Alterations in gene expression in bipolar disorder...... on comprehensive database searches for studies on gene expression in patients with bipolar disorder in specific mood states, was conducted. We searched Medline, Embase, PsycINFO, and The Cochrane Library, supplemented by manually searching reference lists from retrieved publications. Results:  A total of 17...

  12. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    Science.gov (United States)

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  13. Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease.

    Science.gov (United States)

    Hu, Yang; Zhang, Junli; Jia, Hongge; Sosso, Davide; Li, Ting; Frommer, Wolf B; Yang, Bing; White, Frank F; Wang, Nian; Jones, Jeffrey B

    2014-01-28

    Citrus bacterial canker (CBC) disease occurs worldwide and incurs considerable costs both from control measures and yield losses. Bacteria that cause CBC require one of six known type III transcription activator-like (TAL) effector genes for the characteristic pustule formation at the site of infection. Here, we show that Xanthomonas citri subspecies citri strain Xcc306, with the type III TAL effector gene pthA4 or with the distinct yet biologically equivalent gene pthAw from strain XccA(w), induces two host genes, CsLOB1 and CsSWEET1, in a TAL effector-dependent manner. CsLOB1 is a member of the Lateral Organ Boundaries (LOB) gene family of transcription factors, and CsSWEET1 is a homolog of the SWEET sugar transporter and rice disease susceptibility gene. Both TAL effectors drive expression of CsLOB1 and CsSWEET1 promoter reporter gene fusions when coexpressed in citrus or Nicotiana benthamiana. Artificially designed TAL effectors directed to sequences in the CsLOB1 promoter region, but not the CsSWEET1 promoter, promoted pustule formation and higher bacterial leaf populations. Three additional distinct TAL effector genes, pthA*, pthB, and pthC, also direct pustule formation and expression of CsLOB1. Unlike pthA4 and pthAw, pthB and pthC do not promote the expression of CsSWEET1. CsLOB1 expression was associated with the expression of genes associated with cell expansion. The results indicate that CBC-inciting species of Xanthomonas exploit a single host disease susceptibility gene by altering the expression of an otherwise developmentally regulated gene using any one of a diverse set of TAL effector genes in the pathogen populations.

  14. A combination of independent transcriptional regulators shapes bacterial virulence gene expression during infection.

    Directory of Open Access Journals (Sweden)

    Samuel A Shelburne

    2010-03-01

    Full Text Available Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS suggested that the transcriptional regulator catabolite control protein A (CcpA influences many of the same genes as the control of virulence (CovRS two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both DeltaccpA and DeltacovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, DeltaccpA and DeltacovRDeltaccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the DeltaccpA and DeltacovRDeltaccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection.

  15. Antimicrobial Peptide-PNA Conjugates Selectively Targeting Bacterial Genes

    Science.gov (United States)

    2013-07-22

    change, (Good, 2000). In the case of MRSA, RNA polymerase σ⁷⁷ (encoded by gene rpoD) is a conserved prokaryotic factor essential for transcription...silencing technology to bacteria is the inefficient entry of PNAs into the targeted cell due to restrictions imposed by the bacterial membrane . Peptide...AMP and (RW)3, a linear hexameric peptide, both designed in our lab, interact with wall polymers and cause penetration of the cell membrane at sub

  16. Altered motility and duration of bacterial overgrowth in experimental blind loop syndrome.

    Science.gov (United States)

    Justus, P G; Mcherron, L E; Ward, T T

    1984-07-01

    To better understand the pathogenesis of the increased motility previously described in the blind loop rat, we studied the relationship between duration of bacterial overgrowth and both myoelectric activity and bacterial flora in this model. Myoelectric studies and quantitative bacterial cultures were performed on self-filling and self-emptying (control) blind loop rats one, two, and three weeks postoperatively. All self-filling blind loop rats had greater random action potential activity and higher frequencies of migrating action potential complexes than controls (P less than 0.05). One-week self-filling blind loop rats had a higher frequency of migrating action potential complexes (P less than 0.05) and a higher ratio of counts of Escherichia coli to Bacteroides species (P less than 0.05) than the two- or three-week self-filling blind loop groups. Thus, qualitative changes in myoelectric activity occur during the development of bacterial overgrowth in the blind loop rat which may reflect evolving alterations in the bacterial flora.

  17. Mining Bacterial Genomes for Secondary Metabolite Gene Clusters.

    Science.gov (United States)

    Adamek, Martina; Spohn, Marius; Stegmann, Evi; Ziemert, Nadine

    2017-01-01

    With the emergence of bacterial resistance against frequently used antibiotics, novel antibacterial compounds are urgently needed. Traditional bioactivity-guided drug discovery strategies involve laborious screening efforts and display high rediscovery rates. With the progress in next generation sequencing methods and the knowledge that the majority of antibiotics in clinical use are produced as secondary metabolites by bacteria, mining bacterial genomes for secondary metabolites with antimicrobial activity is a promising approach, which can guide a more time and cost-effective identification of novel compounds. However, what sounds easy to accomplish, comes with several challenges. To date, several tools for the prediction of secondary metabolite gene clusters are available, some of which are based on the detection of signature genes, while others are searching for specific patterns in gene content or regulation.Apart from the mere identification of gene clusters, several other factors such as determining cluster boundaries and assessing the novelty of the detected cluster are important. For this purpose, comparison of the predicted secondary metabolite genes with different cluster and compound databases is necessary. Furthermore, it is advisable to classify detected clusters into gene cluster families. So far, there is no standardized procedure for genome mining; however, different approaches to overcome all of these challenges exist and are addressed in this chapter. We give practical guidance on the workflow for secondary metabolite gene cluster identification, which includes the determination of gene cluster boundaries, addresses problems occurring with the use of draft genomes, and gives an outlook on the different methods for gene cluster classification. Based on comprehensible examples a protocol is set, which should enable the readers to mine their own genome data for interesting secondary metabolites.

  18. The genetic alteration of retinoblastoma gene in esophageal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Jae Il; Shim, Yung Mok; Kim, Chang Min [Korea Cancer Center Hospital of Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1994-12-01

    Retinoblastoma(RB) gene is the prototype of tumor suppressor gene and it`s alteration have been frequently observed in a large number of human tumors. To investigate the role of RB in esophageal cancer, we studied 36 esophageal cancer tissues with Southern blot analysis to detect gross LOH and PCR-SSCP method to find minute LOH and mutation, if any. In the cases with abnormalities, the nucleotide sequence analysis was performed. Allelic loss of chromosome 13q14 occurred in 20 out of 32 informative cases (62.5%) by Southern analysis. Furthermore, PCR-LOH added three positive cases. Mobility shift by PCR-SSCP was observed in one case at exon 22, which showed 1 bp deletion in codon 771 of RB gene resulting in frame shift mutation. Besides, nine PCR-band alteration in tumor tissue compared with normal tissue were observed in exon 14 and 22, but mutation was not found on sequencing analysis suggesting the epigenetic alteration in tumor tissue. Analysis of the clinical data did not show any difference depending upon RB alteration. However, the total incidence of RB gene may play an important role in the development of esophageal cancer. The main genetic alteration of RB gene was deletion detected by Southern blot and one bp deletion leading to frame shift was also observed. 8 figs, 5 tabs. (Author).

  19. Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; WU Chao; LIU Mei; LIU Xu-ri; Hu Guo-cheng; SI Hua-min; SUN Zong-xiu; LIU Wen-zhen; Fu Ya-ping

    2011-01-01

    Antimierobial peptide is a polypeptide with antimicrobial activity.Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp (Fenneropenaeus Chinensis) were integrated into Oryza sativa L.subsp.japonica cv.Aichi ashahi by Agrobacterium mediated transformation system.PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in T0 generation,respectively.RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation,and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants.Four Np3 and Np5 transgenic lines in T1 generation were inoculated with ×anthomonas oryzae pv.oryzae strain CR4,and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4.The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2,Zhe 173 and OS-225.It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

  20. Altered functionality of anti-bacterial antibodies in patients with chronic hepatitis C virus infection.

    Directory of Open Access Journals (Sweden)

    Anne Lamontagne

    Full Text Available Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0 immunoglobulin G molecule (IgG that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease.The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined.Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure.Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease.

  1. PCR-DGGE analysis of the bacterial composition of a kaolin slurry showing altered rheology.

    Science.gov (United States)

    Papp, Ildikó; Balázs, Margit; Tombácz, Etelka; Babcsán, Norbert; Kesserű, Péter; Kiss, István; Szvetnik, Attila

    2012-04-01

    Kaolin is an important industrial raw material and a basis of a range of different products. Microbial spoilage is a detrimental process observed especially in kaolin slurries, leading to low quality products and economic loss. Although the alteration of kaolin slurries in ceramic industry was observed, the process and the microbial background have not been analyzed in details. This study provides the first data using a cultivation independent molecular biological approach (PCR-DGGE) regarding the bacterial composition of an altered kaolin slurry. The results show that potential exopolymer (EPS) producer bacteria (e.g. Acinetobacter, Pseudomonas) appear in the altered kaolin slurry, which may have an important role in the modification of kaolin slurries.

  2. Alteration textures in terrestrial volcanic glass and the associated bacterial community.

    Science.gov (United States)

    Cockell, C S; Olsson-Francis, K; Herrera, A; Meunier, A

    2009-01-01

    Alteration textures were examined in subglacial (hyaloclastite) deposits at Valafell, Southern Iceland. Pitted and 'elongate' alteration features are observed in the glass similar to granular and tubular features reported previously in deep-ocean basaltic glasses, but elongate features generally did not have a length to width ratio greater than five. Elongate features were found in only 7% of surfaces. Crystalline basalt clasts, which are incorporated into the hyaloclastite, did not display elongate structures. Pitted alteration features were poorly defined in crystalline basalt, comprising only 4% of the surface compared to 47% in the case of basaltic glass. Examination of silica-rich glass (obsidian) and rhyolite similarly showed poorly defined pitted textures that comprised less than 15% of the surface and no elongate features were observed. These data highlight the differences in alteration textures between terrestrial basaltic glass and previously studied deep-ocean and subsurface basaltic glass, and the important role of mineralogy in controlling the type and abundance of alteration features. The hyaloclastite contains a diverse and abundant bacterial population, as determined by 16S rDNA analysis, which could be involved in weathering the glass. Despite the presence of phototrophs, we show that they were not involved in the production of most alteration textures in the basaltic glass materials we examined.

  3. Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development.

    Science.gov (United States)

    Andreote, Fernando D; Mendes, Rodrigo; Dini-Andreote, Francisco; Rossetto, Priscilla B; Labate, Carlos A; Pizzirani-Kleiner, Aline A; van Elsas, Jan Dirck; Azevedo, João L; Araújo, Welington L

    2008-05-01

    The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium-plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant

  4. Alteration of bacterial communities and organic matter in microbial fuel cells (MFCs) supplied with soil and organic fertilizer.

    Science.gov (United States)

    Mocali, Stefano; Galeffi, Carlo; Perrin, Elena; Florio, Alessandro; Migliore, Melania; Canganella, Francesco; Bianconi, Giovanna; Di Mattia, Elena; Dell'Abate, Maria Teresa; Fani, Renato; Benedetti, Anna

    2013-02-01

    The alteration of the organic matter (OM) and the composition of bacterial community in microbial fuel cells (MFCs) supplied with soil (S) and a composted organic fertilizer (A) was examined at the beginning and at the end of 3 weeks of incubation under current-producing as well as no-current-producing conditions. Denaturing gradient gel electrophoresis revealed a significant alteration of the microbial community structure in MFCs generating electricity as compared with no-current-producing MFCs. The genetic diversity of cultivable bacterial communities was assessed by random amplified polymorphic DNA (RAPD) analysis of 106 bacterial isolates obtained by using both generic and elective media. Sequencing of the 16S rRNA genes of the more representative RAPD groups indicated that over 50.4% of the isolates from MFCs fed with S were Proteobacteria, 25.1% Firmicutes, and 24.5% Actinobacteria, whereas in MFCs supplied with A 100% of the dominant species belonged to γ-Proteobacteria. The chemical analysis performed by fractioning the OM and using thermal analysis showed that the amount of total organic carbon contained in the soluble phase of the electrochemically active chambers significantly decreased as compared to the no-current-producing systems, whereas the OM of the solid phase became more humified and aromatic along with electricity generation, suggesting a significant stimulation of a humification process of the OM. These findings demonstrated that electroactive bacteria are commonly present in aerobic organic substrates such as soil or a fertilizer and that MFCs could represent a powerful tool for exploring the mineralization and humification processes of the soil OM.

  5. Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data

    Directory of Open Access Journals (Sweden)

    Tintle Nathan L

    2012-08-01

    Full Text Available Abstract Background Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. Results We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Conclusions Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

  6. Gyramides prevent bacterial growth by inhibiting DNA gyrase and altering chromosome topology.

    Science.gov (United States)

    Rajendram, Manohary; Hurley, Katherine A; Foss, Marie H; Thornton, Kelsey M; Moore, Jared T; Shaw, Jared T; Weibel, Douglas B

    2014-06-20

    Antibiotics targeting DNA gyrase have been a clinical success story for the past half-century, and the emergence of bacterial resistance has fueled the search for new gyrase inhibitors. In this paper we demonstrate that a new class of gyrase inhibitors, the gyramides, are bacteriostatic agents that competitively inhibit the ATPase activity of Escherichia coli gyrase and produce supercoiled DNA in vivo. E. coli cells treated with gyramide A have abnormally localized, condensed chromosomes that blocks DNA replication and interrupts chromosome segregation. The resulting alterations in DNA topology inhibit cell division through a mechanism that involves the SOS pathway. Importantly, gyramide A is a specific inhibitor of gyrase and does not inhibit the closely related E. coli enzyme topoisomerase IV. E. coli mutants with reduced susceptibility to gyramide A do not display cross-resistance to ciprofloxacin and novobiocin. The results demonstrate that the gyramides prevent bacterial growth by a mechanism in which the topological state of chromosomes is altered and halts DNA replication and segregation. The specificity and activity of the gyramides for inhibiting gyrase makes these compounds important chemical tools for studying the mechanism of gyrase and the connection between DNA topology and bacterial cell division.

  7. Chronic N-amended soils exhibit an altered bacterial community structure in Harvard Forest, MA, USA.

    Science.gov (United States)

    Turlapati, Swathi A; Minocha, Rakesh; Bhiravarasa, Premsai S; Tisa, Louis S; Thomas, William K; Minocha, Subhash C

    2013-02-01

    At the Harvard Forest, Petersham, MA, the impact of 20 years of annual ammonium nitrate application to the mixed hardwood stand on soil bacterial communities was studied using 16S rRNA genes pyrosequencing. Amplification of 16S rRNA genes was done using DNA extracted from 30 soil samples (three treatments × two horizons × five subplots) collected from untreated (control), low N-amended (50 kg ha(-1) year(-1)) and high N-amended (150 kg ha(-1) year(-1)) plots. A total of 1.3 million sequences were processed using qiime. Although Acidobacteria represented the most abundant phylum based on the number of sequences, Proteobacteria were the most diverse in terms of operational taxonomic units (OTUs). UniFrac analyses revealed that the bacterial communities differed significantly among soil horizons and treatments. Microsite variability among the five subplots was also evident. Nonmetric multidimensional scaling ordination of normalized OTU data followed by permutational manova further confirmed these observations. Richness indicators and indicator species analyses revealed higher bacterial diversity associated with N amendment. Differences in bacterial diversity and community composition associated with the N treatments were also observed at lower phylogenetic levels. Only 28-35% of the 6 936 total OTUs identified were common to three treatments, while the rest were specific to one treatment or common to two.

  8. Deoxygenation alters bacterial diversity and community composition in the ocean's largest oxygen minimum zone.

    Science.gov (United States)

    Beman, J Michael; Carolan, Molly T

    2013-01-01

    Oceanic oxygen minimum zones (OMZs) have a central role in biogeochemical cycles and are expanding as a consequence of climate change, yet how deoxygenation will affect the microbial communities that control these cycles is unclear. Here we sample across dissolved oxygen gradients in the oceans' largest OMZ and show that bacterial richness displays a unimodal pattern with decreasing dissolved oxygen, reaching maximum values on the edge of the OMZ and decreasing within it. Rare groups on the OMZ margin are abundant at lower dissolved oxygen concentrations, including sulphur-cycling Chromatiales, for which 16S rRNA was amplified from extracted RNA. Microbial species distribution models accurately replicate community patterns based on multivariate environmental data, demonstrate likely changes in distributions and diversity in the eastern tropical North Pacific Ocean, and highlight the sensitivity of key bacterial groups to deoxygenation. Through these mechanisms, OMZ expansion may alter microbial composition, competition, diversity and function, all of which have implications for biogeochemical cycling in OMZs.

  9. Bacterial chitinolytic communities respond to chitin and pH alteration in soil

    DEFF Research Database (Denmark)

    Kielak, Anna; Cretoiu, Mariana; Semenov, Alexander

    2013-01-01

    in moderately acid soil in which the level of chitin, next to the pH, was altered. Examination of chitinase activities revealed fast responses to the added crude chitin, with peaks of enzymatic activity occurring on day 7. PCR-denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA and chi...... understanding of the response of the soil bacterial communities to chitin and are of use for both the understanding of soil suppressiveness and the possible mining of soil for novel enzymes....

  10. Performance of resistance gene pyramids to races of rice bacterial blight in Zhejiang Province

    Institute of Scientific and Technical Information of China (English)

    ZHENGKangle; ZHUANGJieyun; WANGHanrong

    1998-01-01

    The effect of gene pyramiding on resistance to bacterial blight (BB) in rice was evahlated among the IR24-based near isogenic lines conraining single resistance gene and gene pyramids containing two, three or lour resistancegenes (see table).

  11. Significant alteration of soil bacterial communities and organic carbon decomposition by different long-term fertilization management conditions of extremely low-productivity arable soil in South China.

    Science.gov (United States)

    Xun, Weibing; Zhao, Jun; Xue, Chao; Zhang, Guishan; Ran, Wei; Wang, Boren; Shen, Qirong; Zhang, Ruifu

    2016-06-01

    Different fertilization managements of red soil, a kind of Ferralic Cambisol, strongly affected the soil properties and associated microbial communities. The association of the soil microbial community and functionality with long-term fertilization management in the unique low-productivity red soil ecosystem is important for both soil microbial ecology and agricultural production. Here, 454 pyrosequencing analysis of 16S recombinant ribonucleic acid genes and GeoChip4-NimbleGen-based functional gene analysis were used to study the soil bacterial community composition and functional genes involved in soil organic carbon degradation. Long-term nitrogen-containing chemical fertilization-induced soil acidification and fertility decline and significantly altered the soil bacterial community, whereas long-term organic fertilization and fallow management improved the soil quality and maintained the bacterial diversity. Short-term quicklime remediation of the acidified soils did not change the bacterial communities. Organic fertilization and fallow management supported eutrophic ecosystems, in which copiotrophic taxa increased in relative abundance and have a higher intensity of labile-C-degrading genes. However, long-term nitrogen-containing chemical fertilization treatments supported oligotrophic ecosystems, in which oligotrophic taxa increased in relative abundance and have a higher intensity of recalcitrant-C-degrading genes but a lower intensity of labile-C-degrading genes. Quicklime application increased the relative abundance of copiotrophic taxa and crop production, although these effects were utterly inadequate. This study provides insights into the interaction of soil bacterial communities, soil functionality and long-term fertilization management in the red soil ecosystem; these insights are important for improving the fertility of unique low-productivity red soil.

  12. Altered expression of immune-related genes in children with Down syndrome.

    Directory of Open Access Journals (Sweden)

    Bruna Lancia Zampieri

    Full Text Available Individuals with Down syndrome (DS have a high incidence of immunological alterations with increased susceptibility to bacterial and viral infections and high frequency of different types of hematologic malignancies and autoimmune disorders. In the current study, we profiled the expression pattern of 92 immune-related genes in peripheral blood mononuclear cells (PBMCs of two different groups, children with DS and control children, to identify differentially expressed genes that might be of pathogenetic importance for the development and phenotype of the immunological alterations observed in individuals with DS. PBMCs samples were obtained from six DS individuals with karyotypically confirmed full trisomy 21 and six healthy control individuals (ages 2-6 years. Gene expression was profiled in duplicate according to the manufacturer's instructions provided by commercially available TaqMan Human Immune Array representing 92 immune function genes and four reference genes on a 96-plex gene card. A set of 17 differentially expressed genes, not located on chromosome 21 (HSA21, involved in immune and inflammatory pathways was identified including 13 genes (BCL2, CCL3, CCR7, CD19, CD28, CD40, CD40LG, CD80, EDN1, IKBKB, IL6, NOS2 and SKI significantly down-regulated and four genes (BCL2L1, CCR2, CCR5 and IL10 significantly up-regulated in children with DS. These findings highlight a list of candidate genes for further investigation into the molecular mechanism underlying DS pathology and reinforce the secondary effects of the presence of a third copy of HSA21.

  13. Soil fungal:bacterial ratios are linked to altered carbon cycling

    Directory of Open Access Journals (Sweden)

    Ashish A. Malik

    2016-08-01

    Full Text Available Despite several lines of observational evidence, there is a lack of consensus on whether higher fungal:bacterial (F:B ratios directly cause higher soil carbon (C storage. We employed RNA sequencing, protein profiling and isotope tracer techniques to evaluate whether differing F:B ratios are associated with differences in C storage. A mesocosm 13C labeled foliar litter decomposition experiment was performed in two soils that were similar in their physico-chemical properties but differed in microbial community structure, specifically their F:B ratio (determined by PLFA analyses, RNA sequencing and protein profiling; all three corroborating each other. Following litter addition, we observed a consistent increase in abundance of fungal phyla; and greater increases in the fungal dominated soil; implicating the role of fungi in litter decomposition. Litter derived 13C in respired CO2 was consistently lower, and residual 13C in bulk SOM was higher in high F:B soil demonstrating greater C storage potential in the fungal:bacterial dominated soil. We conclude that in this soil system, the increased abundance of fungi in both soils and the altered C cycling patterns in the fungal:bacterial dominated soils highlight the significant role of fungi in litter decomposition and indicate that F:B ratios are linked to higher C storage potential.

  14. Bioaugmentation of Hydrogenispora ethanolica LX-B affects hydrogen production through altering indigenous bacterial community structure.

    Science.gov (United States)

    Yang, Zhiman; Guo, Rongbo; Shi, Xiaoshuang; He, Shuai; Wang, Lin; Dai, Meng; Qiu, Yanling; Dang, Xiaoxiao

    2016-07-01

    Bioaugmentation can facilitate hydrogen production from complex organic substrates, but it still is unknown how indigenous microbial communities respond to the added bacteria. Here, using a Hydrogenispora ethanolica LX-B (named as LX-B) bioaugmentation experiments, the distribution of metabolites and the responses of indigenous bacterial communities were investigated via batch cultivation (BC) and repeated batch cultivation (RBC). In BC the LX-B/sludge ratio of 0.12 achieved substantial high hydrogen yield, which was over twice that of control. In RBC one-time bioaugmentation and repeated batch bioaugmentation of LX-B resulted in the hydrogen yield that was average 1.2-fold and 0.8-fold higher than that in control, respectively. This improved hydrogen production performance mainly benefited from a shift in composition of the indigenous bacterial community caused by LX-B bioaugmentation. The findings represented an important step in understanding the relationship between bioaugmentation, a shift in bacterial communities, and altered bioreactor performance.

  15. Transport of magnesium by a bacterial Nramp-related gene.

    Directory of Open Access Journals (Sweden)

    Jung-Ho Shin

    2014-06-01

    Full Text Available Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5-2.0 mM. Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.

  16. Evolvability and hierarchy in rewired bacterial gene networks

    Science.gov (United States)

    Isalan, Mark; Lemerle, Caroline; Michalodimitrakis, Konstantinos; Beltrao, Pedro; Horn, Carsten; Raineri, Emanuele; Garriga-Canut, Mireia; Serrano, Luis

    2009-01-01

    Sequencing DNA from several organisms has revealed that duplication and drift of existing genes have primarily molded the contents of a given genome. Though the effect of knocking out or over-expressing a particular gene has been studied in many organisms, no study has systematically explored the effect of adding new links in a biological network. To explore network evolvability, we constructed 598 recombinations of promoters (including regulatory regions) with different transcription or σ-factor genes in Escherichia coli, added over a wild-type genetic background. Here we show that ~95% of new networks are tolerated by the bacteria, that very few alter growth, and that expression level correlates with factor position in the wild-type network hierarchy. Most importantly, we find that certain networks consistently survive over the wild-type under various selection pressures. Therefore new links in the network are rarely a barrier for evolution and can even confer a fitness advantage. PMID:18421347

  17. Five-week dietary exposure to dry diets alters the faecal bacterial populations in the domestic cat (Felis catus).

    Science.gov (United States)

    Bermingham, Emma N; Kittelmann, Sandra; Henderson, Gemma; Young, Wayne; Roy, Nicole C; Thomas, David G

    2011-10-01

    The effects of wet (canned) or dry (kibbled) diets on faecal bacterial populations in the cat were investigated in eight domestic short-haired cats (four males and four females; averaging 6 years of age and 3.4 kg) in a nested design. The cats were fed ad libitum a commercially available wet diet (moisture 82.0 %, crude protein 51.7 %, fat 28.9 %, carbohydrate (CHO) 8.9 % and ash 10.6 % DM) for 5 weeks. On the fifth week, individual feed intakes and faecal outputs were determined. Fresh faecal samples were collected twice daily, mixed for homogeneity, subsampled and stored at - 85 °C until analysis. The cats were then switched to a commercially available dry diet (moisture 8.5 %, crude protein 33.0 %, fat 11.0 %, CHO 49.4 % and ash 6.6 % DM) for 5 weeks, and fresh faeces were sampled as described previously. Energy intake tended to be higher in cats fed dry diets (P 0.05). Denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA genes amplified from DNA extracted from faeces was performed. The unweighted pair group method with arithmetic mean cluster analysis of bacterial community profiles using Pearson's correlation revealed diet-specific clustering when the same cats were fed on either a dry or a wet diet (dissimilarity between the groups, 88.6 %; P Pelomonas and Fusobacteriaceae were influenced by a short-term change in diet format. This suggests that 5-week dietary exposure is sufficient to alter gastrointestinal microflora.

  18. Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

    Science.gov (United States)

    Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  19. Mating alters gene expression patterns in Drosophila melanogaster male heads

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    Ellis Lisa L

    2010-10-01

    Full Text Available Abstract Background Behavior is a complex process resulting from the integration of genetic and environmental information. Drosophila melanogaster rely on multiple sensory modalities for reproductive success, and mating causes physiological changes in both sexes that affect reproductive output or behavior. Some of these effects are likely mediated by changes in gene expression. Courtship and mating alter female transcript profiles, but it is not known how mating affects male gene expression. Results We used Drosophila genome arrays to identify changes in gene expression profiles that occur in mated male heads. Forty-seven genes differed between mated and control heads 2 hrs post mating. Many mating-responsive genes are highly expressed in non-neural head tissues, including an adipose tissue called the fat body. One fat body-enriched gene, female-specific independent of transformer (fit, is a downstream target of the somatic sex-determination hierarchy, a genetic pathway that regulates Drosophila reproductive behaviors as well as expression of some fat-expressed genes; three other mating-responsive loci are also downstream components of this pathway. Another mating-responsive gene expressed in fat, Juvenile hormone esterase (Jhe, is necessary for robust male courtship behavior and mating success. Conclusions Our study demonstrates that mating causes changes in male head gene expression profiles and supports an increasing body of work implicating adipose signaling in behavior modulation. Since several mating-induced genes are sex-determination hierarchy target genes, additional mating-responsive loci may be downstream components of this pathway as well.

  20. Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

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    Cora S. Thiel

    2015-01-01

    Full Text Available Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes” are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1 which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  1. In vitro maturation alters gene expression in bovine oocytes.

    Science.gov (United States)

    Adona, Paulo R; Leal, Cláudia L V; Biase, Fernando H; De Bem, Tiago H; Mesquita, Lígia G; Meirelles, Flávio V; Ferraz, André L; Furlan, Luiz R; Monzani, Paulo S; Guemra, Samuel

    2016-08-01

    Gene expression profiling of in vivo- and in vitro-matured bovine oocytes can identify transcripts related to the developmental potential of oocytes. Nonetheless, the effects of in vitro culturing oocytes are yet to be fully understood. We tested the effects of in vitro maturation on the transcript profile of oocytes collected from Bos taurus indicus cows. We quantified the expression of 1488 genes in in vivo- and in vitro-matured oocytes. Of these, 51 genes were up-regulated, whereas 56 were down-regulated (≥2-fold) in in vivo-matured oocytes in comparison with in vitro-matured oocytes. Quantitative real-time polymerase chain reaction (PCR) of nine genes confirmed the microarray results of differential expression between in vivo- and in vitro-matured oocytes (EZR, EPN1, PSEN2, FST, IGFBP3, RBBP4, STAT3, FDPS and IRS1). We interrogated the results for enrichment of Gene Ontology categories and overlap with protein-protein interactions. The results revealed that the genes altered by in vitro maturation are mostly related to the regulation of oocyte metabolism. Additionally, analysis of protein-protein interactions uncovered two regulatory networks affected by the in vitro culture system. We propose that the differentially expressed genes are candidates for biomarkers of oocyte competence. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence.

  2. Wheat and Rice Growth Stages and Fertilization Regimes Alter Soil Bacterial Community Structure, But Not Diversity.

    Science.gov (United States)

    Wang, Jichen; Xue, Chao; Song, Yang; Wang, Lei; Huang, Qiwei; Shen, Qirong

    2016-01-01

    Maintaining soil fertility and the microbial communities that determine fertility is critical to sustainable agricultural strategies, and the use of different organic fertilizer (OF) regimes represents an important practice in attempts to preserve soil quality. However, little is known about the dynamic response of bacterial communities to fertilization regimes across crop growth stages. In this study, we examined microbial community structure and diversity across eight representative growth stages of wheat-rice rotation under four different fertilization treatments: no nitrogen fertilizer (NNF), chemical fertilizer (CF), organic-inorganic mixed fertilizer (OIMF), and OF. Quantitative PCR (QPCR) and high-throughput sequencing of bacterial 16S rRNA gene fragments revealed that growth stage as the best predictor of bacterial community abundance and structure. Additionally, bacterial community compositions differed between wheat and rice rotations. Relative to soils under wheat rotation, soils under rice rotation contained higher relative abundances (RA) of anaerobic and mesophilic microbes and lower RA of aerophilic microbes. With respect to fertilization regime, NNF plots had a higher abundance of nitrogen-fixing Cyanobacteria. OIMF had a lower abundance of ammonia-oxidizing Thaumarchaeota compared with CF. Application of chemical fertilizers (CF and OIMF treatments) significantly increased the abundance of some generally oligotrophic bacteria such those belonging to the Acidobacteria, while more copiotrophic of the phylum Proteobacteria increased with OF application. A high correlation coefficient was found when comparing RA of Acidobacteria based upon QPCR vs. sequence analysis, yet poor correlations were found for the α- and β- Proteobacteria, highlighting the caution required when interpreting these molecular data. In total, crop, fertilization scheme and plant developmental stage all influenced soil microbial community structure, but not total levels of alpha

  3. Wheat and Rice Growth Stages and Fertilization Regimes Alter Soil Bacterial Community Structure, but Not Diversity

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    Jichen Wang

    2016-08-01

    Full Text Available Maintaining soil fertility and the microbial communities that determine fertility is critical to sustainable agricultural strategies, and the use of different organic fertilizer regimes represents an important practice in attempts to preserve soil quality. However, little is known about the dynamic response of bacterial communities to fertilization regimes across crop growth stages. In this study, we examined microbial community structure and diversity across eight representative growth stages of wheat-rice rotation under four different fertilization treatments: no nitrogen fertilizer (NNF, chemical fertilizer (CF, organic-inorganic mixed fertilizer (OIMF and organic fertilizer (OF. Quantitative PCR (QPCR and high-throughput sequencing of bacterial 16S rRNA gene fragments revealed that growth stage as the best predictor of bacterial community abundance and structure. Additionally, bacterial community compositions differed between wheat and rice rotations. Relative to soils under wheat rotation, soils under rice rotation contained higher relative abundances (RA of anaerobic and mesophilic microbes and lower RA of aerophilic microbes. With respect to fertilization regime, NNF plots had a higher abundance of nitrogen–fixing Cyanobacteria. OIMF had a lower abundance of ammonia-oxidizing Thaumarchaeota compared with CF. Application of chemical fertilizers (CF and OIMF treatments significantly increased the abundance of some generally oligotrophic bacteria such those belonging to the Acidobacteria, while more copiotrophic of the phylum Proteobacteria increased with organic fertilizer application. A high correlation coefficient was found when comparing RA of Acidobacteria based upon QPCR versus sequence analysis, yet poor correlations were found for the Alpha- and Beta- Proteobacteria, highlighting the caution required when interpreting these molecular data. In total, crop, fertilization scheme and plant developmental stage all influenced soil

  4. Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering Gene Expression in Circulating Leukocytes.

    Science.gov (United States)

    Barr, Tasha; Girke, Thomas; Sureshchandra, Suhas; Nguyen, Christina; Grant, Kathleen; Messaoudi, Ilhem

    2016-01-01

    Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections, whereas moderate alcohol consumption decreases the incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations > 80 mg/dl) suppressed, whereas moderate alcohol consumption (blood ethanol concentrations consumption. To uncover the molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post-modified vaccinia Ankara vaccination, the earliest time point at which we detected differences in T cell and Ab responses. Overall, chronic heavy alcohol consumption reduced the expression of immune genes involved in response to infection and wound healing and increased the expression of genes associated with the development of lung inflammatory disease and cancer. In contrast, chronic moderate alcohol consumption upregulated the expression of genes involved in immune response and reduced the expression of genes involved in cancer. To uncover mechanisms underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate the expression of mRNAs differentially expressed in our data set.

  5. Mechanisms of post-transcriptional gene regulation in bacterial biofilms

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    Viveka eVadyvaloo

    2014-03-01

    Full Text Available Abstract Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria.

  6. Exposure to bacterial signals does not alter pea aphids' survival upon a second challenge or investment in production of winged offspring.

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    Bas ter Braak

    Full Text Available Pea aphids have an obligate nutritional symbiosis with the bacteria Buchneraaphidicola and frequently also harbor one or more facultative symbionts. Aphids are also susceptible to bacterial pathogen infections, and it has been suggested that aphids have a limited immune response towards such pathogen infections compared to other, more well-studied insects. However, aphids do possess at least some of the genes known to be involved in bacterial immune responses in other insects, and immune-competent hemocytes. One possibility is that immune priming with microbial elicitors could stimulate immune protection against subsequent bacterial infections, as has been observed in several other insect systems. To address this hypothesis we challenged aphids with bacterial immune elicitors twenty-four hours prior to live bacterial pathogen infections and then compared their survival rates to aphids that were not pre-exposed to bacterial signals. Using two aphid genotypes, we found no evidence for immune protection conferred by immune priming during infections with either Serratia marcescens or with Escherichia coli. Immune priming was not altered by the presence of facultative, beneficial symbionts in the aphids. In the absence of inducible immune protection, aphids may allocate energy towards other defense traits, including production of offspring with wings that could escape deteriorating conditions. To test this, we monitored the ratio of winged to unwinged offspring produced by adult mothers of a single clone that had been exposed to bacterial immune elicitors, to live E. coli infections or to no challenge. We found no correlation between immune challenge and winged offspring production, suggesting that this mechanism of defense, which functions upon exposure to fungal pathogens, is not central to aphid responses to bacterial infections.

  7. Mathematical modeling of bacterial track-altering motors: Track cleaving through burnt-bridge ratchets

    Science.gov (United States)

    Shtylla, Blerta; Keener, James P.

    2015-04-01

    The generation of directed movement of cellular components frequently requires the rectification of Brownian motion. Molecular motor enzymes that use ATP to walk on filamentous tracks are typically involved in cell transport, however, a track-altering motor can arise when an enzyme interacts with and alters its track. In Caulobacter crescentus and other bacteria, an active DNA partitioning (Par) apparatus is employed to segregate replicated chromosome regions to specific locations in dividing cells. The Par apparatus is composed of two proteins: ParA, an ATPase that can form polymeric structures on the nucleoid, and ParB, a protein that can bind and destabilize ParA structures. It has been proposed that the ParB-mediated alteration of ParA structures could be responsible for generating the directed movement of DNA during bacterial division. How precisely these actions are coordinated and translated into directed movement is not clear. In this paper we consider the C. crescentus segregation apparatus as an example of a track altering motor that operates using a so-called burnt-bridge mechanism. We develop and analyze mathematical models that examine how diffusion and ATP-hydrolysis-mediated monomer removal (or cleaving) can be combined to generate directed movement. Using a mean first passage approach, we analytically calculate the effective ParA track-cleaving velocities, effective diffusion coefficient, and other higher moments for the movement a ParB protein cluster that breaks monomers away at random locations on a single ParA track. Our model results indicate that cleaving velocities and effective diffusion constants are sensitive to ParB-induced ATP hydrolysis rates. Our analytical results are in excellent agreement with stochastic simulation results.

  8. Soil bacterial community composition altered by increased nutrient availability in Arctic tundra soils

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    Akihiro eKoyama

    2014-10-01

    Full Text Available The pool of soil organic carbon (SOC in the Arctic is disproportionally large compared to those in other biomes. This large quantity of SOC accumulated over millennia due to slow rates of decomposition relative to net primary productivity. Decomposition is constrained by low temperatures and nutrient concentrations, which limit soil microbial activity. We investigated how nutrients limit bacterial and fungal biomass and community composition in organic and mineral soils within moist acidic tussock tundra ecosystems. We sampled two experimental arrays of moist acidic tussock tundra that included fertilized and non-fertilized control plots. One array included plots that had been fertilized annually since 1989 and the other since 2006. Fertilization significantly altered overall bacterial community composition and reduced evenness, to a greater degree in organic than mineral soils, and in the 1989 compared to the 2006 site. The relative abundance of copiotrophic α-proteobacteria and β-proteobacteria was higher in fertilized than control soils, and oligotrophic Acidobacteria were less abundant in fertilized than control soils at the 1989 site. Fungal community composition was less sensitive to increased nutrient availability, and fungal responses to fertilization were not consistent between soil horizons and sites. We detected two ectomycorrhizal genera, Russula and Cortinarius spp., associated with shrubs. Their relative abundance was not affected by fertilization despite increased dominance of their host plants in the fertilized plots. Our results indicate that fertilization, which has been commonly used to simulate warming in Arctic tundra, has limited applicability for investigating fungal dynamics under warming.

  9. Canine Mammary Carcinomas: A Comparative Analysis of Altered Gene Expression

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    Farruk M. Lutful Kabir

    2015-12-01

    Full Text Available Breast cancer represents the second most frequent neoplasm in humans and sexually intact female dogs after lung and skin cancers, respectively. Many similar features in human and dog cancers including, spontaneous development, clinical presentation, tumor heterogeneity, disease progression and response to conventional therapies have supported development of this comparative model as an alternative to mice. The highly conserved similarities between canine and human genomes are also key to this comparative analysis, especially when compared to the murine genome. Studies with canine mammary tumor (CMT models have shown a strong genetic correlation with their human counterparts, particularly in terms of altered expression profiles of cell cycle regulatory genes, tumor suppressor and oncogenes and also a large group of non-coding RNAs or microRNAs (miRNAs. Because CMTs are considered predictive intermediate models for human breast cancer, similarities in genetic alterations and cancer predisposition between humans and dogs have raised further interest. Many cancer-associated genetic defects critical to mammary tumor development and oncogenic determinants of metastasis have been reported and appear to be similar in both species. Comparative analysis of deregulated gene sets or cancer signaling pathways has shown that a significant proportion of orthologous genes are comparably up- or down-regulated in both human and dog breast tumors. Particularly, a group of cell cycle regulators called cyclin-dependent kinase inhibitors (CKIs acting as potent tumor suppressors are frequently defective in CMTs. Interestingly, comparative analysis of coding sequences has also shown that these genes are highly conserved in mammals in terms of their evolutionary divergence from a common ancestor. Moreover, co-deletion and/or homozygous loss of the INK4A/ARF/INK4B (CDKN2A/B locus, encoding three members of the CKI tumor suppressor gene families (p16/INK4A, p14ARF and p15

  10. Genes but not genomes reveal bacterial domestication of Lactococcus lactis.

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    Delphine Passerini

    Full Text Available BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE. METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable

  11. Interplay of gene expression noise and ultrasensitive dynamics affects bacterial operon organization.

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    J Christian J Ray

    Full Text Available Bacterial chromosomes are organized into polycistronic cotranscribed operons, but the evolutionary pressures maintaining them are unclear. We hypothesized that operons alter gene expression noise characteristics, resulting in selection for or against maintaining operons depending on network architecture. Mathematical models for 6 functional classes of network modules showed that three classes exhibited decreased noise and 3 exhibited increased noise with same-operon cotranscription of interacting proteins. Noise reduction was often associated with a decreased chance of reaching an ultrasensitive threshold. Stochastic simulations of the lac operon demonstrated that the predicted effects of transcriptional coupling hold for a complex network module. We employed bioinformatic analysis to find overrepresentation of noise-minimizing operon organization compared with randomized controls. Among constitutively expressed physically interacting protein pairs, higher coupling frequencies appeared at lower expression levels, where noise effects are expected to be dominant. Our results thereby suggest an important role for gene expression noise, in many cases interacting with an ultrasensitive switch, in maintaining or selecting for operons in bacterial chromosomes.

  12. Gene Expression Profiling of Biological Pathway Alterations by Radiation Exposure

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    Kuei-Fang Lee

    2014-01-01

    Full Text Available Though damage caused by radiation has been the focus of rigorous research, the mechanisms through which radiation exerts harmful effects on cells are complex and not well-understood. In particular, the influence of low dose radiation exposure on the regulation of genes and pathways remains unclear. In an attempt to investigate the molecular alterations induced by varying doses of radiation, a genome-wide expression analysis was conducted. Peripheral blood mononuclear cells were collected from five participants and each sample was subjected to 0.5 Gy, 1 Gy, 2.5 Gy, and 5 Gy of cobalt 60 radiation, followed by array-based expression profiling. Gene set enrichment analysis indicated that the immune system and cancer development pathways appeared to be the major affected targets by radiation exposure. Therefore, 1 Gy radioactive exposure seemed to be a critical threshold dosage. In fact, after 1 Gy radiation exposure, expression levels of several genes including FADD, TNFRSF10B, TNFRSF8, TNFRSF10A, TNFSF10, TNFSF8, CASP1, and CASP4 that are associated with carcinogenesis and metabolic disorders showed significant alterations. Our results suggest that exposure to low-dose radiation may elicit changes in metabolic and immune pathways, potentially increasing the risk of immune dysfunctions and metabolic disorders.

  13. A new experimental approach for studying bacterial genomic island evolution identifies island genes with bacterial host-specific expression patterns

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    Nickerson Cheryl A

    2006-01-01

    Full Text Available Abstract Background Genomic islands are regions of bacterial genomes that have been acquired by horizontal transfer and often contain blocks of genes that function together for specific processes. Recently, it has become clear that the impact of genomic islands on the evolution of different bacterial species is significant and represents a major force in establishing bacterial genomic variation. However, the study of genomic island evolution has been mostly performed at the sequence level using computer software or hybridization analysis to compare different bacterial genomic sequences. We describe here a novel experimental approach to study the evolution of species-specific bacterial genomic islands that identifies island genes that have evolved in such a way that they are differentially-expressed depending on the bacterial host background into which they are transferred. Results We demonstrate this approach by using a "test" genomic island that we have cloned from the Salmonella typhimurium genome (island 4305 and transferred to a range of Gram negative bacterial hosts of differing evolutionary relationships to S. typhimurium. Systematic analysis of the expression of the island genes in the different hosts compared to proper controls allowed identification of genes with genera-specific expression patterns. The data from the analysis can be arranged in a matrix to give an expression "array" of the island genes in the different bacterial backgrounds. A conserved 19-bp DNA site was found upstream of at least two of the differentially-expressed island genes. To our knowledge, this is the first systematic analysis of horizontally-transferred genomic island gene expression in a broad range of Gram negative hosts. We also present evidence in this study that the IS200 element found in island 4305 in S. typhimurium strain LT2 was inserted after the island had already been acquired by the S. typhimurium lineage and that this element is likely not

  14. Transcriptome-Level Signatures in Gene Expression and Gene Expression Variability during Bacterial Adaptive Evolution

    Science.gov (United States)

    Erickson, Keesha E.; Otoupal, Peter B.

    2017-01-01

    ABSTRACT Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation. Here, we research the regulation of adaptive resistance by investigating transcriptome profiles of Escherichia coli upon adaptation to disparate toxins, including antibiotics and biofuels. We locate potential target genes via conventional gene expression analysis as well as using a new analysis technique examining differential gene expression variability. By investigating trends across the diverse adaptation conditions, we identify a focused set of genes with conserved behavior, including those involved in cell motility, metabolism, membrane structure, and transport, and several genes of unknown function. To validate the biological relevance of the observed changes, we synthetically perturb gene expression using clustered regularly interspaced short palindromic repeat (CRISPR)-dCas9. Manipulation of select genes in combination with antibiotic treatment promotes adaptive resistance as demonstrated by an increased degree of antibiotic tolerance and heterogeneity in MICs. We study the mechanisms by which identified genes influence adaptation and find that select differentially variable genes have the potential to impact metabolic rates, mutation rates, and motility. Overall, this work provides evidence for a complex nongenetic response, encompassing shifts in gene expression and gene expression variability, which underlies adaptive resistance. IMPORTANCE Even initially sensitive bacteria can rapidly thwart antibiotic treatment

  15. Microbial taxa and functional genes shift in degraded soil with bacterial wilt

    Science.gov (United States)

    Zhang, Hongchun; Wang, Rui; Chen, Shu; Qi, Gaofu; He, Zhili; Zhao, Xiuyun

    2017-01-01

    Soil degradation is a serious global problem, but little is known about how soil microbial communities respond to soil degradation as well as their feedback to ecosystem functioning. In this study, we found the microbial community composition, structure and functional potential significantly altered in the degraded soils with bacterial wilt (termed as degraded soils). Compared with healthy soils, OTU richness of beneficial microorganisms were significantly decreased, but OTU richness of pathogenic microorganisms were significantly increased in the degraded soils. Functional gene array (GeoChip 5.0) analysis showed the functional metabolic potential of genes involved in stress, virulence, sulfur cycle, metal resistance, degradation of plant cell wall was significantly increased in the degraded soils. Increased functional metabolic potential of these genes may be related to the acidification and severe plant disease of degraded soils. Biological activity of degraded soils was obviously decreased with weakened soil enzyme activities when compared to the healthy soils. Soil pH and enzyme activities were negatively correlated with the abundance of genes involved in sulfur cycle, virulence, and stress responses. This study provides new insights into our understanding of soil microbial community responses to soil degradation. PMID:28051173

  16. Long term repeated fire disturbance alters soil bacterial diversity but not the abundance in an Australian wet sclerophyll forest.

    Science.gov (United States)

    Shen, Ju-pei; Chen, C R; Lewis, Tom

    2016-01-20

    Effects of fire on biogeochemical cycling in terrestrial ecosystem are widely acknowledged, while few studies have focused on the bacterial community under the disturbance of long-term frequent prescribed fire. In this study, three treatments (burning every two years (B2), burning every four years (B4) and no burning (B0)) were applied for 38 years in an Australian wet sclerophyll forest. Results showed that bacterial alpha diversity (i.e. bacterial OTU) in the top soil (0-10 cm) was significantly higher in the B2 treatment compared with the B0 and B4 treatments. Non-metric multidimensional analysis (NMDS) of bacterial community showed clear separation of the soil bacterial community structure among different fire frequency regimes and between the depths. Different frequency fire did not have a substantial effect on bacterial composition at phylum level or bacterial 16S rRNA gene abundance. Soil pH and C:N ratio were the major drivers for bacterial community structure in the most frequent fire treatment (B2), while other factors (EC, DOC, DON, MBC, NH4(+), TC and TN) were significant in the less frequent burning and no burning treatments (B4 and B0). This study suggested that burning had a dramatic impact on bacterial diversity but not abundance with more frequent fire.

  17. Long term repeated fire disturbance alters soil bacterial diversity but not the abundance in an Australian wet sclerophyll forest

    Science.gov (United States)

    Shen, Ju-Pei; Chen, C. R.; Lewis, Tom

    2016-01-01

    Effects of fire on biogeochemical cycling in terrestrial ecosystem are widely acknowledged, while few studies have focused on the bacterial community under the disturbance of long-term frequent prescribed fire. In this study, three treatments (burning every two years (B2), burning every four years (B4) and no burning (B0)) were applied for 38 years in an Australian wet sclerophyll forest. Results showed that bacterial alpha diversity (i.e. bacterial OTU) in the top soil (0–10 cm) was significantly higher in the B2 treatment compared with the B0 and B4 treatments. Non-metric multidimensional analysis (NMDS) of bacterial community showed clear separation of the soil bacterial community structure among different fire frequency regimes and between the depths. Different frequency fire did not have a substantial effect on bacterial composition at phylum level or bacterial 16S rRNA gene abundance. Soil pH and C:N ratio were the major drivers for bacterial community structure in the most frequent fire treatment (B2), while other factors (EC, DOC, DON, MBC, NH4+, TC and TN) were significant in the less frequent burning and no burning treatments (B4 and B0). This study suggested that burning had a dramatic impact on bacterial diversity but not abundance with more frequent fire.

  18. Altered Colonic Bacterial Fermentation as a Potential Pathophysiological Factor in Irritable Bowel Syndrome.

    Science.gov (United States)

    Ringel-Kulka, Tamar; Choi, Chang Hwan; Temas, Daniel; Kim, Ari; Maier, Daniele M; Scott, Karen; Galanko, Joseph A; Ringel, Yehuda

    2015-09-01

    Dysbiosis leading to abnormal intestinal fermentation has been suggested as a possible etiological mechanism in irritable bowel syndrome (IBS). We aimed to investigate the location and magnitude of altered intestinal bacterial fermentation in IBS and its clinical subtypes. IBS patients who satisfied the Rome III criteria (114) and 33 healthy controls (HC) were investigated. Intestinal fermentation was assessed using two surrogate measures: intestinal intraluminal pH and fecal short-chain fatty acids (SCFAs). Intraluminal pH and intestinal transit times were measured in the small and large bowel using a wireless motility capsule (SmartPill) in 47 IBS and 10 HC. Fecal SCFAs including acetate, propionate, butyrate, and lactate were analyzed by capillary gas chromatography in all enrolled subjects. Correlations between intestinal pH, fecal SCFAs, intestinal transit time, and IBS symptom scores were analyzed. Colonic intraluminal pH levels were significantly lower in IBS patients compared with HC (total colonic pH, 6.8 for IBS vs. 7.3 for HC, P=0.042). There were no differences in total and segmental pH levels in the small bowel between IBS patients and HC (6.8 vs. 6.8, P=not significant). The intraluminal colonic pH differences were consistent in all IBS subtypes. Total SCFA level was significantly lower in C-IBS patients than in D-IBS and M-IBS patients and HC. The total SCFA level in all IBS subjects was similar with that of HC. Colonic pH levels correlated positively with colon transit time (CTT) and IBS symptoms severity. Total fecal SCFAs levels correlated negatively with CTT and positively with stool frequency. Colonic intraluminal pH is decreased, suggesting higher colonic fermentation, in IBS patients compared with HC. Fecal SCFAs are not a sensitive marker to estimate intraluminal bacterial fermentation.

  19. Clinicopathologic implications of NF1 gene alterations in diffuse gliomas.

    Science.gov (United States)

    Vizcaíno, M Adelita; Shah, Smit; Eberhart, Charles G; Rodriguez, Fausto J

    2015-09-01

    Recent studies have identified somatic alterations in the gene encoding for neurofibromin (NF1) in a subset of glioblastoma (GBM), usually associated with the mesenchymal molecular subtype. To understand the significance of NF1 genetic alterations in diffuse gliomas in general, we evaluated public databases and tested for NF1 copy number alterations in a cohort using fluorescence in situ hybridization. NF1 genetic loss (homozygous NF1 deletions or mutations with predicted functional consequences) was present in 30 (of 281) (11%) GBM and 21 (of 286) (7%) lower-grade gliomas in The Cancer Genome Atlas data. Furthermore, NF1 loss was associated with worse overall and disease-specific survival in the lower-grade glioma, but not GBM, Group in The Cancer Genome Atlas cohort. IDH1 or 2 mutations co-existed in lower-grade gliomas with NF1 loss (36%) but not in GBM. In our cohort studied by fluorescence in situ hybridization, NF1/17q (n = 2) or whole Ch17 (n = 3) losses were only identified in the GBM group (5/86 [6%]). Tumors with NF1/Ch17 loss were predominantly adult GBM (4/5); lacked EGFR amplification (0/4), strong p53 immunolabeling (1/5), or IDH1 (R132H) protein expression (0/5); but expressed the mesenchymal marker podoplanin in 4/5. NF1 genetic loss occurs in a subset of diffuse gliomas, and its significance deserves further exploration.

  20. Clinicopathologic implications of NF1 gene alterations in diffuse gliomas☆

    Science.gov (United States)

    Vizcaíno, M. Adelita; Shah, Smit; Eberhart, Charles G.; Rodriguez, Fausto J.

    2015-01-01

    Summary Recent studies have identified somatic alterations in the gene encoding for neurofibromin (NF1) in a subset of glioblastoma (GBM), usually associated with the mesenchymal molecular subtype. To understand the significance of NF1 genetic alterations in diffuse gliomas in general, we evaluated public databases and tested for NF1 copy number alterations in a cohort using fluorescence in situ hybridization. NF1 genetic loss (homozygous NF1 deletions or mutations with predicted functional consequences) was present in 30 (of 281) (11%) GBM and 21 (of 286) (7%) lower-grade gliomas in The Cancer Genome Atlas data. Furthermore, NF1 loss was associated with worse overall and disease-specific survival in the lower-grade glioma, but not GBM, Group in The Cancer Genome Atlas cohort. IDH1 or 2 mutations co-existed in lower-grade gliomas with NF1 loss (36%) but not in GBM. In our cohort studied by fluorescence in situ hybridization, NF1/17q (n = 2) or whole Ch17 (n = 3) losses were only identified in the GBM group (5/86 [6%]). Tumors with NF1/Ch17 loss were predominantly adult GBM (4/5); lacked EGFR amplification (0/4), strong p53 immunolabeling (1/5), or IDH1 (R132H) protein expression (0/5); but expressed the mesenchymal marker podoplanin in 4/5. NF1 genetic loss occurs in a subset of diffuse gliomas, and its significance deserves further exploration. PMID:26190195

  1. Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    WEI-DONG JI; JIA-KUN CHEN; JIA-CHUN LU; ZHONG-LIANG WU; FEI YI; SU-MEI FENG

    2006-01-01

    To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immoral human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P<0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

  2. Altering the thermal resistance of foodborne bacterial pathogens with an eggshell membrane waste by-product.

    Science.gov (United States)

    Poland, A L; Sheldon, B W

    2001-04-01

    Eggshells from egg-breaking operations are a significant waste disposal problem. Thus, the development of value-added by-products from this waste would be welcomed by the industry. The ability of extracted eggshell membranes containing, several bacteriolytic enzymes (i.e., lysozyme and beta-N-acetylglucosaminidase) or other membrane components to alter the thermal resistance of gram-positive and gram-negative bacterial pathogens was evaluated. Mid-log phase cells of Salmonella Enteritidis (SE), Salmonella Typhimurium (ST), Escherichia coli O157:H7 (EC), Listeria monocytogenes Scott A (LM), and Staphylococcus aureus (SA) were suspended in 100 ml of 0.1% peptone water (pH 6.9, 10(7-8) CFU/ml) containing either 0 (control) or 10 g of an eggshell membrane extract and incubated at 37 degrees C for 45 min. Following exposure, membrane-free samples (1.5 ml) were heated in a 56 degrees C (LM, SA), 54 degrees C (SE, ST), or 52 degrees C (EC) water bath from 0 to 14 min in sealed glass reaction vials (12 by 32 mm), and the survivors were recovered on brain heart infusion agar. Population reductions ranging from 27.6% (SA) to 99.8% (LM) (ST, 43.8%; SE, 47.5%; EC, 71.8%) were observed for cells treated for 45 min with extracted membrane, as compared to controls. D-value reductions ranging from 0 (LM) to 87.2% (SE) (SA, 36.7%; EC, 83.3%; ST, 86.3%) were observed when membrane-treated cells were subsequently heat inactivated. The effects of exposure pH, time, temperature, and organic load on membrane activity were also evaluated with Salmonella Typhimurium. Exposure pH (5.0 versus 6.9), time (15 versus 45 min), and temperature (4 degrees C versus 37 degrees C) did not significantly reduce the impact of eggshell membranes on D-values. However, the presence of organic matter (0.1% peptone water versus skim milk) significantly reduced the thermal resistance-reducing capacity of the membranes. These preliminary findings provide information on the potential use of extracted eggshell

  3. Blood gene expression profiles suggest altered immune function associated with symptoms of generalized anxiety disorder.

    Science.gov (United States)

    Wingo, Aliza P; Gibson, Greg

    2015-01-01

    Prospective epidemiological studies found that generalized anxiety disorder (GAD) can impair immune function and increase risk for cardiovascular disease or events. Mechanisms underlying the physiological reverberations of anxiety, however, are still elusive. Hence, we aimed to investigate molecular processes mediating effects of anxiety on physical health using blood gene expression profiles of 336 community participants (157 anxious and 179 control). We examined genome-wide differential gene expression in anxiety, as well as associations between nine major modules of co-regulated transcripts in blood gene expression and anxiety. No significant differential expression was observed in women, but 631 genes were differentially expressed between anxious and control men at the false discovery rate of 0.1 after controlling for age, body mass index, race, and batch effect. Gene set enrichment analysis (GSEA) revealed that genes with altered expression levels in anxious men were involved in response of various immune cells to vaccination and to acute viral and bacterial infection, and in a metabolic network affecting traits of metabolic syndrome. Further, we found one set of 260 co-regulated genes to be significantly associated with anxiety in men after controlling for the relevant covariates, and demonstrate its equivalence to a component of the stress-related conserved transcriptional response to adversity profile. Taken together, our results suggest potential molecular pathways that can explain negative effects of GAD observed in epidemiological studies. Remarkably, even mild anxiety, which most of our participants had, was associated with observable changes in immune-related gene expression levels. Our findings generate hypotheses and provide incremental insights into molecular mechanisms mediating negative physiological effects of GAD.

  4. Primary study on the resistance to bacterial blight (X. oryzae) in Cecropin B gene transgenic rices

    Institute of Scientific and Technical Information of China (English)

    HUAZhihua; HUANGDanian; XUERui; WANGXiaoling; GAOZhenya

    1998-01-01

    Bacterial blight (BB) is one of the major diseases to rice. Antibacterial Cecropin B gene has been cloned and transformed into rice. We studied the resistance to bacterial blight in Cecropin B gene tronsgenic rices.

  5. AI-2 biosynthesis module in a magnetic nanofactory alters bacterial response via localized synthesis and delivery.

    Science.gov (United States)

    Fernandes, Rohan; Bentley, William E

    2009-02-01

    Nanofactories are nano-dimensioned and comprised of modules serving various functions that alter the response of targeted cells when deployed by locally synthesizing and delivering cargo to the surfaces of the targeted cells. In its basic form, a nanofactory consists of a minimum of two functional modules: a cell capture module and a synthesis module. In this work, magnetic nanofactories that alter the response of targeted bacteria by the localized synthesis and delivery of the "universal" bacterial quorum sensing signal molecule autoinducer AI-2 are demonstrated. The magnetic nanofactories consist of a cell capture module (chitosan-mag nanoparticles) and an AI-2 biosynthesis module that contains both AI-2 biosynthetic enzymes Pfs and LuxS on a fusion protein (His-LuxS-Pfs-Tyr, HLPT) assembled together. HLPT is hypothesized to be more efficient than its constituent enzymes (used separately) at conversion of the substrate SAH to product AI-2 on account of the proximity of the two enzymes within the fusion protein. HLPT is demonstrated to be more active than the constituent enzymes, Pfs and LuxS, over a wide range of experimental conditions. The magnetic nanofactories (containing bound HLPT) are also demonstrated to be more active than free, unbound HLPT. They are also shown to elicit an increased response in targeted Escherichia coli cells, due to the localized synthesis and delivery of AI-2, when compared to the response produced by the addition of AI-2 directly to the cells. Studies investigating the universality of AI-2 and unraveling AI-2 based quorum sensing in bacteria using magnetic nanofactories are envisioned. The prospects of using such multi-modular nanofactories in developing the next generation of antimicrobials based on intercepting and interrupting quorum sensing based signaling are discussed.

  6. Altered choroid plexus gene expression in major depressive disorder

    Directory of Open Access Journals (Sweden)

    Cortney Ann Turner

    2014-04-01

    Full Text Available Given the emergent interest in biomarkers for mood disorders, we assessed gene expression in the choroid plexus, the region that produces cerebrospinal fluid (CSF, in individuals with major depressive disorder (MDD. Genes that are expressed in the choroid plexus (CP can be secreted into the CSF and may be potential biomarker candidates. Given that we have previously shown that fibroblast growth factor family members are differentially expressed in post-mortem brain of subjects with MDD and the CP is a known source of growth factors in the brain, we posed the question whether growth factor dysregulation would be found in the CP of subjects with MDD. We performed laser capture microscopy of the choroid plexus at the level of the hippocampus in subjects with MDD and psychiatrically normal controls. We then extracted, amplified, labeled and hybridized the cRNA to Illumina BeadChips to assess gene expression. In controls, the most highly abundant known transcript was transthyretin. Moreover, half of the 14 most highly expressed transcripts in controls encode ribosomal proteins. Using BeadStudio software, we identified 169 transcripts differentially expressed (p< 0.05 between control and MDD samples. Using pathway analysis we noted that the top network altered in subjects with MDD included multiple members of the transforming growth factor-beta (TGFβ pathway. Quantitative real-time PCR (qRT-PCR confirmed downregulation of several transcripts that interact with the extracellular matrix in subjects with MDD. These results suggest that there may be an altered cytoskeleton in the choroid plexus in MDD subjects that may lead to a disrupted blood-CSF-brain barrier.

  7. Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

    Science.gov (United States)

    Abuisneineh, Fida; Fahrenbach, John P.; Zhang, Yuan; MacLeod, Heather; Dellefave, Lisa; Pytel, Peter; Selig, Sara; Labno, Christine M.; Reddy, Karen; Singh, Harinder; McNally, Elizabeth

    2010-01-01

    Background Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression. Methods/Findings To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction. Conclusions These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered. PMID:21179469

  8. Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation.

    Directory of Open Access Journals (Sweden)

    Stephanie K Mewborn

    Full Text Available BACKGROUND: Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression. METHODS/FINDINGS: To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction. CONCLUSIONS: These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.

  9. Computational design of a Zn2+ receptor that controls bacterial gene expression

    Science.gov (United States)

    Dwyer, M. A.; Looger, L. L.; Hellinga, H. W.

    2003-09-01

    The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a -galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.

  10. Impact of lux gene insertion on bacterial surface properties and transport.

    Science.gov (United States)

    Chen, Gang; Srinivasa Ranga, Vijay Penagonda; Mao, Yongjun; Chen, Kevin; Qiao, Hanzi

    2008-03-01

    Genetic markers have been in popular use for tracing microbial movement in the environment. However, the impact of genetic marker insertion on microbial surface properties and consequent transport is often ignored. For this research, we investigated the impact of luminescence-based genetic marker insertion on bacterial surface properties and transport. Typical Gram-positive bacterial strains of Lactobacillus casei, Streptococcus mitis and Micrococcus luteus were used as model bacterial strains in this research. We manipulated gene transfer to observe the impact of lux gene insertion on bacterial surface properties based on contact angle measurements, and we conducted column experiments to evaluate the impact of lux gene insertion on bacterial transport. After lux gene insertion, bacterial interactions with the porous media increased, demonstrating stronger deposition potential in the porous media. Accordingly, retention of the daughter strains increased. Lux gene insertion also resulted in an increase in bacterial dispersion and equilibrium adsorption in the porous media. The bacterial deposition coefficient was found to correlate with the free energy of interactions between bacteria and the porous media.

  11. Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma

    Science.gov (United States)

    Singhania, Akul; Rupani, Hitasha; Jayasekera, Nivenka; Lumb, Simon; Hales, Paul; Gozzard, Neil; Davies, Donna E.

    2017-01-01

    Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy. PMID:28045928

  12. Altered gene expression in human placenta after suspected preterm labour.

    Science.gov (United States)

    Oros, D; Strunk, M; Breton, P; Paules, C; Benito, R; Moreno, E; Garcés, M; Godino, J; Schoorlemmer, J

    2017-07-01

    Suspected preterm labour occurs in around 9% of pregnancies. However, almost two-thirds of women admitted for threatened preterm labour ultimately deliver at term and are considered risk-free for fetal development. We examined placental and umbilical cord blood samples from preterm or term deliveries after threatened preterm labour as well as term deliveries without threatened preterm labour. We quantitatively analysed the mRNA expression of inflammatory markers (IL6, IFNγ, and TNFα) and modulators of angiogenesis (FGF2, PGF, VEGFA, VEGFB, and VEGFR1). A total of 132 deliveries were analysed. Preterm delivery and term delivery after suspected preterm labour groups showed similar increases in TNFα expression compared with the term delivery control group in umbilical cord blood samples. Placental samples from preterm and term deliveries after suspected preterm labour exhibited significantly increased expression of TNFα and IL6 and decreased expression of IFNγ. Suspected preterm labour was also associated with altered expression of angiogenic factors, although not all differences reached statistical significance. We found gene expression patterns indicative of inflammation in human placentas after suspected preterm labour regardless of whether the deliveries occurred preterm or at term. Similarly, a trend towards altered expression of angiogeneic factors was not limited to preterm birth. These findings suggest that the biological mechanisms underlying threatened preterm labour affect pregnancies independently of gestational age at birth. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  14. Bacterial genes in the aphid genome: absence of functional gene transfer from Buchnera to its host.

    Directory of Open Access Journals (Sweden)

    Naruo Nikoh

    2010-02-01

    Full Text Available Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria, which have highly reduced genomes (420-650 kb, raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD-carboxypeptidases (LdcA1, LdcA2,psiLdcA, five rare lipoprotein As (RlpA1-5, N-acetylmuramoyl-L-alanine amidase (AmiD, 1,4-beta-N-acetylmuramidase (bLys, DNA polymerase III alpha chain (psiDnaE, and ATP synthase delta chain (psiAtpH. Buchnera was the apparent source of two highly truncated pseudogenes (psiDnaE and psiAtpH. Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria. At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5 are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the

  15. Hard-Wired Control of Bacterial Processes by Chromosomal Gene Location

    NARCIS (Netherlands)

    Slager, Jelle; Veening, Jan-Willem

    2016-01-01

    Bacterial processes, such as stress responses and cell differentiation, are controlled at many different levels. While some factors, such as transcriptional regulation, are well appreciated, the importance of chromosomal gene location is often underestimated or even completely neglected. A

  16. Hard-Wired Control of Bacterial Processes by Chromosomal Gene Location

    NARCIS (Netherlands)

    Slager, Jelle; Veening, Jan-Willem

    2016-01-01

    Bacterial processes, such as stress responses and cell differentiation, are controlled at many different levels. While some factors, such as transcriptional regulation, are well appreciated, the importance of chromosomal gene location is often underestimated or even completely neglected. A combinati

  17. Evaluating bacterial gene-finding HMM structures as probabilistic logic programs

    DEFF Research Database (Denmark)

    Mørk, Søren; Holmes, Ian

    2012-01-01

    , a probabilistic dialect of Prolog. Results: We evaluate Hidden Markov Model structures for bacterial protein-coding gene potential, including a simple null model structure, three structures based on existing bacterial gene finders and two novel model structures. We test standard versions as well as ADPH length......Motivation: Probabilistic logic programming offers a powerful way to describe and evaluate structured statistical models. To investigate the practicality of probabilistic logic programming for structure learning in bioinformatics, we undertook a simplified bacterial gene-finding benchmark in PRISM...... modeling and three-state versions of the five model structures. The models are all represented as probabilistic logic programs and evaluated using the PRISM machine learning system in terms of statistical information criteria and gene-finding prediction accuracy, in two bacterial genomes. Neither of our...

  18. Molecular methods for bacterial genotyping and analyzed gene regions

    Directory of Open Access Journals (Sweden)

    İbrahim Halil Yıldırım1, Seval Cing Yıldırım2, Nadir Koçak3

    2011-06-01

    Full Text Available Bacterial strain typing is an important process for diagnosis, treatment and epidemiological investigations. Current bacterial strain typing methods may be classified into two main categories: phenotyping and genotyping. Phenotypic characters are the reflection of genetic contents. Genotyping, which refers discrimination of bacterial strains based on their genetic content, has recently become widely used for bacterial strain typing. The methods already used in genotypingof bacteria are quite different from each other. In this review we tried to summarize the basic principles of DNA-based methods used in genotyping of bacteria and describe some important DNA regions that are used in genotyping of bacteria. J Microbiol Infect Dis 2011;1(1:42-46.

  19. Phylogenetic and functional alterations in bacterial community compositions in broiler ceca as a result of mannan oligosaccharide supplementation.

    Science.gov (United States)

    Corrigan, A; de Leeuw, Marcel; Penaud-Frézet, Stéphanie; Dimova, Diliana; Murphy, R A

    2015-05-15

    This study focused on identifying reproducible effects of dietary supplementation with a mannan oligosaccharide (MOS) on the broiler cecal bacterial community structure and function in a commercial production setting. Two separate trials, each with a control and a supplemented group, were carried out in the same commercial location and run concurrently. Approximately 10,000 birds from the same commercial hatchery were mirror imaged into each of four commercial broiler sheds and fed either a control or supplemented diet. Cecal contents were obtained on days 7, 21, and 35 posthatch from 12 randomly caught broilers from each group. Bacterial pyrosequencing was performed on all samples, with approximately 250,000 sequences obtained per treatment per time point. The predominant phyla identified at all three time points in both trials were Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Tenericutes, representing >99% of all sequences. MOS supplementation altered the bacterial community composition from 7 days supplementation through 35 days supplementation. Bacteroidetes appeared to be replacing Firmicutes as a result of supplementation, with the most noticeable effects after 35 days. The effects of supplementation were reproducible across both trials. PICRUSt was used to identify differences between the functional potentials of the bacterial communities as a result of MOS supplementation. Using level 3 KEGG ortholog function predictions, differences between control and supplemented groups were observed, with very strong segregation noted on day 35 posthatch in both trials. This indicated that alterations of bacterial communities as a result of MOS are likely to alter the functional capability of the cecum. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Systematically Altering Bacterial SOS Activity under Stress Reveals Therapeutic Strategies for Potentiating Antibiotics.

    Science.gov (United States)

    Mo, Charlie Y; Manning, Sara A; Roggiani, Manuela; Culyba, Matthew J; Samuels, Amanda N; Sniegowski, Paul D; Goulian, Mark; Kohli, Rahul M

    2016-01-01

    The bacterial SOS response is a DNA damage repair network that is strongly implicated in both survival and acquired drug resistance under antimicrobial stress. The two SOS regulators, LexA and RecA, have therefore emerged as potential targets for adjuvant therapies aimed at combating resistance, although many open questions remain. For example, it is not well understood whether SOS hyperactivation is a viable therapeutic approach or whether LexA or RecA is a better target. Furthermore, it is important to determine which antimicrobials could serve as the best treatment partners with SOS-targeting adjuvants. Here we derived Escherichia coli strains that have mutations in either lexA or recA genes in order to cover the full spectrum of possible SOS activity levels. We then systematically analyzed a wide range of antimicrobials by comparing the mean inhibitory concentrations (MICs) and induced mutation rates for each drug-strain combination. We first show that significant changes in MICs are largely confined to DNA-damaging antibiotics, with strains containing a constitutively repressed SOS response impacted to a greater extent than hyperactivated strains. Second, antibiotic-induced mutation rates were suppressed when SOS activity was reduced, and this trend was observed across a wider spectrum of antibiotics. Finally, perturbing either LexA or RecA proved to be equally viable strategies for targeting the SOS response. Our work provides support for multiple adjuvant strategies, while also suggesting that the combination of an SOS inhibitor with a DNA-damaging antibiotic could offer the best potential for lowering MICs and decreasing acquired drug resistance. IMPORTANCE Our antibiotic arsenal is becoming depleted, in part, because bacteria have the ability to rapidly adapt and acquire resistance to our best agents. The SOS pathway, a widely conserved DNA damage stress response in bacteria, is activated by many antibiotics and has been shown to play central role in

  1. Short-term failure of IUD removal to alter bacterial flora in a patient with chronic anaerobic endometritis.

    Science.gov (United States)

    Monif, G R

    1986-10-01

    A patient with cytological and clinical evidence of IUD-associated anaerobic endometritis was studied with quantitative and qualitative bacteriological techniques. With the exception of elimination of Actinomyces israelii from the deep endocervical/endometrial culture, IUD removal induced minimal qualitative changes in the bacterial flora of the endocervical/endometrial sample during the 35 days the patient was monitored. The interposition of menstruation did not significantly alter either the quantitative or qualitative interrelationship of the bacteria present.

  2. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

    Directory of Open Access Journals (Sweden)

    Luthey-Schulten Zaida

    2009-07-01

    Full Text Available Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (HGT of S4 during bacterial evolution. Results In this study we present the complex evolutionary history of ribosomal protein S4 using 660 bacterial genomes from 16 major bacterial phyla. According to conserved characteristics in the sequences, S4 can be classified into C+ (zinc-binding and C- (zinc-free variants, with 26 genomes (mainly from the class Clostridia containing genes for both. A maximum likelihood phylogenetic tree of the S4 sequences was incongruent with the standard bacterial phylogeny, indicating a departure from strict vertical inheritance. Further analysis using the genome content near the S4 genes, which are usually located in a conserved gene cluster, showed not only that HGT of the C- gene had occurred at various stages of bacterial evolution, but also that both the C- and C+ genes were present before the individual phyla diverged. To explain the latter, we theorize that a gene pool existed early in bacterial evolution from which bacteria could sample S4 gene variants, according to environmental conditions. The distribution of the C+/- variants for seven other zinc-binding ribosomal proteins in these 660 bacterial genomes is consistent with that seen for S4 and may shed light on the evolutionary pressures involved. Conclusion The complex history presented for "core" protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution

  3. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    Science.gov (United States)

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  4. Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen

    KAUST Repository

    Ravasi, Timothy

    2016-01-24

    Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. Here we describe the application of RNA sequencing using samples obtained from in vitro infection assays to simultaneously quantify both host and bacterial pathogen gene expression changes, as well as general approaches that can be undertaken to interpret the RNA sequencing data that is generated. These methods can be used to provide insights into host TLR-regulated transcriptional responses to microbial challenge, as well as pathogen subversion mechanisms against such responses.

  5. Using bacterial genomes and essential genes for the development of new antibiotics.

    Science.gov (United States)

    Fields, Francisco R; Lee, Shaun W; McConnell, Michael J

    2017-06-15

    The shrinking antibiotic development pipeline together with the global increase in antibiotic resistant infections requires that new molecules with antimicrobial activity are developed. Traditional empirical screening approaches of natural and non-natural compounds have identified the majority of antibiotics that are currently available, however this approach has produced relatively few new antibiotics over the last few decades. The vast amount of bacterial genome sequence information that has become available since the sequencing of the first bacterial genome more than 20years ago holds potential for contributing to the discovery of novel antimicrobial compounds. Comparative genomic approaches can identify genes that are highly conserved within and between bacterial species, and thus may represent genes that participate in key bacterial processes. Whole genome mutagenesis studies can also identify genes necessary for bacterial growth and survival under different environmental conditions, making them attractive targets for the development of novel inhibitory compounds. In addition, transcriptomic and proteomic approaches can be used to characterize RNA and protein levels on a cellular scale, providing information on bacterial physiology that can be applied to antibiotic target identification. Finally, bacterial genomes can be mined to identify biosynthetic pathways that produce many intrinsic antimicrobial compounds and peptides. In this review, we provide an overview of past and current efforts aimed at using bacterial genomic data in the discovery and development of novel antibiotics. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. The Arbuscular Mycorrhizal Fungus Funneliformis mosseae Alters Bacterial Communities in Subtropical Forest Soils during Litter Decomposition

    Directory of Open Access Journals (Sweden)

    Heng Gui

    2017-06-01

    Full Text Available Bacterial communities and arbuscular mycorrhizal fungi (AMF co-occur in the soil, however, the interaction between these two groups during litter decomposition remains largely unexplored. In order to investigate the effect of AMF on soil bacterial communities, we designed dual compartment microcosms, where AMF (Funneliformis mosseae was allowed access (AM to, or excluded (NM from, a compartment containing forest soil and litterbags. Soil samples from this compartment were analyzed at 0, 90, 120, 150, and 180 days. For each sample, Illumina sequencing was used to assess any changes in the soil bacterial communities. We found that most of the obtained operational taxonomic units (OTUs from both treatments belonged to the phylum of Proteobacteria, Acidobacteria, and Actinobacteria. The community composition of bacteria at phylum and class levels was slightly influenced by both time and AMF. In addition, time and AMF significantly affected bacterial genera (e.g., Candidatus Solibacter, Dyella, Phenylobacterium involved in litter decomposition. Opposite to the bacterial community composition, we found that overall soil bacterial OTU richness and diversity are relatively stable and were not significantly influenced by either time or AMF inoculation. OTU richness at phylum and class levels also showed consistent results with overall bacterial OTU richness. Our study provides new insight into the influence of AMF on soil bacterial communities at the genus level.

  7. Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

    Science.gov (United States)

    Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

    2012-04-01

    Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

  8. Mercury alters the bacterial community structure and diversity in soil even at concentrations lower than the guideline values.

    Science.gov (United States)

    Mahbub, Khandaker Rayhan; Subashchandrabose, Suresh Ramraj; Krishnan, Kannan; Naidu, Ravi; Megharaj, Mallavarapu

    2017-03-01

    This study evaluated the effect of inorganic mercury (Hg) on bacterial community and diversity in different soils. Three soils-neutral, alkaline and acidic-were spiked with six different concentrations of Hg ranging from 0 to 200 mg kg(-1) and aged for 90 days. At the end of the ageing period, 18 samples from three different soils were investigated for bacterial community structure and soil physicochemical properties. Illumina MiSeq-based 16s ribosomal RNA (rRNA) amplicon sequencing revealed the alteration in the bacterial community between un-spiked control soils and Hg-spiked soils. Among the bacterial groups, Actinobacteria (22.65%) were the most abundant phyla in all samples followed by Proteobacteria (21.95%), Bacteroidetes (4.15%), Firmicutes (2.9%) and Acidobacteria (2.04%). However, the largest group showing increased abundance with higher Hg doses was the unclassified group (45.86%), followed by Proteobacteria. Mercury had a considerable negative impact on key soil functional bacteria such as ammonium oxidizers and nitrifiers. Canonical correspondence analysis (CCA) indicated that among the measured soil properties, Hg had a major influence on bacterial community structure. Furthermore, nonlinear regression analysis confirmed that Hg significantly decreased soil bacterial alpha diversity in lower organic carbon containing neutral and alkaline soils, whereas in acidic soil with higher organic carbon there was no significant correlation. EC20 values obtained by a nonlinear regression analysis indicated that Hg significantly decreased soil bacterial diversity in concentrations lower than several guideline values.

  9. Magnetotactic Bacterial Cages as Safe and Smart Gene Delivery Vehicles

    KAUST Repository

    Alsaiari, Shahad K.

    2016-07-27

    In spite of the huge advances in the area of synthetic carriers, their efficiency still poorly compares to natural vectors. Herein, we report the use of unmodified magnetotactic bacteria as a guidable delivery vehicle for DNA functionalized gold nanoparticles (AuNPs). High cargo loading is established under anaerobic conditions (bacteria is alive) through endocytosis where AuNPs are employed as transmembrane proteins mimics (facilitate endocytosis) as well as imaging agents to verify and quantify loading and release. The naturally bio-mineralized magnetosomes, within the bacteria, induce heat generation inside bacteria through magnetic hyperthermia. Most importantly after exposing the system to air (bacteria is dead) the cell wall stays intact providing an efficient bacterial vessel. Upon incubation with THP-1 cells, the magnetotactic bacterial cages (MBCs) adhere to the cell wall and are directly engulfed through the phagocytic activity of these cells. Applying magnetic hyperthermia leads to the dissociation of the bacterial microcarrier and eventual release of cargo.

  10. Antipsychotic pathway genes with expression altered in opposite direction by antipsychotics and amphetamine.

    Science.gov (United States)

    Ko, Françoise; Tallerico, Teresa; Seeman, Philip

    2006-08-01

    To develop a new strategy for identifying possible psychotic- or antipsychotic-related pathway genes, rats were treated with clinical doses of haloperidol and clozapine for 4 days, and the altered expression of genes was compared with the genes altered in expression after amphetamine sensitization. The objective was to identify genes with expression altered in the same direction by haloperidol and clozapine but in the opposite direction in the amphetamine-sensitized rat striatum. These criteria were met by 21 genes, consisting of 15 genes upregulated by amphetamine, and 6 genes downregulated by amphetamine. Of the 21 genes, 15 are not presently identified, and only 3 genes (cathepsin K, GRK6, and a gene with accession number AI177589) are located in chromosome regions known to be associated with schizophrenia.

  11. Monocyte chemotactic protein-1 gene polymorphism and spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Levent; Filik

    2010-01-01

    I read with great interest the article by Gbele et al published in issue 44 of World J Gastroenterol 2009.The results of their study indicate that-2518 Monocyte chemotactic protein-1(MCP-1)genotype AA is a risk factor for spontaneous bacterial peritonitis in patients with alcoholic cirrhosis.However,there are some items that need to be discussed.

  12. Exploring the relationship between fractal features and bacterial essential genes

    Science.gov (United States)

    Yong-Ming, Yu; Li-Cai, Yang; Qian, Zhou; Lu-Lu, Zhao; Zhi-Ping, Liu

    2016-06-01

    Essential genes are indispensable for the survival of an organism in optimal conditions. Rapid and accurate identifications of new essential genes are of great theoretical and practical significance. Exploring features with predictive power is fundamental for this. Here, we calculate six fractal features from primary gene and protein sequences and then explore their relationship with gene essentiality by statistical analysis and machine learning-based methods. The models are applied to all the currently available identified genes in 27 bacteria from the database of essential genes (DEG). It is found that the fractal features of essential genes generally differ from those of non-essential genes. The fractal features are used to ascertain the parameters of two machine learning classifiers: Naïve Bayes and Random Forest. The area under the curve (AUC) of both classifiers show that each fractal feature is satisfactorily discriminative between essential genes and non-essential genes individually. And, although significant correlations exist among fractal features, gene essentiality can also be reliably predicted by various combinations of them. Thus, the fractal features analyzed in our study can be used not only to construct a good essentiality classifier alone, but also to be significant contributors for computational tools identifying essential genes. Project supported by the Shandong Provincial Natural Science Foundation, China (Grant No. ZR2014FM022).

  13. Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment

    LENUS (Irish Health Repository)

    Jones, Robert T

    2010-05-12

    Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C) and human (37°C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of

  14. Photorhabdus adhesion modification protein (Pam binds extracellular polysaccharide and alters bacterial attachment

    Directory of Open Access Journals (Sweden)

    Joyce Susan A

    2010-05-01

    Full Text Available Abstract Background Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. Results A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28°C and human (37°C temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. Conclusions We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect

  15. The plant pathogenic fungus Gaeumannomyces graminis var. tritici improves bacterial growth and triggers early gene regulations in the biocontrol strain Pseudomonas fluorescens Pf29Arp.

    Science.gov (United States)

    Barret, M; Frey-Klett, P; Boutin, M; Guillerm-Erckelboudt, A-Y; Martin, F; Guillot, L; Sarniguet, A

    2009-01-01

    In soil, some antagonistic rhizobacteria contribute to reduce root diseases caused by phytopathogenic fungi. Direct modes of action of these bacteria have been largely explored; however, commensal interaction also takes place between these microorganisms and little is known about the influence of filamentous fungi on bacteria. An in vitro confrontation bioassay between the pathogenic fungus Gaeumannomyces graminis var. tritici (Ggt) and the biocontrol bacterial strain Pseudomonas fluorescens Pf29Arp was set up to analyse bacterial transcriptional changes induced by the fungal mycelium at three time-points of the interaction before cell contact and up until contact. For this, a Pf29Arp shotgun DNA microarray was constructed. Specifity of Ggt effect was assessed in comparison with one of two other filamentous fungi, Laccaria bicolor and Magnaporthe grisea. During a commensal interaction, Ggt increased the growth rate of Pf29Arp. Before contact, Ggt induced bacterial genes involved in mycelium colonization. At contact, genes encoding protein of stress response and a patatin-like protein were up-regulated. Among all the bacterial genes identified, xseB was specifically up-regulated at contact by Ggt but down-regulated by the other fungi. Data showed that the bacterium sensed the presence of the fungus early, but the main gene alteration occurred during bacterial-fungal cell contact.

  16. Bacterial community and arsenic functional genes diversity in arsenic contaminated soils from different geographic locations

    Science.gov (United States)

    Gu, Yunfu; D. Van Nostrand, Joy; Wu, Liyou; He, Zhili; Qin, Yujia; Zhao, Fang-Jie; Zhou, Jizhong

    2017-01-01

    To understand how soil microbial communities and arsenic (As) functional genes respond to soil arsenic (As) contamination, five soils contaminated with As at different levels were collected from diverse geographic locations, incubated for 54 days under flooded conditions, and examined by both MiSeq sequencing of 16S rRNA gene amplicons and functional gene microarray (GeoChip 4.0). The results showed that both bacterial community structure and As functional gene structure differed among geographical locations. The diversity of As functional genes correlated positively with the diversity of 16S rRNA genes (Pcontaminated with different levels of As at different geographic locations, and the impact of environmental As contamination on the soil bacterial community. PMID:28475654

  17. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

    Directory of Open Access Journals (Sweden)

    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  18. Autism associated gene, engrailed2, and flanking gene levels are altered in post-mortem cerebellum.

    Directory of Open Access Journals (Sweden)

    Jiyeon Choi

    Full Text Available BACKGROUND: Previous genetic studies demonstrated association between the transcription factor engrailed2 (EN2 and Autism Spectrum Disorder (ASD. Subsequent molecular analysis determined that the EN2 ASD-associated haplotype (rs1861972-rs1861973 A-C functions as a transcriptional activator to increase gene expression. EN2 is flanked by 5 genes, serotonin receptor5a (HTR5A, insulin induced gene1 (INSIG1, canopy1 homolog (CNPY1, RNA binding motif protein33 (RBM33, and sonic hedgehog (SHH. These flanking genes are co-expressed with EN2 during development and coordinate similar developmental processes. To investigate if mRNA levels for these genes are altered in individuals with autism, post-mortem analysis was performed. METHODS: qRT-PCR quantified mRNA levels for EN2 and the 5 flanking genes in 78 post-mortem cerebellar samples. mRNA levels were correlated with both affection status and rs1861972-rs1861973 genotype. Molecular analysis investigated whether EN2 regulates flanking gene expression. RESULTS: EN2 levels are increased in affected A-C/G-T individuals (p = .0077. Affected individuals also display a significant increase in SHH and a decrease in INSIG1 levels. Rs1861972-rs1861973 genotype is correlated with significant increases for SHH (A-C/G-T and CNPY1 (G-T/G-T levels. Human cell line over-expression and knock-down as well as mouse knock-out analysis are consistent with EN2 and SHH being co-regulated, which provides a possible mechanism for increased SHH post-mortem levels. CONCLUSIONS: EN2 levels are increased in affected individuals with an A-C/G-T genotype, supporting EN2 as an ASD susceptibility gene. SHH, CNPY1, and INSIG1 levels are also significantly altered depending upon affection status or rs1861972-rs1861973 genotype. Increased EN2 levels likely contribute to elevated SHH expression observed in the post-mortem samples.

  19. Bacterial blight of cotton

    Directory of Open Access Journals (Sweden)

    Aïda JALLOUL

    2015-04-01

    Full Text Available Bacterial blight of cotton (Gossypium ssp., caused by Xanthomonas citri pathovar malvacearum, is a severe disease occurring in all cotton-growing areas. The interactions between host plants and the bacteria are based on the gene-for-gene concept, representing a complex resistance gene/avr gene system. In light of the recent data, this review focuses on the understanding of these interactions with emphasis on (1 the genetic basis for plant resistance and bacterial virulence, (2 physiological mechanisms involved in the hypersensitive response to the pathogen, including hormonal signaling, the oxylipin pathway, synthesis of antimicrobial molecules and alteration of host cell structures, and (3 control of the disease.

  20. Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

    Science.gov (United States)

    Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

    2015-08-01

    Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications.

  1. Dissecting specific and global transcriptional regulation of bacterial gene expression

    NARCIS (Netherlands)

    Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

    2013-01-01

    Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an experiment

  2. Genomic Analyses of Bacterial Porin-Cytochrome Gene Clusters

    Directory of Open Access Journals (Sweden)

    Liang eShi

    2014-11-01

    Full Text Available The porin-cytochrome (Pcc protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c-type cytochrome (c-Cyt and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteria from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr gene clusters of other Fe(III-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III and Mn(IV oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III and Mn(IV oxides.

  3. Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux

    Directory of Open Access Journals (Sweden)

    Hinds Jason

    2008-10-01

    Full Text Available Abstract Background Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes. Results The microarray consists of 4958 reporters from 151 bacterial species and include genes for the identification of individual bacterial species as well as mobile genetic elements (transposons, plasmid and phage, virulence genes and antibiotic resistance genes. The ASP microarray was validated with nineteen bacterial pathogens species, including Francisella tularensis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecium and Stenotrophomonas maltophilia. The ASP microarray identified these bacteria, and provided information on potential antibiotic resistance (eg sufamethoxazole resistance and sulfonamide resistance and virulence determinants including genes likely to be acquired by horizontal gene transfer (e.g. an alpha-haemolysin. Conclusion The ASP microarray has potential in the clinic as a diagnostic tool, as a research tool for both known and emerging pathogens, and as an early warning system for pathogenic bacteria that have been recently modified either naturally or deliberately.

  4. A functional gene array for detection of bacterial virulence elements.

    Directory of Open Access Journals (Sweden)

    Crystal Jaing

    Full Text Available Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.

  5. The percentage of bacterial genes on leading versus lagging strands is influenced by multiple balancing forces

    Science.gov (United States)

    Mao, Xizeng; Zhang, Han; Yin, Yanbin; Xu, Ying

    2012-01-01

    The majority of bacterial genes are located on the leading strand, and the percentage of such genes has a large variation across different bacteria. Although some explanations have been proposed, these are at most partial explanations as they cover only small percentages of the genes and do not even consider the ones biased toward the lagging strand. We have carried out a computational study on 725 bacterial genomes, aiming to elucidate other factors that may have influenced the strand location of genes in a bacterium. Our analyses suggest that (i) genes of some functional categories such as ribosome have higher preferences to be on the leading strands; (ii) genes of some functional categories such as transcription factor have higher preferences on the lagging strands; (iii) there is a balancing force that tends to keep genes from all moving to the leading and more efficient strand and (iv) the percentage of leading-strand genes in an bacterium can be accurately explained based on the numbers of genes in the functional categories outlined in (i) and (ii), genome size and gene density, indicating that these numbers implicitly contain the information about the percentage of genes on the leading versus lagging strand in a genome. PMID:22735706

  6. Arabidopsis gene expression patterns are altered during spaceflight

    Science.gov (United States)

    Paul, Anna-Lisa; Popp, Michael P.; Gurley, William B.; Guy, Charles; Norwood, Kelly L.; Ferl, Robert J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K., Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613-621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman®). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced - but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment

  7. A functional gene array for detection of bacterial virulence elements

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C

    2007-11-01

    We report our development of the first of a series of microarrays designed to detect pathogens with known mechanisms of virulence and antibiotic resistance. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. To validate our approach, we developed a first generation array targeting genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for microorganism detection and discrimination, measured the required target concentration, and assessed tolerance for mismatches between probe and target sequences. Mismatch tolerance is a priority for this application, due to DNA sequence variability among members of gene families. Arrays were created using the NimbleGen Maskless Array Synthesizer at Lawrence Livermore National Laboratory. Purified genomic DNA from combinations of one or more of the four target organisms, pure cultures of four related organisms, and environmental aerosol samples with spiked-in genomic DNA were hybridized to the arrays. Based on the success of this prototype, we plan to design further arrays in this series, with the goal of detecting all known virulence and antibiotic resistance gene families in a greatly expanded set of organisms.

  8. Pseudomonas aeruginosa serA Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers

    Science.gov (United States)

    Yasuda, Masashi; Nagata, Syouya; Yamane, Satoshi; Kunikata, Chinami; Kida, Yutaka; Kuwano, Koichi; Suezawa, Chigusa; Okuda, Jun

    2017-01-01

    To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function. PMID:28046014

  9. Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid.

    Science.gov (United States)

    Akutsu, Tomoko; Motani, Hisako; Watanabe, Ken; Iwase, Hirotaro; Sakurada, Koichi

    2012-05-01

    To preliminarily evaluate the applicability of bacterial DNA as a marker for the forensic identification of vaginal fluid, we developed and performed PCR-based detection of 16S ribosomal RNA genes of Lactobacillus spp. dominating the vagina and of bacterial vaginosis-related bacteria from DNA extracted from body fluids and stains. As a result, 16S ribosomal RNA genes of Lactobacillus crispatus, Lactobacillus jensenii and Atopobium vaginae were specifically detected in vaginal fluid and female urine samples. Bacterial genes detected in female urine might have originated from contaminated vaginal fluid. In addition, those of Lactobacillus iners, Lactobacillus gasseri and Gardnerella vaginalis were also detected in non-vaginal body fluids such as semen. Because bacterial genes were successfully amplified in DNA samples extracted by using the general procedure for animal tissues without any optional treatments, DNA samples prepared for the identification of vaginal fluid can also be used for personal identification. In conclusion, 16S ribosomal RNA genes of L. crispatus, L. jensenii and A. vaginae could be effective markers for forensic identification of vaginal fluid.

  10. Grain-rich diets altered the colonic fermentation and mucosa-associated bacterial communities and induced mucosal injuries in goats.

    Science.gov (United States)

    Ye, Huimin; Liu, Junhua; Feng, Panfei; Zhu, Weiyun; Mao, Shengyong

    2016-02-04

    Remarkably little information is available about the impact of high-grain (HG) feeding on colonic mucosa-associated bacteria and mucosal morphology. In the present study, 12 male goats were randomly assigned to either a hay diet (n = 6) or an HG diet (65% grain; n = 6) to characterise the changes in the composition of the bacterial community in colonic mucosa and the mucosal morphology of the colon. The results showed that HG feeding decreased the colonic pH and increased the concentrations of total short chain fatty acids and lipopolysaccharides in colonic digesta. The principal coordinate analysis results showed that the HG diet altered the colonic mucosal bacterial communities, with an increase in the abundance of genus Blautia and a decrease in the abundance of genera Bacillus, Enterococcus, and Lactococcus. The HG-fed goats showed sloughing of the surface layer epithelium, intercellular tight junction erosion, cell mitochondrial damage, and upregulation of the relative mRNA expression of IL-2 and IFN-γ in colonic mucosa. Collectively, our data indicate that HG feeding induced changes in colonic mucosal morphology and cytokines expression that might be caused by excessive fermentation and dramatic shifts in the bacterial populations in the colon.

  11. Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

    Directory of Open Access Journals (Sweden)

    Carlos F Solis

    Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

  12. Interplay of Noisy Gene Expression and Dynamics Explains Patterns of Bacterial Operon Organization

    Science.gov (United States)

    Igoshin, Oleg

    2011-03-01

    Bacterial chromosomes are organized into operons -- sets of genes co-transcribed into polycistronic messenger RNA. Hypotheses explaining the emergence and maintenance of operons include proportional co-regulation, horizontal transfer of intact ``selfish'' operons, emergence via gene duplication, and co-production of physically interacting proteins to speed their association. We hypothesized an alternative: operons can reduce or increase intrinsic gene expression noise in a manner dependent on the post-translational interactions, thereby resulting in selection for or against operons in depending on the network architecture. We devised five classes of two-gene network modules and show that the effects of operons on intrinsic noise depend on class membership. Two classes exhibit decreased noise with co-transcription, two others reveal increased noise, and the remaining one does not show a significant difference. To test our modeling predictions we employed bioinformatic analysis to determine the relationship gene expression noise and operon organization. The results confirm the overrepresentation of noise-minimizing operon architectures and provide evidence against other hypotheses. Our results thereby suggest a central role for gene expression noise in selecting for or maintaining operons in bacterial chromosomes. This demonstrates how post-translational network dynamics may provide selective pressure for organizing bacterial chromosomes, and has practical consequences for designing synthetic gene networks. This work is supported by National Institutes of Health grant 1R01GM096189-01.

  13. Bacterial Human Virulence Genes across Diverse Habitats As Assessed by In silico Analysis of Environmental Metagenomes

    DEFF Research Database (Denmark)

    Søborg, Ditte A; Hendriksen, Niels B; Kilian, Mogens

    2016-01-01

    and glacial ice. Homologs to 16 bacterial human virulence genes, involved in urinary tract infections, gastrointestinal diseases, skin diseases, and wound and systemic infections, showed global ubiquity. A principal component analysis did not demonstrate clear trends across the metagenomes with respect...... to occurrence and frequency of observed gene homologs. Full-length (>95%) homologs of several virulence genes were identified, and translated sequences of the environmental and clinical genes were up to 50-100% identical. Furthermore, phylogenetic analyses indicated deep branching positions of some...

  14. Importance of rare gene copy number alterations for personalized tumor characterization and survival analysis.

    Science.gov (United States)

    Seifert, Michael; Friedrich, Betty; Beyer, Andreas

    2016-10-03

    It has proven exceedingly difficult to ascertain rare copy number alterations (CNAs) that may have strong effects in individual tumors. We show that a regulatory network inferred from gene expression and gene copy number data of 768 human cancer cell lines can be used to quantify the impact of patient-specific CNAs on survival signature genes. A focused analysis of tumors from six tissues reveals that rare patient-specific gene CNAs often have stronger effects on signature genes than frequent gene CNAs. Further comparison to a related network-based approach shows that the integration of indirectly acting gene CNAs significantly improves the survival analysis.

  15. Altered gene expression profiles in mouse tetraploid blastocysts.

    Science.gov (United States)

    Park, Mi-Ryung; Hwang, Kyu-Chan; Bui, Hong-Thuy; Cho, Ssang-Goo; Park, Chankyu; Song, Hyuk; Oh, Jae-Wook; Kim, Jin-Hoi

    2012-01-01

    In this study, it was demonstrated that tetraploid-derived blastocyst embryos had very few Oct4-positive cells at the mid-blastocyst stage and that the inner cell mass at biomarkers Oct4, Sox2 and Klf4 was expressed at less than 10% of the level observed in diploid blastocysts. In contrast, trophectoderm-related gene transcripts showed an approximately 10 to 40% increase. Of 32,996 individual mouse genes evaluated by microarray, 50 genes were differentially expressed between tetraploid or diploid and parthenote embryos at the blastocyst stage (Ptetraploid-derived blastocysts, whereas 22 were more highly downregulated. However, some genes involved in receptor activity, cell adhesion molecule, calcium ion binding, protein biosynthesis, redox processes, transport, and transcription showed a significant decrease or increase in gene expression in the tetraploid-derived blastocyst embryos. Thus, microarray analysis can be used as a tool to screen for underlying defects responsible for the development of tetraploid-derived embryos.

  16. Identifying essential genes in bacterial metabolic networks with machine learning methods

    Directory of Open Access Journals (Sweden)

    Eils Roland

    2010-05-01

    Full Text Available Abstract Background Identifying essential genes in bacteria supports to identify potential drug targets and an understanding of minimal requirements for a synthetic cell. However, experimentally assaying the essentiality of their coding genes is resource intensive and not feasible for all bacterial organisms, in particular if they are infective. Results We developed a machine learning technique to identify essential genes using the experimental data of genome-wide knock-out screens from one bacterial organism to infer essential genes of another related bacterial organism. We used a broad variety of topological features, sequence characteristics and co-expression properties potentially associated with essentiality, such as flux deviations, centrality, codon frequencies of the sequences, co-regulation and phyletic retention. An organism-wise cross-validation on bacterial species yielded reliable results with good accuracies (area under the receiver-operator-curve of 75% - 81%. Finally, it was applied to drug target predictions for Salmonella typhimurium. We compared our predictions to the viability of experimental knock-outs of S. typhimurium and identified 35 enzymes, which are highly relevant to be considered as potential drug targets. Specifically, we detected promising drug targets in the non-mevalonate pathway. Conclusions Using elaborated features characterizing network topology, sequence information and microarray data enables to predict essential genes from a bacterial reference organism to a related query organism without any knowledge about the essentiality of genes of the query organism. In general, such a method is beneficial for inferring drug targets when experimental data about genome-wide knockout screens is not available for the investigated organism.

  17. Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

    Science.gov (United States)

    Buse, Helen Y; Lu, Jingrang; Ashbolt, Nicholas J

    2015-01-01

    Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems.

  18. Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity.

    Science.gov (United States)

    Yasir, Muhammad; Aslam, Zubair; Kim, Seon Won; Lee, Seon-Woo; Jeon, Che Ok; Chung, Young Ryun

    2009-10-01

    Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.

  19. Both msa Genes in Renibacterium salmoninarum Are Needed for Full Virulence in Bacterial Kidney Disease

    OpenAIRE

    Coady, Alison M; Murray, Anthony L.; Elliott, Diane G.; Rhodes, Linda D.

    2006-01-01

    Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in...

  20. Transposon-induced nuclear mutations that alter chloroplast gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, A.

    1992-01-01

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  1. Genetic diversity of bacterial communities and gene transfer agents in northern South China Sea.

    Directory of Open Access Journals (Sweden)

    Fu-Lin Sun

    Full Text Available Pyrosequencing of the 16S ribosomal RNA gene (rDNA amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA major capsid gene (g5 was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS, temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments.

  2. Centrosomal and mitotic abnormalities in cell lines derived from papillary thyroid cancer harboring specific gene alterations

    National Research Council Canada - National Science Library

    Maric, Irena; Viaggi, Silvia; Caria, Paola; Frau, Daniela V; Degan, Paolo; Vanni, Roberta

    2011-01-01

    .... We investigated the centrosome status and mitotic abnormalities in three thyroid carcinoma-derived cell lines, each maintaining the specific, biologically relevant gene alteration harbored by the parental tumors...

  3. Measurement of bacterial gene expression in vivo by laser capture microdissection and quantitative real-time RT-PCR

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Jensen, Tim Kåre; Angen, Øystein

    2007-01-01

    Due to the relative small number of bacterial pathogens present in an infected host, exploration of pathogen gene expression in vivo is challenging. This study reports the development of a protocol for quantifying bacterial gene expression in vivo in Actinobacillus pleuropneumoniae using laser ca...... capture microdissection and real-time quantitative RT-PCR....

  4. Genetic Alterations in Familial Breast Cancer: Mapping and Cloning Genes Other Than BRCAl

    Science.gov (United States)

    1997-09-01

    predispose to breast cancer . These mutations are always in the context of Cowden’s Syndrome, and do not appear in families with brest cancer in the...AD AWARD NUMBER DAMD17-94-J-4307 TITLE: Genetic Alterations in Familial Breast Cancer : Mapping and Cloning Genes Other Than BRCA1 PRINCIPAL...Aug97-) Genetic Alterations in Familial Breast Cancer : Mapping and Cloning Genes Other than BRCA1 6. AUTHOR{S) Mary-Clair King, Ph.D. 7

  5. Alteration of plant meristem function by manipulation of the Retinoblastoma-like plant RRB gene

    Science.gov (United States)

    Durfee, Tim; Feiler, Heidi; Gruissem, Wilhelm; Jenkins, Susan; Roe, Judith; Zambryski, Patricia

    2007-01-16

    This invention provides methods and compositions for altering the growth, organization, and differentiation of plant tissues. The invention is based on the discovery that, in plants, genetically altering the levels of Retinoblastoma-related gene (RRB) activity produces dramatic effects on the growth, proliferation, organization, and differentiation of plant meristem.

  6. Alteration of plant meristem function by manipulation of the Retinoblastoma-like plant RRB gene

    Energy Technology Data Exchange (ETDEWEB)

    Durfee, Tim (Madison, WI); Feiler, Heidi (Albany, CA); Gruissem, Wilhelm (Forch, CH); Jenkins, Susan (Martinez, CA); Roe, Judith (Manhattan, KS); Zambryski, Patricia (Berkeley, CA)

    2007-01-16

    This invention provides methods and compositions for altering the growth, organization, and differentiation of plant tissues. The invention is based on the discovery that, in plants, genetically altering the levels of Retinoblastoma-related gene (RRB) activity produces dramatic effects on the growth, proliferation, organization, and differentiation of plant meristem.

  7. Watershed Urbanization Alters the Composition and Function of Stream Bacterial Communities

    Science.gov (United States)

    Wang, Si-Yi; Sudduth, Elizabeth B.; Wallenstein, Matthew D.; Wright, Justin P.; Bernhardt, Emily S.

    2011-01-01

    Watershed urbanization leads to dramatic changes in draining streams, with urban streams receiving a high frequency of scouring flows, together with the nutrient, contaminant, and thermal pollution associated with urbanization. These changes are known to cause significant losses of sensitive insect and fish species from urban streams, yet little is known about how these changes affect the composition and function of stream microbial communities. Over the course of two years, we repeatedly sampled sediments from eight central North Carolina streams affected to varying degrees by watershed urbanization. For each stream and sampling date, we characterized both overall and denitrifying bacterial communities and measured denitrification potentials. Denitrification is an ecologically important process, mediated by denitrifying bacteria that use nitrate and organic carbon as substrates. Differences in overall and denitrifying bacterial community composition were strongly associated with the gradient in urbanization. Denitrification potentials, which varied widely, were not significantly associated with substrate supply. By incorporating information on the community composition of denitrifying bacteria together with substrate supply in a linear mixed-effects model, we explained 45% of the variation in denitrification potential (p-valuedenitrification. PMID:21857975

  8. Alterations of the USP26 gene in Caucasian men.

    Science.gov (United States)

    Stouffs, Katrien; Lissens, Willy; Tournaye, Herman; Van Steirteghem, André; Liebaers, Inge

    2006-12-01

    The Ubiquitin Specific Protease 26 gene is a testis-specific gene that is located on the X chromosome. Sequence variants of this gene were previously reported in men with azoospermia caused by defects at the level of spermatogenesis. Especially a cluster of three changes (c.370_371insACA, c.494T>C and c.1423C>T) was frequently observed. To further define the role of this cluster of sequence variants in the USP26 gene, we have now analysed 202 control samples and 146 patients of Caucasian origin with cryptozoospermia or oligozoospermia. The detection method was based on a restriction reaction, by which the change c.494T>C can be detected. In none of the patients, the change c.494T>C was observed. Only in one man with normal spermatogenesis this sequence variant was detected. Sequencing can confirm the presence of the three changes of the USP26 gene. These data indicate that the cluster of changes is not restricted to men with severe testicular dysfunction.

  9. Gain and loss of phototrophic genes revealed by comparison of two Citromicrobium bacterial genomes.

    Directory of Open Access Journals (Sweden)

    Qiang Zheng

    Full Text Available Proteobacteria are thought to have diverged from a phototrophic ancestor, according to the scattered distribution of phototrophy throughout the proteobacterial clade, and so the occurrence of numerous closely related phototrophic and chemotrophic microorganisms may be the result of the loss of genes for phototrophy. A widespread form of bacterial phototrophy is based on the photochemical reaction center, encoded by puf and puh operons that typically are in a 'photosynthesis gene cluster' (abbreviated as the PGC with pigment biosynthesis genes. Comparison of two closely related Citromicrobial genomes (98.1% sequence identity of complete 16S rRNA genes, Citromicrobium sp. JL354, which contains two copies of reaction center genes, and Citromicrobium strain JLT1363, which is chemotrophic, revealed evidence for the loss of phototrophic genes. However, evidence of horizontal gene transfer was found in these two bacterial genomes. An incomplete PGC (pufLMC-puhCBA in strain JL354 was located within an integrating conjugative element, which indicates a potential mechanism for the horizontal transfer of genes for phototrophy.

  10. Altering the growth conditions of Gluconacetobacter xylinus to maximize the yield of bacterial cellulose.

    Science.gov (United States)

    Ruka, Dianne R; Simon, George P; Dean, Katherine M

    2012-06-20

    An extensive matrix of different growth conditions including media, incubation time, inoculum volume, surface area and media volume were investigated in order to maximize the yield of bacterial cellulose produced by Gluconacetobacter xylinus, which will be used as reinforcement material to produce fully biodegradable composites. Crystallinity was shown to be controllable depending on the media and conditions employed. Samples with significant difference in crystallinity in a range from 50% to 95% were produced. Through experimental design, the yield of cellulose was maximized; primarily this involved reactor surface area design, optimized media and the use of mannitol being the highest cellulose-producing carbon source. Increasing the volume of the media did achieve a higher cellulose yield, however this increase was not found to be cost or time effective.

  11. FREQUENT STRUCTURE ALTERATIONS OF p53 GENE IN NASOPHARYNGEAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    龙江斌; 区宝祥; 梁启万; 李辉梅

    1998-01-01

    By southern hybridization with 1.8 kb cDNA probe,a high freqnency (40.5%) of structural abnormality of p 53 gene was observed in primary nasopharyngeal carcinoma (NPC) biopsies. The regioas of exons 1 to 4 of the gene were examined by poiymerase chain reaction-single strand conformation polymorphism, no point nmtation was found. Because very low rate of point mutation had been reported in exons 5 to 8,we considered that structural ahnormality in the region of exons 1 to 8 of the gene might be uncommon in NPC. The speetrophotometer scaaning analysis of outoradiograms and rehybridization investigation of nitrocellulose filter with exon 11 probe indicated that most of structure aberrations we observed might be rearrangement occurring in exon ll.

  12. Long acting β2-agonist and corticosteroid restore airway glandular cell function altered by bacterial supernatant

    Directory of Open Access Journals (Sweden)

    Nawrocki-Raby Béatrice

    2010-01-01

    Full Text Available Abstract Background Staphylococcus aureus releases virulence factors (VF that may impair the innate protective functions of airway cells. The aim of this study was to determine whether a long-acting β2 adrenergic receptor agonist (salmeterol hydroxynaphthoate, Sal combined with a corticosteroid (fluticasone propionate, FP was able to regulate ion content and cytokine expression by airway glandular cells after exposure to S. aureus supernatant. Methods A human airway glandular cell line was incubated with S. aureus supernatant for 1 h and then treated with the combination Sal/FP for 4 h. The expression of actin and CFTR proteins was analyzed by immunofluorescence. Videomicroscopy was used to evaluate chloride secretion and X-ray microanalysis to measure the intracellular ion and water content. The pro-inflammatory cytokine expression was assessed by RT-PCR and ELISA. Results When the cells were incubated with S. aureus supernatant and then with Sal/FP, the cellular localisation of CFTR was apical compared to the cytoplasmic localisation in cells incubated with S. aureus supernatant alone. The incubation of airway epithelial cells with S. aureus supernatant reduced by 66% the chloride efflux that was fully restored by Sal/FP treatment. We also observed that Sal/FP treatment induced the restoration of ion (Cl and S and water content within the intracellular secretory granules of airway glandular cells and reduced the bacterial supernatant-dependent increase of pro-inflammatory cytokines IL8 and TNFα. Conclusions Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting β2 adrenergic receptor agonist after bacterial infection restores the airway glandular cell function. Abnormal mucus induced by defective ion transport during pulmonary infection could benefit from treatment with a combination of β2 adrenergic receptor agonist and glucocorticoid.

  13. The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

    Science.gov (United States)

    Valle-García, David; Griffiths, Lyra M; Dyer, Michael A; Bernstein, Emily; Recillas-Targa, Félix

    2014-01-01

    The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

  14. Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA

    OpenAIRE

    Takamatsu, Nobuhiko; Ishikawa, Masayuki; Meshi, Tetsuo; Okada, Yoshimi

    1987-01-01

    We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were ...

  15. Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease

    Science.gov (United States)

    Coady, A.M.; Murray, A.L.; Elliott, D.G.; Rhodes, L.D.

    2006-01-01

    Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile Chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression. Copyright ?? 2006, American Society for Microbiology. All Rights Reserved.

  16. Protecting the herd: the remarkable effectiveness of the bacterial meningitis polysaccharide-protein conjugate vaccines in altering transmission dynamics.

    Science.gov (United States)

    Stephens, David S

    2011-01-01

    Interrupting human-to-human transmission of the agents (Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae) of bacterial meningitis by new capsular polysaccharide-protein conjugate vaccines (PPCVs) has proven to be a remarkable (and unanticipated) contributor to vaccine effectiveness. Herd immunity accounts for ∼50% of the protection by meningococcal serogroup C PPCVs, pneumococcal PPCV7, and H. influenzae b PPCVs. Nasopharyngeal carriage can be reduced ≥75% for vaccine serotypes; the decrease in carriage is correlated with disease reduction in unvaccinated individuals, and the impact of herd immunity lasts for years. Based on these data, models for using herd immunity in vaccine-based prevention strategies are underway for control of meningitis in sub-Saharan Africa. Although the immunologic basis of herd immunity and impact on microbial biology need more study, protecting the unvaccinated by altering pathogen transmission dynamics is a powerful effect of PPCVs and increasingly important in vaccine introduction, implementation, and evaluation strategies.

  17. Nursing frequency alters circadian patterns of mammary gene expression in lactating mice

    Science.gov (United States)

    Milking frequency impacts lactation in dairy cattle and in rodent models of lactation. The role of circadian gene expression in this process is unknown. The hypothesis tested was that changing nursing frequency alters the circadian patterns of mammary gene expression. Mid-lactation CD1 mice were stu...

  18. Gene expression alterations in brains of mice infected with three strains of scrapie

    Directory of Open Access Journals (Sweden)

    Race Richard E

    2006-05-01

    Full Text Available Abstract Background Transmissible spongiform encephalopathies (TSEs or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD in humans, bovine spongiform encephalopathy (BSE in cattle, chronic wasting disease (CWD in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML. Results In symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function. Conclusion These studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs.

  19. Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge.

    Science.gov (United States)

    Shrestha, Sony; Park, Yoonseong; Stanley, David; Kim, Yonggyun

    2010-03-01

    We propose that expression of four genes encoding secretory phospholipases A(2) (sPLA(2)) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA(2)-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA(2) activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA(2) and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA(2)s. The recombinant sPLA(2)s were inhibited by sPLA(2) inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA(2) genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA(2)s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA(2)s showed the presence of the sPLA(2) enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA(2) genes that mediate nodulation reactions strongly supports our hypothesis that sPLA(2)s are central enzymes in insect cellular immune reactions. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  20. Laccase activity is proportional to the abundance of bacterial laccase-like genes in soil from subtropical arable land.

    Science.gov (United States)

    Feng, Shuzhen; Su, Yirong; Dong, Mingzhe; He, Xunyang; Kumaresan, Deepak; O'Donnell, Anthony G; Wu, Jinshui; Chen, Xiangbi

    2015-12-01

    Laccase enzymes produced by both soil bacteria and fungi play important roles in refractory organic matter turnover in terrestrial ecosystems. We investigated the abundance and diversity of fungal laccase genes and bacterial laccase-like genes in soil from subtropical arable lands, and identified which microbial group was associated with laccase activity. Compared with fungal laccase genes, the bacterial laccase-like genes had greater abundance, richness and Shannon-Wiener diversity. More importantly, laccase activity can be explained almost exclusively by the bacterial laccase-like genes, and their abundance had significant linear relationship with laccase activity. Thus, bacterial laccase-like gene has great potential to be used as a sensitive indicator of laccase enzyme for refractory organic matter turnover in subtropical arable lands.

  1. Phylogeny of bacterial and archaeal genomes using conserved genes: supertrees and supermatrices.

    Directory of Open Access Journals (Sweden)

    Jenna Morgan Lang

    Full Text Available Over 3000 microbial (bacterial and archaeal genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA, as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a "primary concordance" tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes.

  2. Symbiotic bacterial communities in ants are modified by invasion pathway bottlenecks and alter host behavior.

    Science.gov (United States)

    Lester, Philip J; Sébastien, Alexandra; Suarez, Andrew V; Barbieri, Rafael F; Gruber, Monica A M

    2017-03-01

    Biological invasions are a threat to global biodiversity and provide unique opportunities to study ecological processes. Population bottlenecks are a common feature of biological invasions and the severity of these bottlenecks is likely to be compounded as an invasive species spreads from initial invasion sites to additional locations. Despite extensive work on the genetic consequences of bottlenecks, we know little about how they influence microbial communities of the invaders themselves. Due to serial bottlenecks, invasive species may lose microbial symbionts including pathogenic taxa (the enemy release hypothesis) and/or may accumulate natural enemies with increasing time after invasion (the pathogen accumulation and invasive decline hypothesis). We tested these alternate hypotheses by surveying bacterial communities of Argentine ants (Linepithema humile). We found evidence for serial symbiont bottlenecks: the bacterial community richness declined over the invasion pathway from Argentina to New Zealand. The abundance of some genera, such as Lactobacillus, also significantly declined over the invasion pathway. Argentine ants from populations in the United States shared the most genera with ants from their native range in Argentina, while New Zealand shared the least (120 vs. 57, respectively). Nine genera were present in all sites around the globe possibly indicating a core group of obligate microbes. In accordance with the pathogen accumulation and invasive decline hypothesis, Argentine ants acquired genera unique to each specific invaded country. The United States had the most unique genera, though even within New Zealand these ants acquired symbionts. In addition to our biogeographic sampling, we administered antibiotics to Argentine ants to determine if changes in the micro-symbiont community could influence behavior and survival in interspecific interactions. Treatment with the antibiotics spectinomycin and kanamycin only slightly increased Argentine ant

  3. Phosphodiesterase-4 inhibition alters gene expression and improves isoniazid-mediated clearance of Mycobacterium tuberculosis in rabbit lungs.

    Directory of Open Access Journals (Sweden)

    Selvakumar Subbian

    2011-09-01

    Full Text Available Tuberculosis (TB treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4 inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH. Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.

  4. Neurotoxocarosis alters myelin protein gene transcription and expression.

    Science.gov (United States)

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  5. Global brain gene expression analysis links glutamatergic and GABAergic alterations to suicide and major depression.

    Directory of Open Access Journals (Sweden)

    Adolfo Sequeira

    Full Text Available BACKGROUND: Most studies investigating the neurobiology of depression and suicide have focused on the serotonergic system. While it seems clear that serotonergic alterations play a role in the pathogenesis of these major public health problems, dysfunction in additional neurotransmitter systems and other molecular alterations may also be implicated. Microarray expression studies are excellent screening tools to generate hypotheses about additional molecular processes that may be at play. In this study we investigated brain regions that are known to be implicated in the neurobiology of suicide and major depression are likely to represent valid global molecular alterations. METHODOLOGY/PRINCIPAL FINDINGS: We performed gene expression analysis using the HG-U133AB chipset in 17 cortical and subcortical brain regions from suicides with and without major depression and controls. Total mRNA for microarray analysis was obtained from 663 brain samples isolated from 39 male subjects, including 26 suicide cases and 13 controls diagnosed by means of psychological autopsies. Independent brain samples from 34 subjects and animal studies were used to control for the potential confounding effects of comorbidity with alcohol. Using a Gene Ontology analysis as our starting point, we identified molecular pathways that may be involved in depression and suicide, and performed follow-up analyses on these possible targets. Methodology included gene expression measures from microarrays, Gene Score Resampling for global ontological profiling, and semi-quantitative RT-PCR. We observed the highest number of suicide specific alterations in prefrontal cortical areas and hippocampus. Our results revealed alterations of synaptic neurotransmission and intracellular signaling. Among these, Glutamatergic (GLU and GABAergic related genes were globally altered. Semi-quantitative RT-PCR results investigating expression of GLU and GABA receptor subunit genes were consistent with

  6. Bacterial gene regulation in diauxic and non-diauxic growth.

    Science.gov (United States)

    Narang, Atul; Pilyugin, Sergei S

    2007-01-21

    When bacteria are grown in a batch culture containing a mixture of two growth-limiting substrates, they exhibit a rich spectrum of substrate consumption patterns including diauxic growth, simultaneous consumption, and bistable growth. In previous work, we showed that a minimal model accounting only for enzyme induction and dilution captures all the substrate consumption patterns [Narang, A., 1998a. The dynamical analogy between microbial growth on mixtures of substrates and population growth of competing species. Biotechnol. Bioeng. 59, 116-121, Narang, A., 2006. Comparitive analysis of some models of gene regulation in mixed-substrate microbial growth, J. Theor. Biol. 242, 489-501]. In this work, we construct the bifurcation diagram of the minimal model, which shows the substrate consumption pattern at any given set of parameter values. The bifurcation diagram explains several general properties of mixed-substrate growth. (1) In almost all the cases of diauxic growth, the "preferred" substrate is the one that, by itself, supports a higher specific growth rate. In the literature, this property is often attributed to the optimality of regulatory mechanisms. Here, we show that the minimal model, which accounts for induction and growth only, displays the property under fairly general conditions. This suggests that the higher growth rate of the preferred substrate is an intrinsic property of the induction and dilution kinetics. It can be explained mechanistically without appealing to optimality principles. (2) The model explains the phenotypes of various mutants containing lesions in the regions encoding for the operator, repressor, and peripheral enzymes. A particularly striking phenotype is the "reversal of the diauxie" in which the wild-type and mutant strains consume the very same two substrates in opposite order. This phenotype is difficult to explain in terms of molecular mechanisms, such as inducer exclusion or CAP activation, but it turns out to be a natural

  7. Altered gene expression in the dorsolateral prefrontal cortex of individuals with schizophrenia.

    Science.gov (United States)

    Guillozet-Bongaarts, A L; Hyde, T M; Dalley, R A; Hawrylycz, M J; Henry, A; Hof, P R; Hohmann, J; Jones, A R; Kuan, C L; Royall, J; Shen, E; Swanson, B; Zeng, H; Kleinman, J E

    2014-04-01

    The underlying pathology of schizophrenia (SZ) is likely as heterogeneous as its symptomatology. A variety of cortical and subcortical regions, including the prefrontal cortex, have been implicated in its pathology, and a number of genes have been identified as risk factors for disease development. We used in situ hybridization (ISH) to examine the expression of 58 genes in the dorsolateral prefrontal cortex (DLPFC, comprised of Brodmann areas 9 and 46) from 19 individuals with a premorbid diagnosis of SZ and 33 control individuals. Genes were selected based on: (1) previous identification as risk factors for SZ; (2) cell type markers or (3) laminar markers. Cell density and staining intensity were compared in the DLPFC, as well as separately in Brodmann areas 9 and 46. The expression patterns of a variety of genes, many of which are associated with the GABAergic system, were altered in SZ when compared with controls. Additional genes, including C8orf79 and NR4A2, showed alterations in cell density or staining intensity between the groups, highlighting the need for additional studies. Alterations were, with only a few exceptions, limited to Brodmann area 9, suggesting regional specificity of pathology in the DLPFC. Our results agree with previous studies on the GABAergic involvement in SZ, and suggest that areas 9 and 46 may be differentially affected in the disease. This study also highlights additional genes that may be altered in SZ, and indicates that these potentially interesting genes can be identified by ISH and high-throughput image analysis techniques.

  8. Does the appearance of drug resistance during therapy alter bacterial susceptibility to opsonophagocytosis?

    Science.gov (United States)

    Gemmell, C G

    1996-01-01

    Coagulase-negative staphylococci (CNS) are common causes of infection in patients undergoing chronic ambulatory peritoneal dialysis (CAPD). Their ability to survive intracellularly within peritoneal macrophages and to persist within the peritoneum during antibiotic therapy has led to the development of drug resistance during treatment. Strains of Staphylococcus epidermidis (SE) and Staphytococcus haemolyticus (SH) have been isolated from patients with CAPD during treatment with ciprofloxacin. The respective MIC values pre-and post-therapy were SE-0.25 and 128 mg/L and SH-0.50 and 64 mg/L. The susceptibility of each isolate to opsonophagocytosis was measured in vitro using isolated polymorphonuclear leucocytes (PMN) derived from fresh human blood donations. The bacteria were radiolabelled during growth, opsonised in either 1 or 10% serum and their uptake measured No differences were seen between the pre- and post therapy isolates when using 10% serum as opsonic source (18 vs. 21%); with 1% serum the corresponding values were lower (5 and 8% respectively). Similarly their ability to generate a respiratory burst as measured by chemiluminescence (CL) in the phagocytic cells was not diminished in the strains which had developed resistance to ciprofloxacin. The mean CL response to the strains isolated at outset of therapy ranged from 0.35-0.45 cpsc, and to the resistant strains following therapy from 0.36-0.50 cpsc. It is clear from the present investigation that although the bacterial strain became at least 10 times more resistant to ciprofloxacin during therapy, no change in their susceptibility to phagocytosis occurred refuting the idea that the emergence of drug resistant strains during therapy results in "super-bugs" of greater virulence.

  9. [Numeric alterations in the dys gene and their association with clinical features].

    Science.gov (United States)

    Mampel, Alejandra; Echeverría, María Inés; Vargas, Ana Lía; Roque, María

    2011-01-01

    The Duchenne/Becker muscular dystrophy is a hereditary miopathy with a recessive sex-linked pattern. The related gene is called DYS and the coded protein plays a crucial role in the anchorage between the cytoskeleton and the cellular membrane in muscle cells. Different clinical manifestations are observed depending on the impact of the genetic alteration on the protein. The global register of mutations reveals an enhanced frequency for deletions/duplications of one or more exons affecting the DYS gene. In the present work, numeric alterations have been studied in the 79 exons of the DYS gene. The study has been performed on 59 individuals, including 31 independent cases and 28 cases with a familial link. The applied methodology was Multiplex Ligation Dependent Probe Amplification (MLPA). In the 31 independent cases clinical data were established: i.e. the clinical score, the Raven test percentiles, and the creatininphosphokinase (CPK) blood values. Our results reveal a 61.3% frequency of numeric alterations affecting the DYS gene in our population, provoking all of them a reading frame shift. The rate for de novo mutations was identified as 35.2%. Alterations involving a specific region of one exon were observed with high frequency, affecting a specific region. A significant association was found between numeric alterations and a low percentile for the Raven test. These data contribute to the local knowledge of genetic alterations and their phenotypic impact for the Duchenne/Becker disease.

  10. Mitochondrial genes are altered in blood early in Alzheimer's disease.

    Science.gov (United States)

    Lunnon, Katie; Keohane, Aoife; Pidsley, Ruth; Newhouse, Stephen; Riddoch-Contreras, Joanna; Thubron, Elisabeth B; Devall, Matthew; Soininen, Hikka; Kłoszewska, Iwona; Mecocci, Patrizia; Tsolaki, Magda; Vellas, Bruno; Schalkwyk, Leonard; Dobson, Richard; Malik, Afshan N; Powell, John; Lovestone, Simon; Hodges, Angela

    2017-01-07

    Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species.

  11. Acidic Conditions in the NHE2-/- Mouse Intestine Result in an Altered Mucosa-Associated Bacterial Population with Changes in Mucus Oligosaccharides

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    Melinda A. Engevik

    2013-12-01

    Full Text Available Background: The mechanisms bacteria use to proliferate and alter the normal bacterial composition remain unknown. The ability to link changes in the intestinal micro-environment, such as ion composition and pH, to bacterial proliferation is clinically advantageous for diseases that involve an altered gut microbiota, such as Inflammatory Bowel Disease, obesity and diabetes. In human and mouse intestine, the apical Na+/H+ exchangers NHE2 and NHE3 affect luminal Na+, water, and pH. Loss of NHE2 results in acidic luminal pH. Since acid resistance systems in gram-positive bacteria are well documented, we hypothesize that gram-positive bacteria would increase in representation in the acidic NHE2-/- intestine. Methods: Intestinal ion composition was measured by fame photometry and chloridometry and pH measured electrochemically. DNA extracted from intestinal flushes or from mucosal scrapings was analyzed by qRT-PCR to examine luminal and mucosa-associated bacterial populations. Epithelial mucus oligosaccharide patterns were examined by histology with FIT-C labeled lectins. Results: Although total luminal and mucosa-associated bacteria were unchanged in NHE2-/- intestine, gram-positive bacterial phyla were increased in the mucosa-associated bacterial population in a region-specific manner. The genera Clostridium and Lactobacillus were increased in the cecum and colon which corresponded to changes in NHE2-/- mucus oligosaccharide composition of mannose, N-acetyglucosamine, N-acetygalactosamine and galactose. Conclusions: Together these data indicate that changes in ion transport induce region-specific bacterial changes, which alter host mucus oligosaccharide patterns. These host-bacterial interactions provide a possible mechanism of niche-development and shed insight on how certain groups proliferate in changing environments and maintain their proliferation by altering the host.

  12. Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis

    Institute of Scientific and Technical Information of China (English)

    徐永忠; 谢建平; 李瑶; 乐军; 陈建平; 淳于利娟; 王洪海

    2003-01-01

    Objective To examine the global effects of Mycobacterium tuberculosis (M.tuberculosis) infection on macrophages. Methods The gene expression profiling of macrophage U937, in response to infection with M.tuberculosis H37Ra, was monitored using a high-density cDNA microarray. Results M.tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M.tuberculosis infection and intracellular survival. Conclusions M.tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M.tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

  13. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1990-12-31

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  14. Altered phenotypes in plants transformed with chimeric tobacco peroxidase genes

    Energy Technology Data Exchange (ETDEWEB)

    Lagrimini, L.M.

    1990-01-01

    Peroxidases have been implicated in a variety of secondary metabolic reactions including lignification, cross-linking of cell wall polysaccharides, oxidation of indole-3-acetic acid, regulation of cell elongation, wound-healing, phenol oxidation, and pathogen defense. However, due to the many different isoenzymes and even more potential substrates, it has proven difficult to verify actual physiological roles for peroxidase. We are studying the molecular biology of the tobacco peroxidase genes, and have utilized genetic engineering techniques to produce transgenic plants which differ only in their expression of an individual peroxidase isoenzyme. Many of the in planta functions for any individual isoenzyme may be predicted through the morphological and physiological analysis of transformed plants.

  15. Gene expression analysis during cassava defense response to bacterial blight disease

    OpenAIRE

    2006-01-01

    Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is a destructive disease in the South América and África and yield losses range between 12 and 100%. Cytochemistry and biochemistry of defense response to CBB have been well studied. However, the response of the plant to pathogen attack at the molecular and cellular level remains uncharacterized. Identification of genes associated with defense responses is one of most critical steps leading to the elucidation ...

  16. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles

    OpenAIRE

    Piel, Jörn

    2002-01-01

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were ...

  17. Antibiotic resistance genes occurrence and bacterial community composition in the Liuxi River

    Directory of Open Access Journals (Sweden)

    Wenguang eXiong

    2014-12-01

    Full Text Available Antibiotic resistance genes (ARGs in the environment have paid great concern due to their health risk. We investigated antibiotics concentrations (tetracyclines, sulfonamides and fluoroquinolones, ARGs abundances (tetracycline, sulfonamide and plasmid-mediated quinolone resistance (PMQR genes, and bacterial community composition in sediment and water samples in the Liuxi River, China. Antibiotics concentrations were determined by ultra-performance liquid chromatography-electrospray tandem mass spectrometry. ARGs abundances were quantified by a culture-independent method. Bacterial community composition was analyzed by metagenomic approach based on Ion Torrent Personal Genome Machine platform. Antibiotics concentrations were at the levels of 1.19 to 622 ug kg-1 in sediment samples and below the limit of detection to 127 ng L-1 in water samples. Relative abundances (ARGs copies/16S rRNA gene copies of detected ARGs were at the range of 10-5 to 10-2. The dominant phyla were Proteobacteria, Bacteroidetes and Verrucomicrobia in sediment samples, and were Proteobacteria, Actinobacteria and Bacteroidetes in water samples. The results indicated that the river environment was contaminated by antibiotics and may be as a reservoir of ARGs. This study provided quantitative data on antibiotics, ARGs and bacterial community composition in the Liuxi River, a geographical location different from the reported studies.

  18. Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris).

    Science.gov (United States)

    Erler, Silvio; Popp, Mario; Lattorff, H Michael G

    2011-03-29

    The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT). There is a lack of immune genes in social insects (e.g. honeybees) when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals). The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge.Antimicrobial peptides (AMP) (abaecin, defensin 1, hymenoptaecin) were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish) and JNK pathway (basket). Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment.Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the transcription

  19. Dynamics of immune system gene expression upon bacterial challenge and wounding in a social insect (Bombus terrestris.

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    Silvio Erler

    Full Text Available The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT. There is a lack of immune genes in social insects (e.g. honeybees when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals. The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge.Antimicrobial peptides (AMP (abaecin, defensin 1, hymenoptaecin were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish and JNK pathway (basket. Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment.Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

  20. Evolution of variation in presence and absence of genes in bacterial pathways

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    Francis Andrew R

    2012-04-01

    Full Text Available Abstract Background Bacterial genomes exhibit a remarkable degree of variation in the presence and absence of genes, which probably extends to the level of individual pathways. This variation may be a consequence of the significant evolutionary role played by horizontal gene transfer, but might also be explained by the loss of genes through mutation. A challenge is to understand why there would be variation in gene presence within pathways if they confer a benefit only when complete. Results Here, we develop a mathematical model to study how variation in pathway content is produced by horizontal transfer, gene loss and partial exposure of a population to a novel environment. Conclusions We discuss the possibility that variation in gene presence acts as cryptic genetic variation on which selection acts when the appropriate environment occurs. We find that a high level of variation in gene presence can be readily explained by decay of the pathway through mutation when there is no longer exposure to the selective environment, or when selection becomes too weak to maintain the genes. In the context of pathway variation the role of horizontal gene transfer is probably the initial introduction of a complete novel pathway rather than in building up the variation in a genome without the pathway.

  1. Bacterial plasmid-mediated quinolone resistance genes in aquatic environments in China

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    Yan, Lei; Liu, Dan; Wang, Xin-Hua; Wang, Yunkun; Zhang, Bo; Wang, Mingyu; Xu, Hai

    2017-01-01

    Emerging antimicrobial resistance is a major threat to human’s health in the 21st century. Understanding and combating this issue requires a full and unbiased assessment of the current status on the prevalence of antimicrobial resistance genes and their correlation with each other and bacterial groups. In aquatic environments that are known reservoirs for antimicrobial resistance genes, we were able to reach this goal on plasmid-mediated quinolone resistance (PMQR) genes that lead to resistance to quinolones and possibly also to the co-emergence of resistance to β-lactams. Novel findings were made that qepA and aac-(6′)-Ib genes that were previously regarded as similarly abundant with qnr genes are now dominant among PMQR genes in aquatic environments. Further statistical analysis suggested that the correlation between PMQR and β-lactam resistance genes in the environment is still weak, that the correlations between antimicrobial resistance genes could be weakened by sufficient wastewater treatment, and that the prevalence of PMQR has been implicated in environmental, pathogenic, predatory, anaerobic, and more importantly, human symbiotic bacteria. This work provides a comprehensive analysis of PMQR genes in aquatic environments in Jinan, China, and provides information with which combat with the antimicrobial resistance problem may be fought. PMID:28094345

  2. Prion disease induced alterations in gene expression in spleen and brain prior to clinical symptoms

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    Hyeon O Kim

    2008-09-01

    Full Text Available Hyeon O Kim1, Greg P Snyder1, Tyler M Blazey1, Richard E Race2, Bruce Chesebro2, Pamela J Skinner11Department of Veterinary and Biomedical Sciences, University of Minnesota, USA; 2NIH Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, Montana, USAAbstract: Prion diseases are fatal neurodegenerative disorders that affect animals and humans. There is a need to gain understanding of prion disease pathogenesis and to develop diagnostic assays to detect prion diseases prior to the onset of clinical symptoms. The goal of this study was to identify genes that show altered expression early in the disease process in the spleen and brain of prion disease-infected mice. Using Affymetrix microarrays, we identified 67 genes that showed increased expression in the brains of prion disease-infected mice prior to the onset of clinical symptoms. These genes function in many cellular processes including immunity, the endosome/lysosome system, hormone activity, and the cytoskeleton. We confirmed a subset of these gene expression alterations using other methods and determined the time course in which these changes occur. We also identified 14 genes showing altered expression prior to the onset of clinical symptoms in spleens of prion disease infected mice. Interestingly, four genes, Atp1b1, Gh, Anp32a, and Grn, were altered at the very early time of 46 days post-infection. These gene expression alterations provide insights into the molecular mechanisms underlying prion disease pathogenesis and may serve as surrogate markers for the early detection and diagnosis of prion disease.Keywords: prion disease, microarrays, gene expression

  3. Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

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    Mariana Gomes Germano

    2012-05-01

    Full Text Available The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE and their biochar (BC. Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (α-ARHD bacterial gene were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF and agriculture (AG -, and the biochar (SF_BC and AG_BC, respectively. Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.

  4. Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS.

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    Rosa C Baños

    2009-06-01

    Full Text Available Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

  5. Chromosomal Integration and Expression of Two Bacterial alpha-Acetolactate Decarboxylase Genes in Brewer's Yeast.

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    Blomqvist, K; Suihko, M L; Knowles, J; Penttilä, M

    1991-10-01

    A bacterial gene encoding alpha-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the alpha-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the alpha-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.

  6. Themes and Variations: Regulation of RpoN-Dependent Flagellar Genes across Diverse Bacterial Species

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    Jennifer Tsang

    2014-01-01

    Full Text Available Flagellar biogenesis in bacteria is a complex process in which the transcription of dozens of structural and regulatory genes is coordinated with the assembly of the flagellum. Although the overall process of flagellar biogenesis is conserved among bacteria, the mechanisms used to regulate flagellar gene expression vary greatly among different bacterial species. Many bacteria use the alternative sigma factor σ54 (also known as RpoN to transcribe specific sets of flagellar genes. These bacteria include members of the Epsilonproteobacteria (e.g., Helicobacter pylori and Campylobacter jejuni, Gammaproteobacteria (e.g., Vibrio and Pseudomonas species, and Alphaproteobacteria (e.g., Caulobacter crescentus. This review characterizes the flagellar transcriptional hierarchies in these bacteria and examines what is known about how flagellar gene regulation is linked with other processes including growth phase, quorum sensing, and host colonization.

  7. Impacts of Alterations of Organic Inputs on the Bacterial Community within the sediments of Wind Cave, South Dakota, USA

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    Chelius Marisa K.

    2009-01-01

    Full Text Available Wind Cave (WICA in the Black Hills of South Dakota, like many mostly dry caves in temperate regions is an energy-starved system.The biotic communities that reside in these systems are low in diversity and simple in structure, and sensitive to changes in externalinputs of organic matter. Caves open to tourist traffic offer an opportunity to study the impacts of organic matter amendments in theform of human and rodent hair and dander, clothing lint, material from rodent activity (nesting materials and feces, and algal growthin and around artificial lighting. This study reports on the impacts of carbon amendments from humans and rodents on the bacterialand archaeal communities within the sediments of WICA from annual surveys and from a manipulative study that added lint (‘L’;cellulose plus rodent dander and rodent hair, rodent feces (‘F’, and a combination of both (‘LF’. The survey confirmed that bacterialbiomass was higher in regions of the cave with the highest rates of lint (hair and natural clothing fibers input. The manipulative studyfound that organic amendments in the forms of lint (L and rodent feces (F altered the WICA bacterial community structure in bothabundance and diversity, with the combined lint and feces (LF amendment having the most significant response. The high similarityof the LF and L communities suggests that the cave bacterial community is more carbon than nitrogen limited. The implication ofcave development to management practices is immediate and practical. Even small amounts of lint and organic matter foreign tocave bacteria significantly compromise the integrity of the endemic community resulting in the replacement of undescribed speciesby assemblages with at best, unknown impacts to natural cave features.

  8. Horizontal gene transfer of a bacterial insect toxin gene into the Epichloë fungal symbionts of grasses

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    Ambrose, Karen V.; Koppenhöfer, Albrecht M.; Belanger, Faith C.

    2014-01-01

    Horizontal gene transfer is recognized as an important factor in genome evolution, particularly when the newly acquired gene confers a new capability to the recipient species. We identified a gene similar to the makes caterpillars floppy (mcf1 and mcf2) insect toxin genes in Photorhabdus, bacterial symbionts of nematodes, in the genomes of the Epichloë fungi, which are intercellular symbionts of grasses. Infection by Epichloë spp. often confers insect resistance to the grass hosts, largely due to the production of fungal alkaloids. A mcf-like gene is present in all of the Epichloë genome sequences currently available but in no other fungal genomes. This suggests the Epichloë genes were derived from a single lineage-specific HGT event. Molecular dating was used to estimate the time of the HGT event at between 7.2 and 58.8 million years ago. The mcf-like coding sequence from Epichloë typhina subsp. poae was cloned and expressed in Escherichia coli. E. coli cells expressing the Mcf protein were toxic to black cutworms (Agrotis ipsilon), whereas E. coli cells containing the vector only were non-toxic. These results suggest that the Epichloë mcf-like genes may be a component, in addition to the fungal alkaloids, of the insect resistance observed in Epichloë-infected grasses. PMID:24990771

  9. Epigenetic and Genetic Alterations Affect the WWOX Gene in Head and Neck Squamous Cell Carcinoma

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    Ekizoglu, Seda; Bulut, Pelin; Karaman, Emin; Kilic, Erkan; Buyru, Nur

    2015-01-01

    Different types of genetic and epigenetic changes are associated with HNSCC. The molecular mechanisms of HNSCC carcinogenesis are still undergoing intensive investigation. WWOX gene expression is altered in many cancers and in a recent work reduced WWOX expression has been associated with miR-134 expression in HNSCC. In this study we investigated the WWOX messenger RNA expression levels in association with the promoter methylation of the WWOX gene and miR-134 expression levels in 80 HNSCC tumor and non-cancerous tissue samples. Our results show that WWOX expression is down-regulated especially in advanced-stage tumor samples or in tumors with SCC. This down-regulation was associated with methylation of the WWOX promoter region but not with miR-134 expression. There was an inverse correlation between the expression level and promoter methylation. We also analyzed whole exons and exon/intron boundries of the WWOX gene by direct sequencing. In our study group we observed 10 different alterations in the coding sequences and 18 different alterations in the non-coding sequences of the WWOX gene in HNSCC tumor samples. These results indicate that the WWOX gene can be functionally inactivated by promoter methylation, epigenetically or by mutations affecting the sequences coding for the enzymatic domain of the gene, functionally. We conclude that inactivation of WWOX gene contributes to the progression of HNSCC. PMID:25612104

  10. Aerobic bacterial microbiota of the conjunctiva in diabetic patients with normal and altered glycated hemoglobin levels in two regions in Brazil

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    Natalia Pimentel Moreno

    2014-12-01

    Full Text Available Purpose: To study the aerobic bacterial microbiota of the conjunctiva in diabetic patients with regard to the management of diabetes, assessed using glycated hemoglobin levels. Methods: A cross-sectional study was conducted using conjunctival smears of diabetic patients from both sexes and with different ages, residing in two different Brazilian cities (Sorocaba and Rio Branco. A control group of non-diabetic patients was also included. The diabetic patients were considered to have controlled diabetes when their glycated hemoglobin level was ≤7% and blood glucose level was ≤126 mg/dL. Patients with non-controlled diabetes were those with glycated hemoglobin levels >7% and blood glucose levels >126 mg/dL. The samples obtained were inoculated in Brain-Heart Infusion broth and in culture media for aerobic bacteria (blood and chocolate agars; bacterial growth was evaluated in a microbiology laboratory. Results: A total of 120 eyes of 120 patients were included in the present study. The percentage of cultures in which bacterial growth was observed was greater in diabetic patients, although the difference was not statistically significant (p=0.103. There was a greater trend toward bacterial growth in the conjunctiva of diabetic patients with altered fasting blood glucose. There was no difference in the frequency of bacterial growth on the conjunctiva between diabetic patients with normal or altered glycated hemoglobin levels. In Sorocaba, conjunctival bacterial growth was similar to that observed in Rio Branco. The microorganism most frequently detected in the present study was Staphylococcus epidermidis, followed by Staphylococcus aureus, Proteus mirabilis, and Escherichia coli. Conclusion: There was no difference between diabetic patients with normal or altered glycated hemoglobin levels. The microorganisms found were similar to those found in studies investigating the conjunctival bacterial flora of diabetic and non-diabetic patients.

  11. Aerobic bacterial microbiota of the conjunctiva in diabetic patients with normal and altered glycated hemoglobin levels in two regions in Brazil.

    Science.gov (United States)

    Moreno, Natalia Pimentel; Moreno, Renaldo Duarte; Sousa, Luciene Barbosa

    2014-01-01

    To study the aerobic bacterial microbiota of the conjunctiva in diabetic patients with regard to the management of diabetes, assessed using glycated hemoglobin levels. A cross-sectional study was conducted using conjunctival smears of diabetic patients from both sexes and with different ages, residing in two different Brazilian cities (Sorocaba and Rio Branco). A control group of non-diabetic patients was also included. The diabetic patients were considered to have controlled diabetes when their glycated hemoglobin level was ≤7% and blood glucose level was ≤126 mg/dL. Patients with non-controlled diabetes were those with glycated hemoglobin levels >7% and blood glucose levels >126 mg/dL. The samples obtained were inoculated in Brain-Heart Infusion broth and in culture media for aerobic bacteria (blood and chocolate agars); bacterial growth was evaluated in a microbiology laboratory. A total of 120 eyes of 120 patients were included in the present study. The percentage of cultures in which bacterial growth was observed was greater in diabetic patients, although the difference was not statistically significant (p=0.103). There was a greater trend toward bacterial growth in the conjunctiva of diabetic patients with altered fasting blood glucose. There was no difference in the frequency of bacterial growth on the conjunctiva between diabetic patients with normal or altered glycated hemoglobin levels. In Sorocaba, conjunctival bacterial growth was similar to that observed in Rio Branco. The microorganism most frequently detected in the present study was Staphylococcus epidermidis, followed by Staphylococcus aureus, Proteus mirabilis, and Escherichia coli. There was no difference between diabetic patients with normal or altered glycated hemoglobin levels. The microorganisms found were similar to those found in studies investigating the conjunctival bacterial flora of diabetic and non-diabetic patients.

  12. Pyramiding blast, bacterial blight and brown planthopper resistance genes in rice restorer lines

    Institute of Scientific and Technical Information of China (English)

    JI Zhi-juan; Yang Shu-dong; ZENG Yu-xiang; LIANG Yan; YANG Chang-deng; QIAN Qian

    2016-01-01

    Rice blast, bacterial blight (BB) and brown planthopper (BPH) are the three main pests of rice. This study investigated pyr-amiding genes resistant to blast, BB and BPH to develop restorer lines. Ten new lines with blast, BB and/or BPH resistance genes were developed using marker-assisted selection (MAS) technique and agronomic trait selection (ATS) method. Only HR13 with resistance genes to blast, BB and BPH was obtained. In addition to blast and BB resistance, four lines (HR39, HR41, HR42, HR43) demonstrated moderate resistance to BPH, but MAS for BPH resistance genes were not conducted in developing these four lines. These data suggested that there were unknown elite BPH resistance genes in the Zhongzu 14 donor parent. A more effective defense was demonstrated in the lines withPi1 andPi2 genes although the weather in 2012 was favorable to disease incidence. Blast resistance of the lines with a single resistance gene,Pita, was easily inlfuenced by the weather. Overal, the information obtained through pyramiding multiple resistance genes on developing the restorer lines is helpful for rice resistance breeding.

  13. Gene expression in gut symbiotic organ of stinkbug affected by extracellular bacterial symbiont.

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    Ryo Futahashi

    Full Text Available The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations.

  14. Chronic mild stress alters circadian expressions of molecular clock genes in the liver.

    Science.gov (United States)

    Takahashi, Kei; Yamada, Tetsuya; Tsukita, Sohei; Kaneko, Keizo; Shirai, Yuta; Munakata, Yuichiro; Ishigaki, Yasushi; Imai, Junta; Uno, Kenji; Hasegawa, Yutaka; Sawada, Shojiro; Oka, Yoshitomo; Katagiri, Hideki

    2013-02-01

    Chronic stress is well known to affect metabolic regulation. However, molecular mechanisms interconnecting stress response systems and metabolic regulations have yet to be elucidated. Various physiological processes, including glucose/lipid metabolism, are regulated by the circadian clock, and core clock gene dysregulation reportedly leads to metabolic disorders. Glucocorticoids, acting as end-effectors of the hypothalamus-pituitary-adrenal (HPA) axis, entrain the circadian rhythms of peripheral organs, including the liver, by phase-shifting core clock gene expressions. Therefore, we examined whether chronic stress affects circadian expressions of core clock genes and metabolism-related genes in the liver using the chronic mild stress (CMS) procedure. In BALB/c mice, CMS elevated and phase-shifted serum corticosterone levels, indicating overactivation of the HPA axis. The rhythmic expressions of core clock genes, e.g., Clock, Npas2, Bmal1, Per1, and Cry1, were altered in the liver while being completely preserved in the hypothalamic suprachiasmatic nuculeus (SCN), suggesting that the SCN is not involved in alterations in hepatic core clock gene expressions. In addition, circadian patterns of glucose and lipid metabolism-related genes, e.g., peroxisome proliferator activated receptor (Ppar) α, Pparγ-1, Pparγ-coactivator-1α, and phosphoenolepyruvate carboxykinase, were also disturbed by CMS. In contrast, in C57BL/6 mice, the same CMS procedure altered neither serum corticosterone levels nor rhythmic expressions of hepatic core clock genes and metabolism-related genes. Thus, chronic stress can interfere with the circadian expressions of both core clock genes and metabolism-related genes in the liver possibly involving HPA axis overactivation. This mechanism might contribute to metabolic disorders in stressful modern societies.

  15. The AS87_04050 gene is involved in bacterial lipopolysaccharide biosynthesis and pathogenicity of Riemerella anatipestifer.

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    Xiaolan Wang

    Full Text Available Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. In this study, we identified a mutant strain RA2640 by Tn4351 transposon mutagenesis, in which the AS87_04050 gene was inactivated by insertion of the transposon. Southern blot analysis indicated that only one insertion was found in the genome of the mutant strain RA2640. SDS-PAGE followed by silver staining showed that the lipopolysaccharide (LPS pattern of mutant strain RA2640 was different from its wild-type strain Yb2, suggesting the LPS was defected. In addition, the phenotype of the mutant strain RA2640 was changed to rough-type, evident by altered colony morphology, autoaggregation ability and crystal violet staining characteristics. Bacterial LPS is a key factor in virulence as well as in both innate and acquired host responses to infection. The rough-type mutant strain RA2640 showed higher sensitivity to antibiotics, disinfectants and normal duck serum, and higher capability of adherence and invasion to Vero cells, compared to its wild-type strain Yb2. Moreover, the mutant strain RA2640 lost the agglutination ability of its wild-type strain Yb2 to R. anatipestifer serotype 2 positive sera, suggesting that the O-antigen is defected. Animal experiments indicated that the virulence of the mutant strain RA2640 was attenuated by more than 100,000-fold, compared to its wild-type strain Yb2. These results suggested that the AS87_04050 gene in R. anatipestifer is associated with the LPS biosynthesis and bacterial pathogenicity.

  16. 16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

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    Jan S Suchodolski

    Full Text Available BACKGROUND: Canine idiopathic inflammatory bowel disease (IBD is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6 and dogs with moderate IBD (n = 7 or severe IBD (n = 7 as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001. Proportions of Fusobacteria (p = 0.010, Bacteroidaceae (p = 0.015, Prevotellaceae (p = 0.022, and Clostridiales (p = 0.019 were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044 and Acinetobacter (p = 0.040, were either more abundant or more frequently identified in IBD dogs. CONCLUSIONS/SIGNIFICANCE: In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

  17. Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition

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    Zhu Zhu

    2016-01-01

    Full Text Available Background. The risk of atherosclerosis is elevated in abnormal lipid metabolism and circadian rhythm disorder. We investigated whether abnormal lighting condition would have influenced the circadian expression of clock genes and clock-controlled lipid metabolism-related genes in ApoE-KO mice. Methods. A mouse model of atherosclerosis with circadian clock genes expression disorder was established using ApoE-KO mice (ApoE-KO LD/DL mice by altering exposure to light. C57 BL/6J mice (C57 mice and ApoE-KO mice (ApoE-KO mice exposed to normal day and night and normal diet served as control mice. According to zeitgeber time samples were acquired, to test atheromatous plaque formation, serum lipids levels and rhythmicity, clock genes, and lipid metabolism-related genes along with Sirtuin 1 (Sirt1 levels and rhythmicity. Results. Atherosclerosis plaques were formed in the aortic arch of ApoE-KO LD/DL mice. The serum lipids levels and oscillations in ApoE-KO LD/DL mice were altered, along with the levels and diurnal oscillations of circadian genes, lipid metabolism-associated genes, and Sirt1 compared with the control mice. Conclusions. Abnormal exposure to light aggravated plaque formation and exacerbated disorders of serum lipids and clock genes, lipid metabolism genes and Sirt1 levels, and circadian oscillation.

  18. Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition.

    Science.gov (United States)

    Zhu, Zhu; Hua, Bingxuan; Shang, Zhanxian; Yuan, Gongsheng; Xu, Lirong; Li, Ermin; Li, Xiaobo; Sun, Ning; Yan, Zuoqin; Qian, Ruizhe; Lu, Chao

    2016-01-01

    Background. The risk of atherosclerosis is elevated in abnormal lipid metabolism and circadian rhythm disorder. We investigated whether abnormal lighting condition would have influenced the circadian expression of clock genes and clock-controlled lipid metabolism-related genes in ApoE-KO mice. Methods. A mouse model of atherosclerosis with circadian clock genes expression disorder was established using ApoE-KO mice (ApoE-KO LD/DL mice) by altering exposure to light. C57 BL/6J mice (C57 mice) and ApoE-KO mice (ApoE-KO mice) exposed to normal day and night and normal diet served as control mice. According to zeitgeber time samples were acquired, to test atheromatous plaque formation, serum lipids levels and rhythmicity, clock genes, and lipid metabolism-related genes along with Sirtuin 1 (Sirt1) levels and rhythmicity. Results. Atherosclerosis plaques were formed in the aortic arch of ApoE-KO LD/DL mice. The serum lipids levels and oscillations in ApoE-KO LD/DL mice were altered, along with the levels and diurnal oscillations of circadian genes, lipid metabolism-associated genes, and Sirt1 compared with the control mice. Conclusions. Abnormal exposure to light aggravated plaque formation and exacerbated disorders of serum lipids and clock genes, lipid metabolism genes and Sirt1 levels, and circadian oscillation.

  19. OpWise: Operons aid the identification of differentially expressed genes in bacterial microarray experiments

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    Arkin Adam P

    2006-01-01

    Full Text Available Abstract Background Differentially expressed genes are typically identified by analyzing the variation between replicate measurements. These procedures implicitly assume that there are no systematic errors in the data even though several sources of systematic error are known. Results OpWise estimates the amount of systematic error in bacterial microarray data by assuming that genes in the same operon have matching expression patterns. OpWise then performs a Bayesian analysis of a linear model to estimate significance. In simulations, OpWise corrects for systematic error and is robust to deviations from its assumptions. In several bacterial data sets, significant amounts of systematic error are present, and replicate-based approaches overstate the confidence of the changers dramatically, while OpWise does not. Finally, OpWise can identify additional changers by assigning genes higher confidence if they are consistent with other genes in the same operon. Conclusion Although microarray data can contain large amounts of systematic error, operons provide an external standard and allow for reasonable estimates of significance. OpWise is available at http://microbesonline.org/OpWise.

  20. The population and evolutionary dynamics of homologous gene recombination in bacterial populations.

    Directory of Open Access Journals (Sweden)

    Bruce R Levin

    2009-08-01

    Full Text Available In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination -- broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT -- plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1 the contribution of HGR to the rate of adaptive evolution in these populations and (2 the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1 HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2 once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent

  1. An improved method for detecting and delineating genomic regions with altered gene expression in cancer

    OpenAIRE

    2008-01-01

    Genomic regions with altered gene expression are a characteristic feature of cancer cells. We present a novel method for identifying such regions in gene expression maps. This method is based on total variation minimization, a classical signal restoration technique. In systematic evaluations, we show that our method combines top-notch detection performance with an ability to delineate relevant regions without excessive over-segmentation, making it a significant advance over existing methods. ...

  2. Altered global gene expression profiles in human gastrointestinal epithelial Caco2 cells exposed to nanosilver

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    Saura C. Sahu

    2016-01-01

    Full Text Available Extensive consumer exposure to food- and cosmetics-related consumer products containing nanosilver is of public safety concern. Therefore, there is a need for suitable in vitro models and sensitive predictive rapid screening methods to assess their toxicity. Toxicogenomic profile showing subtle changes in gene expressions following nanosilver exposure is a sensitive toxicological endpoint for this purpose. We evaluated the Caco2 cells and global gene expression profiles as tools for predictive rapid toxicity screening of nanosilver. We evaluated and compared the gene expression profiles of Caco-2 cells exposed to 20 nm and 50 nm nanosilver at a concentration 2.5 μg/ml. The global gene expression analysis of Caco2 cells exposed to 20 nm nanosilver showed that a total of 93 genes were altered at 4 h exposure, out of which 90 genes were up-regulated and 3 genes were down-regulated. The 24 h exposure of 20 nm silver altered 15 genes in Caco2 cells, out of which 14 were up-regulated and one was down-regulated. The most pronounced changes in gene expression were detected at 4 h. The greater size (50 nm nanosilver at 4 h exposure altered more genes by more different pathways than the smaller (20 nm one. Metallothioneins and heat shock proteins were highly up-regulated as a result of exposure to both the nanosilvers. The cellular pathways affected by the nanosilver exposure is likely to lead to increased toxicity. The results of our study presented here suggest that the toxicogenomic characterization of Caco2 cells is a valuable in vitro tool for assessing toxicity of nanomaterials such as nanosilver.

  3. NF-Y activates genes of metabolic pathways altered in cancer cells.

    Science.gov (United States)

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-12

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

  4. Altered gene expression in highly purified enterocytes from patients with active coeliac disease

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    Jackson John

    2008-08-01

    Full Text Available Abstract Background Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. Results Enterocyte suspensions of high purity (98–99% were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p Conclusion This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease.

  5. Bacterial intra-species gene loss occurs in a largely clocklike manner mostly within a pool of less conserved and constrained genes.

    Science.gov (United States)

    Bolotin, Evgeni; Hershberg, Ruth

    2016-10-13

    Gene loss is a major contributor to the evolution of bacterial gene content. Gene loss may occur as a result of shifts in environment leading to changes in the intensity and/or directionality of selection applied for the maintenance of specific genes. Gene loss may also occur in a more neutral manner, when gene functions are lost that were not subject to strong selection to be maintained, irrespective of changes to environment. Here, we used a pangenome-based approach to investigate patterns of gene loss across 15 bacterial species. We demonstrate that gene loss tends to occur mostly within a pool of genes that are less constrained within species, even in those strains from which they are not lost, and less conserved across bacterial species. Our results indicate that shifts in selection, resulting from shifts in environment are not required to explain the majority of gene loss events occurring within a diverse collection of bacterial species. Caution should therefore be taken when attributing differences in gene content to differences in environment.

  6. Gene expression analysis during cassava defense response to bacterial blight disease

    Directory of Open Access Journals (Sweden)

    Soto-Suárez Mauricio

    2006-12-01

    Full Text Available Cassava bacterial blight (CBB caused by Xanthomonas axonopodis pv. manihotis (Xam is a destructive disease in the South América and África and yield losses range between 12 and 100%. Cytochemistry and biochemistry of defense response to CBB have been well studied. However, the response of the plant to pathogen attack at the molecular and cellular level remains uncharacterized. Identification of genes associated with defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in cassava. In this study, we identified differentially expressed genes during pathogen attack by subtractive hybridization, using the Differential Subtraction Chain method (DSC. A population of cDNA obtained from infected plants was used as ";treatment"; and a population of cDNA obtained from healthy plants was used as ";control";. 1536 clones were isolated from the resistant varieties (MBRA 685 and SG 107-35. Of these, 110 randomly selected clones were sequenced and a homology search was conducted. The sequence analysis showed that 14 cDNA clones shared homology with plant genes involved in defense responses, 70 clones were either homologous to plant genes of unknown function or showed no homology, representing new genes potentially involved in cassava defense responses. A cDNA microarray was constructed by spotting the clones identified from our subtractive libraries. Other clones potentially involved in cassava defense responses were also included. The cassava defense cDNA microarray was used to confirm the differential expression of the clones. Keywords: cassava, bacterial blight, gene expression, subtractive library, microarrays.

  7. Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

    Science.gov (United States)

    Kolotilin, Igor; Koltai, Hinanit; Bar-Or, Carmiya; Chen, Lea; Nahon, Sahadia; Shlomo, Haviva; Levin, Ilan; Reuveni, Moshe

    2011-07-01

    Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed.

  8. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

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    Kaul Rajinder

    2009-11-01

    Full Text Available Abstract Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia

  9. Analysis of gene expression levels in individual bacterial cells without image segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, In Hae; Son, Minjun [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States); Hagen, Stephen J., E-mail: sjhagen@ufl.edu [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  10. Differential gene expression in the honeybee head after a bacterial challenge.

    Science.gov (United States)

    Scharlaken, Bieke; de Graaf, Dirk C; Goossens, Karen; Peelman, Luc J; Jacobs, Frans J

    2008-01-01

    Bidirectional interactions between the immune and nervous systems are well established in vertebrates. Insects show similar neuro-immune-behavioral interactions to those seen in vertebrates. Using quantitative real-time PCR, we present evidence that gene expression in the honeybee head is influenced by activation of the immune system 8h after a bacterial challenge with Escherichia coli. Seven genes were selected for quantitative analysis in order to cover both typical functions of the head such as exocrine secretion (mrjp3 and mrjp4) and olfactory processes (obp17) as well as more general processes such as structural functions (mlc2 and paramyosin), stress response (ERp60) and energy housekeeping (enolase). In this way, we show at the molecular level that the immune system functions as a sensory organ in insects -- as it does in vertebrates -- which signals to the head that a bacterial infection is present, and leads to regulation of expression of several genes in the head by a yet unidentified mechanism.

  11. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles.

    Science.gov (United States)

    Piel, Jörn

    2002-10-29

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were cloned from total DNA of Paederus fuscipes beetles, which use this compound for chemical defense. Sequence analysis of the gene cluster and adjacent regions revealed the presence of ORFs with typical bacterial architecture and homologies. The ped cluster, which is present only in beetle specimens with high pederin content, is located on a 54-kb region bordered by transposase pseudogenes and encodes a mixed modular polyketide synthase/nonribosomal peptide synthetase. Notably, none of the modules contains regions with homology to acyltransferase domains, but two copies of isolated monodomain acyltransferase genes were found at the upstream end of the cluster. In line with an involvement in pederin biosynthesis, the upstream cluster region perfectly mirrors pederin structure. The unexpected presence of additional polyketide synthase/nonribosomal peptide synthetase modules reveals surprising insights into the evolutionary relationship between pederin-type pathways in beetles and sponges.

  12. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  13. Comparing wastewater chemicals, indicator bacteria concentrations, and bacterial pathogen genes as fecal pollution indicators

    Science.gov (United States)

    Haack, S.K.; Duris, J.W.; Fogarty, L.R.; Kolpin, D.W.; Focazio, M.J.; Furlong, E.T.; Meyer, M.T.

    2009-01-01

    The objective of this study was to compare fecal indicator bacteria (FIB) (fecal coliforms, Escherichia coli [EC], and enterococci [ENT]) concentrations with a wide array of typical organic wastewater chemicals and selected bacterial genes as indicators of fecal pollution in water samples collected at or near 18 surface water drinking water intakes. Genes tested included esp (indicating human-pathogenic ENT) and nine genes associated with various animal sources of shiga-toxin-producing EC (STEC). Fecal pollution was indicated by genes and/or chemicals for 14 of the 18 tested samples, with little relation to FIB standards. Of 13 samples with antibiotics were detected in three, and stx1 or stx2 genes (indicating varying animal sources of STEC) were detected in eight. Only the EC eaeA gene was positively correlated with FIB concentrations. Human-source fecal pollution was indicated by the esp gene and the human pharmaceutical carbamazepine in one of the nine samples that met all FIB recreational water quality standards. Escherichia coli rfbO157 and stx2c genes, which are typically associated with cattle sources and are of potential human health significance, were detected in one sample in the absence of tested chemicals. Chemical and gene-based indicators of fecal contamination may be present even when FIB standards are met, and some may, unlike FIB, indicate potential sources. Application of multiple water quality indicators with variable environmental persistence and fate may yield greater confidence in fecal pollution assessment and may inform remediation decisions. Copyright ?? 2009 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved.

  14. Early maternal alcohol consumption alters hippocampal DNA methylation, gene expression and volume in a mouse model.

    Directory of Open Access Journals (Sweden)

    Heidi Marjonen

    Full Text Available The adverse effects of alcohol consumption during pregnancy are known, but the molecular events that lead to the phenotypic characteristics are unclear. To unravel the molecular mechanisms, we have used a mouse model of gestational ethanol exposure, which is based on maternal ad libitum ingestion of 10% (v/v ethanol for the first 8 days of gestation (GD 0.5-8.5. Early neurulation takes place by the end of this period, which is equivalent to the developmental stage early in the fourth week post-fertilization in human. During this exposure period, dynamic epigenetic reprogramming takes place and the embryo is vulnerable to the effects of environmental factors. Thus, we hypothesize that early ethanol exposure disrupts the epigenetic reprogramming of the embryo, which leads to alterations in gene regulation and life-long changes in brain structure and function. Genome-wide analysis of gene expression in the mouse hippocampus revealed altered expression of 23 genes and three miRNAs in ethanol-exposed, adolescent offspring at postnatal day (P 28. We confirmed this result by using two other tissues, where three candidate genes are known to express actively. Interestingly, we found a similar trend of upregulated gene expression in bone marrow and main olfactory epithelium. In addition, we observed altered DNA methylation in the CpG islands upstream of the candidate genes in the hippocampus. Our MRI study revealed asymmetry of brain structures in ethanol-exposed adult offspring (P60: we detected ethanol-induced enlargement of the left hippocampus and decreased volume of the left olfactory bulb. Our study indicates that ethanol exposure in early gestation can cause changes in DNA methylation, gene expression, and brain structure of offspring. Furthermore, the results support our hypothesis of early epigenetic origin of alcohol-induced disorders: changes in gene regulation may have already taken place in embryonic stem cells and therefore can be seen in

  15. Gene expression patterns underlying parasite-induced alterations in host behaviour and life history.

    Science.gov (United States)

    Feldmeyer, Barbara; Mazur, Johanna; Beros, Sara; Lerp, Hannes; Binder, Harald; Foitzik, Susanne

    2016-01-01

    Many parasites manipulate their hosts' phenotype. In particular, parasites with complex life cycles take control of their intermediate hosts' behaviour and life history to increase transmission to their definitive host. The proximate mechanisms underlying these parasite-induced alterations are poorly understood. The cestode Anomotaenia brevis affects the behaviour, life history and morphology of parasitized Temnothorax nylanderi ants and indirectly of their unparasitized nestmates. To gain insights on how parasites alter host phenotypes, we contrast brain gene expression patterns of T. nylanderi workers parasitized with the cestode, their unparasitized nestmates and unparasitized workers from unparasitized colonies. Over 400 differentially expressed genes between the three groups were identified, with most uniquely expressed genes detected in parasitized workers. Among these are genes that can be linked to the increased lifespan of parasitized workers. Furthermore, many muscle (functionality) genes are downregulated in these workers, potentially causing the observed muscular deformations and their inactive behaviour. Alterations in lifespan and activity could be adaptive for the parasite by increasing the likelihood that infected workers residing in acorns are eaten by their definitive host, a woodpecker. Our transcriptome analysis reveals numerous gene expression changes in parasitized workers and their uninfected nestmates and indicates possible routes of parasite manipulation. Although causality still needs to be established, parasite-induced alterations in lifespan and host behaviour appear to be partly explained by morphological muscle atrophy instead of central nervous system interference, which is often the core of behavioural regulation. Results of this study will shed light upon the molecular basis of antagonistic species interactions.

  16. Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

    Directory of Open Access Journals (Sweden)

    Weber Mitch

    2008-03-01

    Full Text Available Abstract Background Women with polycystic ovary syndrome (PCOS are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD, which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a mice, possessing a mutation (Ay in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction. Methods Female LY mice received daily oral doses of either 0.01 mg pioglitazone (n = 4 or an equal volume of vehicle (DMSO; n = 4 for 8 weeks. At the end of treatment, ovaries were removed and DNA microarrays were used to analyze differential gene expression. Results Twenty-seven genes showed at least a two-fold difference in ovarian expression with pioglitazone treatment. These included leptin, angiopoietin, angiopoietin-like 4, Foxa3, PGE1 receptor, resistin-like molecule-alpha (RELM, and actin-related protein 6 homolog (ARP6. For most altered genes, pioglitazone changed levels of expression to those seen in untreated C57BL/6J(a/a non-mutant lean mice. Conclusion TZD administration may influence ovarian function via numerous diverse mechanisms that may or may not be directly related to insulin/IGF signaling.

  17. Genome-wide identification of Streptococcus pneumoniae genes essential for bacterial replication during experimental meningitis

    DEFF Research Database (Denmark)

    Molzen, T E; Burghout, P; Bootsma, H J

    2010-01-01

    Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis...... of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis...

  18. Antibiotics, Antibiotic Resistance Genes, and Bacterial Community Composition in Fresh Water Aquaculture Environment in China.

    Science.gov (United States)

    Xiong, Wenguang; Sun, Yongxue; Zhang, Tong; Ding, Xueyao; Li, Yafei; Wang, Mianzhi; Zeng, Zhenling

    2015-08-01

    Environmental antibiotic resistance has drawn increasing attention due to its great threat to human health. In this study, we investigated concentrations of antibiotics (tetracyclines, sulfonamides and (fluoro)quinolones) and abundances of antibiotic resistance genes (ARGs), including tetracycline resistance genes, sulfonamide resistance genes, and plasmid-mediated quinolone resistance genes, and analyzed bacterial community composition in aquaculture environment in Guangdong, China. The concentrations of sulfametoxydiazine, sulfamethazine, sulfamethoxazole, oxytetracycline, chlorotetracycline, doxycycline, ciprofloxacin, norfloxacin, and enrofloxacin were as high as 446 μg kg(-1) and 98.6 ng L(-1) in sediment and water samples, respectively. The relative abundances (ARG copies/16S ribosomal RNA (rRNA) gene copies) of ARGs (sul1, sul2, sul3, tetM, tetO, tetW, tetS, tetQ, tetX, tetB/P, qepA, oqxA, oqxB, aac(6')-Ib, and qnrS) were as high as 2.8 × 10(-2). The dominant phyla were Proteobacteria, Bacteroidetes, and Firmicutes in sediment samples and Proteobacteria, Actinobacteria and Bacteroidetes in water samples. The genera associated with pathogens were also observed, such as Acinetobacter, Arcobacter, and Clostridium. This study comprehensively investigated antibiotics, ARGs, and bacterial community composition in aquaculture environment in China. The results indicated that fish ponds are reservoirs of ARGs and the presence of potential resistant and pathogen-associated taxonomic groups in fish ponds might imply the potential risk to human health.

  19. Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections.

    Science.gov (United States)

    Pridgeon, Julia W; Aksoy, Mediha; Klesius, Phillip H; Li, Yuehong; Mu, Xingjiang; Srivastava, Kunwar; Reddy, Gopal

    2011-11-15

    To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (ptilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (ptilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (ptilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.

  20. Francisella tularensis subsp. tularensis induces a unique pulmonary inflammatory response: role of bacterial gene expression in temporal regulation of host defense responses.

    Directory of Open Access Journals (Sweden)

    Kathie-Anne Walters

    Full Text Available Pulmonary exposure to Francisella tularensis is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-β and TNF-α, during early infection has led to the suggestion that F. tularensis evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to Francisella infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (Francisella tularensis ssp tularensis SchuS4. Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, Francisella tularensis was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (Yersinia pestis and non-lethal (Legionella pneumophila, Pseudomonas aeruginosa pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of Francisella. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of Francisella-specific transcripts, including those associated with bacterial surface components. In summary, virulent Francisella induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and Francisella transcriptome changes that occur during in vivo infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways.

  1. Alterations of tumor suppressor gene p16INK4a in pancreatic ductal carcinoma

    Directory of Open Access Journals (Sweden)

    Radotra Bishan

    2005-06-01

    Full Text Available Abstract Background Cell cycle inhibitor and tumor suppressor gene p16 / MTS-1 has been reported to be altered in a variety of human tumors. The purpose of the study was to evaluate primary pancreatic ductal adenocarcinomas for potentially inactivating p16 alterations. Methods We investigated the status of p16 gene by polymerase chain reaction (PCR, nonradioisotopic single strand conformation polymorphism (SSCP, DNA sequencing and hypermethylation analysis in 25 primary resected ductal adenocarcinomas. In addition, we investigated p16 protein expression in these cases by immunohistochemistry (IHC using a monoclonal antibody clone (MS-887-PO. Results Out of the 25 samples analyzed and compared to normal pancreatic control tissues, the overall frequency of p16 alterations was 80% (20/25. Aberrant promoter methylation was the most common mechanism of gene inactivation present in 52% (13/25 cases, followed by coding sequence mutations in 16% (4/25 cases and presumably homozygous deletion in 12% (3/25 cases. These genetic alterations correlated well with p16 protein expression as complete loss of p16 protein was found in 18 of 25 tumors (72%. Conclusion These findings confirm that loss of p16 function could be involved in pancreatic cancer and may explain at least in part the aggressive behaviour of this tumor type.

  2. Alterations of tumor suppressor gene p16INK4a in pancreatic ductal carcinoma

    Science.gov (United States)

    Attri, Jyotika; Srinivasan, Radhika; Majumdar, Siddhartha; Radotra, Bishan Dass; Wig, Jaidev

    2005-01-01

    Background Cell cycle inhibitor and tumor suppressor gene p16 / MTS-1 has been reported to be altered in a variety of human tumors. The purpose of the study was to evaluate primary pancreatic ductal adenocarcinomas for potentially inactivating p16 alterations. Methods We investigated the status of p16 gene by polymerase chain reaction (PCR), nonradioisotopic single strand conformation polymorphism (SSCP), DNA sequencing and hypermethylation analysis in 25 primary resected ductal adenocarcinomas. In addition, we investigated p16 protein expression in these cases by immunohistochemistry (IHC) using a monoclonal antibody clone (MS-887-PO). Results Out of the 25 samples analyzed and compared to normal pancreatic control tissues, the overall frequency of p16 alterations was 80% (20/25). Aberrant promoter methylation was the most common mechanism of gene inactivation present in 52% (13/25) cases, followed by coding sequence mutations in 16% (4/25) cases and presumably homozygous deletion in 12% (3/25) cases. These genetic alterations correlated well with p16 protein expression as complete loss of p16 protein was found in 18 of 25 tumors (72%). Conclusion These findings confirm that loss of p16 function could be involved in pancreatic cancer and may explain at least in part the aggressive behaviour of this tumor type. PMID:15985168

  3. Di-(2 ethylhexyl phthalate and flutamide alter gene expression in the testis of immature male rats

    Directory of Open Access Journals (Sweden)

    Yu Frank H

    2009-09-01

    Full Text Available Abstract We previously demonstrated that the androgenic and anti-androgenic effects of endocrine disruptors (EDs alter reproductive function and exert distinct effects on developing male reproductive organs. To further investigate these effects, we used an immature rat model to examine the effects of di-(2 ethylhexyl phthalate (DEHP and flutamide (Flu on the male reproductive system. Immature male SD rats were treated daily with DEHP and Flu on postnatal days (PNDs 21 to 35, in a dose-dependent manner. As results, the weights of the testes, prostate, and seminal vesicle and anogenital distances (AGD decreased significantly in response to high doses of DEHP or Flu. Testosterone (T levels significantly decreased in all DEHP- treated groups, whereas luteinizing hormone (LH plasma levels were not altered by any of the two treatments at PND 36. However, treatment with DEHP or Flu induced histopathological changes in the testes, wherein degeneration and disorders of Leydig cells, germ cells and dilatation of tubular lumen were observed in a dose-dependent manner. Conversely, hyperplasia and denseness of Leydig, Sertoli and germ cells were observed in rats given with high doses of Flu. The results by cDNA microarray analysis indicated that 1,272 genes were up-regulated by more than two-fold, and 1,969 genes were down-regulated in response to DEHP, Flu or both EDs. These genes were selected based on their markedly increased or decreased expression levels. These genes have been also classified on the basis of gene ontology (e.g., steroid hormone biosynthetic process, regulation of transcription, signal transduction, metabolic process, biosynthetic process.... Significant decreases in gene expression were observed in steroidogenic genes (i.e., Star, Cyp11a1 and Hsd3b. In addition, the expression of a common set of target genes, including CaBP1, Vav2, Plcd1, Lhx1 and Isoc1, was altered following exposure to EDs, suggesting that they may be marker genes to

  4. Simultaneous amplification of two bacterial genes: more reliable method of Helicobacter pylori detection in microbial rich dental plaque samples.

    Science.gov (United States)

    Chaudhry, Saima; Idrees, Muhammad; Izhar, Mateen; Butt, Arshad Kamal; Khan, Ayyaz Ali

    2011-01-01

    Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.

  5. Duration of chronic inflammation alters gene expression in muscle from untreated girls with juvenile dermatomyositis

    Directory of Open Access Journals (Sweden)

    Gordish-Dressman Heather

    2008-07-01

    Full Text Available Abstract Background To evaluate the impact of the duration of chronic inflammation on gene expression in skeletal muscle biopsies (MBx from untreated children with juvenile dermatomyositis (JDM and identify genes and biological processes associated with the disease progression, expression profiling data from 16 girls with active symptoms of JDM greater than or equal to 2 months were compared with 3 girls with active symptoms less than 2 months. Results Seventy-nine genes were differentially expressed between the groups with long or short duration of untreated disease. Genes involved in immune responses and vasculature remodelling were expressed at a higher level in muscle biopsies from children with greater or equal to 2 months of symptoms, while genes involved in stress responses and protein turnover were expressed at a lower level. Among the 79 genes, expression of 9 genes showed a significant linear regression relationship with the duration of untreated disease. Five differentially expressed genes – HLA-DQA1, smooth muscle myosin heavy chain, clusterin, plexin D1 and tenomodulin – were verified by quantitative RT-PCR. The chronic inflammation of longer disease duration was also associated with increased DC-LAMP+ and BDCA2+ mature dendritic cells, identified by immunohistochemistry. Conclusion We conclude that chronic inflammation alters the gene expression patterns in muscle of untreated children with JDM. Symptoms lasting greater or equal to 2 months were associated with dendritic cell maturation and anti-angiogenic vascular remodelling, directly contributing to disease pathophysiology.

  6. Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.

    Science.gov (United States)

    Gesta, S; Lolmède, K; Daviaud, D; Berlan, M; Bouloumié, A; Lafontan, M; Valet, P; Saulnier-Blache, J S

    2003-03-01

    Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.

  7. Frequent alteration of the tumor suppressor gene APC in sporadic canine colorectal tumors.

    Science.gov (United States)

    Youmans, Lydia; Taylor, Cynthia; Shin, Edwin; Harrell, Adrienne; Ellis, Angela E; Séguin, Bernard; Ji, Xinglai; Zhao, Shaying

    2012-01-01

    Sporadic canine colorectal cancers (CRCs) should make excellent models for studying the corresponding human cancers. To molecularly characterize canine CRC, we investigated exonic sequence mutations of adenomatous polyposis coli (APC), the best known tumor suppressor gene of human CRC, in 23 sporadic canine colorectal tumors, including 8 adenomas and 15 adenocarcinomas, via exon-resequencing analysis. As a comparison, we also performed the same sequencing analysis on 10 other genes, either located at human 5q22 (the same locus as APC) or 18q21 (also frequently altered in human CRC), or known to play a role in human carcinogenesis. We noted that APC was the most significantly mutated gene in both canine adenomas and adenocarcinomas among the 11 genes examined. Significantly, we detected large deletions of ≥ 10 bases, many clustered near the mutation cluster region, as well as single or two base deletions in ~70% canine tumors of both subtypes. These observations indicate that like in the human, APC is also frequently altered in sporadic colorectal tumors in the dog and its alteration is an early event in canine colorectal tumorigenesis. Our study provides further evidence demonstrating the molecular similarity in pathogenesis between sporadic human and canine CRCs. This work, along with our previous copy number abnormality study, supports that sporadic canine CRCs are valid models of human CRCs at the molecular level.

  8. Altered circadian rhythm and metabolic gene profile in rats subjected to advanced light phase shifts.

    Directory of Open Access Journals (Sweden)

    Laura Herrero

    Full Text Available The circadian clock regulates metabolic homeostasis and its disruption predisposes to obesity and other metabolic diseases. However, the effect of phase shifts on metabolism is not completely understood. We examined whether alterations in the circadian rhythm caused by phase shifts induce metabolic changes in crucial genes that would predispose to obesity. Three-month-old rats were maintained on a standard diet under lighting conditions with chronic phase shifts consisting of advances, delays or advances plus delays. Serum leptin, insulin and glucose levels decreased only in rats subjected to advances. The expression of the clock gene Bmal 1 increased in the hypothalamus, white adipose tissue (WAT, brown adipose tissue (BAT and liver of the advanced group compared to control rats. The advanced group showed an increase in hypothalamic AgRP and NPY mRNA, and their lipid metabolism gene profile was altered in liver, WAT and BAT. WAT showed an increase in inflammation and ER stress and brown adipocytes suffered a brown-to-white transformation and decreased UCP-1 expression. Our results indicate that chronic phase advances lead to significant changes in neuropeptides, lipid metabolism, inflammation and ER stress gene profile in metabolically relevant tissues such as the hypothalamus, liver, WAT and BAT. This highlights a link between alteration of the circadian rhythm and metabolism at the transcriptional level.

  9. VERTICAL HEREDITY VS. HORIZONTAL GENE TRANSFER: A CHALLENGE TO BACTERIAL CLASSIFICATION

    Institute of Scientific and Technical Information of China (English)

    HAO Bailin; QI Ji

    2003-01-01

    The diversity and classification of microbes has been a long-standing issue. Molecular phylogeny of the prokaryotes based on comparison of the 16S rRNA sequences of the small ribosomal subunit has led to a reasonable tree of life in the late 1970s. However, the availability of more and more complete bacterial genomes has brought about complications instead of refinement of the tree. In particular, it turns out that different choice of genes may tell different history. This might be caused by possible horizontal gene transfer (HGT) among species. There is an urgent need to develop phylogenetic methods that make use of whole genome data. We describe a new approach in molecular phylogeny, namely, tree construction based on K-tuple frequency analysis of the genomic sequences. Putting aside the technicalities, we emphasize the transition from randomness to determinism when the string length K increases and try to comment on the challenge mentioned in the title.

  10. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.

    Science.gov (United States)

    Gong, Shiaoching; Zheng, Chen; Doughty, Martin L; Losos, Kasia; Didkovsky, Nicholas; Schambra, Uta B; Nowak, Norma J; Joyner, Alexandra; Leblanc, Gabrielle; Hatten, Mary E; Heintz, Nathaniel

    2003-10-30

    The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.

  11. Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host.

    Science.gov (United States)

    Yamada, Yuuki; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo

    2015-06-01

    Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.

  12. Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure, with Concomitant Bacterial Population Changes.

    Science.gov (United States)

    Wang, Hang; Li, Hongyi; Gilbert, Jack A; Li, Haibo; Wu, Longhua; Liu, Meng; Wang, Liling; Zhou, Qiansheng; Yuan, Junxiang; Zhang, Zhijian

    2015-11-01

    Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P biofertilizer in agroecosystems. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Benchmarking of methods for identification of antimicrobial resistance genes in bacterial whole genome data

    DEFF Research Database (Denmark)

    Clausen, Philip T. L. C.; Zankari, Ea; Aarestrup, Frank Møller;

    2016-01-01

    Next generation sequencing (NGS) may be an alternative to phenotypic susceptibility testing for surveillance and clinical diagnosis. However, current bioinformatics methods may be associated with false positives and negatives. In this study, a novel mapping method was developed and benchmarked...... to two different methods in current use for identification of antibiotic resistance genes in bacterial WGS data. A novel method, KmerResistance, which examines the co-occurrence of k-mers between the WGS data and a database of resistance genes, was developed. The performance of this method was compared...... with two previously described methods; ResFinder and SRST2, which use an assembly/BLAST method and BWA, respectively, using two datasets with a total of 339 isolates, covering five species, originating from the Oxford University Hospitals NHS Trust and Danish pig farms. The predicted resistance...

  14. Altered gene expression pattern in peripheral blood mononuclear cells in patients with acute myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Marek Kiliszek

    Full Text Available BACKGROUND: Despite a substantial progress in diagnosis and therapy, acute myocardial infarction (MI is a major cause of mortality in the general population. A novel insight into the pathophysiology of myocardial infarction obtained by studying gene expression should help to discover novel biomarkers of MI and to suggest novel strategies of therapy. The aim of our study was to establish gene expression patterns in leukocytes from acute myocardial infarction patients. METHODS AND RESULTS: Twenty-eight patients with ST-segment elevation myocardial infarction (STEMI were included. The blood was collected on the 1(st day of myocardial infarction, after 4-6 days, and after 6 months. Control group comprised 14 patients with stable coronary artery disease, without history of myocardial infarction. Gene expression analysis was performed with Affymetrix Human Gene 1.0 ST microarrays and GCS3000 TG system. Lists of genes showing altered expression levels (fold change >1.5, p<0.05 were submitted to Ingenuity Pathway Analysis. Gene lists from each group were examined for canonical pathways and molecular and cellular functions. Comparing acute phase of MI with the same patients after 6 months (stable phase and with control group we found 24 genes with changed expression. In canonical analysis three pathways were highlighted: signaling of PPAR (peroxisome proliferator-activated receptor, IL-10 and IL-6 (interleukin 10 and 6. CONCLUSIONS: In the acute phase of STEMI, dozens of genes from several pathways linked with lipid/glucose metabolism, platelet function and atherosclerotic plaque stability show altered expression. Up-regulation of SOCS3 and FAM20 genes in the first days of myocardial infarction is observed in the vast majority of patients.

  15. Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss.

    Science.gov (United States)

    den Bakker, Henk C; Cummings, Craig A; Ferreira, Vania; Vatta, Paolo; Orsi, Renato H; Degoricija, Lovorka; Barker, Melissa; Petrauskene, Olga; Furtado, Manohar R; Wiedmann, Martin

    2010-12-02

    The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i) a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii) a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii) divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i) a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii) conserved genome size (between 2.8 and 3.2 Mb), and (iii) a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus Listeria thus provides an example of a group of

  16. Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss

    Directory of Open Access Journals (Sweden)

    Barker Melissa

    2010-12-01

    Full Text Available Abstract Background The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. Results To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. Conclusions Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii conserved genome size (between 2.8 and 3.2 Mb, and (iii a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus

  17. Responses of bacterial community and functional marker genes of nitrogen cycling to biochar, compost and combined amendments in soil.

    Science.gov (United States)

    Wu, Haipeng; Zeng, Guangming; Liang, Jie; Chen, Jin; Xu, Jijun; Dai, Juan; Li, Xiaodong; Chen, Ming; Xu, Piao; Zhou, Yaoyu; Li, Fei; Hu, Liang; Wan, Jia

    2016-10-01

    Biochar and compost are seen as two attractive waste management options and are used for soil amendment and pollution remediation. The interaction between biochar and composting may improve the potential benefits of biochar and compost. We investigated soil physicochemical properties, bacterial community, bacterial 16S rRNA, and functional marker genes of nitrogen cycling of the soil remedied with nothing (S), compost (SC), biochar (SB), a mixture of compost and biochar (SBC), composted biochar (SBced), and a composted mixture of biochar and biomass (SBCing). The results were that all amendments (1) increased the bacterial community richness (except SB) and SBCing showed the greatest efficiency; (2) increased the bacterial community diversity (SBCing > SBC > SC > SBced > SB > S); and (3) changed the gene copy numbers of 16S rRNA, nirK, nirS, and nosZ genes of bacteria, ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB). All amendments (except SB) could increase the gene copy number of 16S rRNA, and SBCing had the greatest efficiency. The changes of soil bacterial community richness and diversity and the gene copy numbers of 16S rRNA, nirK, nirS, nosZ, AOA, and AOB would affect carbon and nitrogen cycling of the ecosystem and also implied that BCing had the greatest efficiency on soil amendment.

  18. [Advances in molecular mechanisms of bacterial resistance caused by stress-induced transfer of resistance genes--a review].

    Science.gov (United States)

    Sun, Dongchang; Wang, Bing; Zhu, Lihong

    2013-07-04

    The transfer of resistance gene is one of the most important causes of bacterial resistance. Recent studies reveal that stresses induce the transfer of antibiotic resistance gene through multiple mechanisms. DNA damage stresses trigger bacterial SOS response and induce the transfer of resistance gene mediated by conjugative DNA. Antibiotic stresses induce natural bacterial competence for transformation in some bacteria which lack the SOS system. In addition, our latest studies show that the general stress response regulator RpoS regulates a novel type of resistance gene transfer which is mediated by double-stranded plasmid DNA and occurs exclusively on the solid surface. In this review, we summarized recent advances in SOS dependent and independent stress-induced DNA transfer which is mediated by conjugation and transformation respectively, and the transfer of double-stranded plasmid DNA on the solid surface which is regulated by RpoS. We propose that future work should address how stresses activate the key regulators and how these regulators control the expression of gene transfer related genes. Answers to the above questions would pave the way for searching for candidate targets for controlling bacterial resistance resulted from the transfer of antibiotic genes.

  19. Altered gene expression in blood and sputum in COPD frequent exacerbators in the ECLIPSE cohort.

    Directory of Open Access Journals (Sweden)

    Dave Singh

    Full Text Available Patients with chronic obstructive pulmonary disease (COPD who are defined as frequent exacerbators suffer with 2 or more exacerbations every year. The molecular mechanisms responsible for this phenotype are poorly understood. We investigated gene expression profile patterns associated with frequent exacerbations in sputum and blood cells in a well-characterised cohort. Samples from subjects from the ECLIPSE COPD cohort were used; sputum and blood samples from 138 subjects were used for microarray gene expression analysis, while blood samples from 438 subjects were used for polymerase chain reaction (PCR testing. Using microarray, 150 genes were differentially expressed in blood (>±1.5 fold change, p≤0.01 between frequent compared to non-exacerbators. In sputum cells, only 6 genes were differentially expressed. The differentially regulated genes in blood included downregulation of those involved in lymphocyte signalling and upregulation of pro-apoptotic signalling genes. Multivariate analysis of the microarray data followed by confirmatory PCR analysis identified 3 genes that predicted frequent exacerbations; B3GNT, LAF4 and ARHGEF10. The sensitivity and specificity of these 3 genes to predict the frequent exacerbator phenotype was 88% and 33% respectively. There are alterations in systemic immune function associated with frequent exacerbations; down-regulation of lymphocyte function and a shift towards pro-apoptosis mechanisms are apparent in patients with frequent exacerbations.

  20. Altered gene expression in blood and sputum in COPD frequent exacerbators in the ECLIPSE cohort.

    Science.gov (United States)

    Singh, Dave; Fox, Steven M; Tal-Singer, Ruth; Bates, Stewart; Riley, John H; Celli, Bartolome

    2014-01-01

    Patients with chronic obstructive pulmonary disease (COPD) who are defined as frequent exacerbators suffer with 2 or more exacerbations every year. The molecular mechanisms responsible for this phenotype are poorly understood. We investigated gene expression profile patterns associated with frequent exacerbations in sputum and blood cells in a well-characterised cohort. Samples from subjects from the ECLIPSE COPD cohort were used; sputum and blood samples from 138 subjects were used for microarray gene expression analysis, while blood samples from 438 subjects were used for polymerase chain reaction (PCR) testing. Using microarray, 150 genes were differentially expressed in blood (>±1.5 fold change, p≤0.01) between frequent compared to non-exacerbators. In sputum cells, only 6 genes were differentially expressed. The differentially regulated genes in blood included downregulation of those involved in lymphocyte signalling and upregulation of pro-apoptotic signalling genes. Multivariate analysis of the microarray data followed by confirmatory PCR analysis identified 3 genes that predicted frequent exacerbations; B3GNT, LAF4 and ARHGEF10. The sensitivity and specificity of these 3 genes to predict the frequent exacerbator phenotype was 88% and 33% respectively. There are alterations in systemic immune function associated with frequent exacerbations; down-regulation of lymphocyte function and a shift towards pro-apoptosis mechanisms are apparent in patients with frequent exacerbations.

  1. Identification of genes whose expression is altered by obesity throughout the arterial tree

    Science.gov (United States)

    Jenkins, Nathan T.; Thorne, Pamela K.; Martin, Jeffrey S.; Rector, R. Scott; Davis, J. Wade; Laughlin, M. Harold

    2014-01-01

    We used next-generation RNA sequencing (RNA-Seq) technology on the whole transcriptome to identify genes whose expression is consistently affected by obesity across multiple arteries. Specifically, we examined transcriptional profiles of the iliac artery as well as the feed artery, first, second, and third branch order arterioles in the soleus, gastrocnemius, and diaphragm muscles from obese Otsuka Long-Evans Tokushima Fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats. Within the gastrocnemius and soleus muscles, the number of genes differentially expressed with obesity tended to increase with increasing branch order arteriole number (i.e., decreasing size of the artery). This trend was opposite in the diaphragm. We found a total of 15 genes that were consistently upregulated with obesity (MIS18A, CTRB1, FAM151B, FOLR2, PXMP4, OAS1B, SREBF2, KLRA17, SLC25A44, SNX10, SLFN3, MEF2BNB, IRF7, RAD23A, LGALS3BP) and five genes that were consistently downregulated with obesity (C2, GOLGA7, RIN3, PCP4, CYP2E1). A small fraction (∼9%) of the genes affected by obesity was modulated across all arteries examined. In conclusion, the present study identifies a select number of genes (i.e., 20 genes) whose expression is consistently altered throughout the arterial network in response to obesity and provides further insight into the heterogeneous vascular effects of obesity. Although there is no known direct function of the majority of 20 genes related to vascular health, the obesity-associated upregulation of SREBF2, LGALS3BP, IRF7, and FOLR2 across all arteries is suggestive of an unfavorable vascular phenotypic alteration with obesity. These data may serve as an important resource for identifying novel therapeutic targets against obesity-related vascular complications. PMID:25271210

  2. Identification of genes whose expression is altered by obesity throughout the arterial tree.

    Science.gov (United States)

    Padilla, Jaume; Jenkins, Nathan T; Thorne, Pamela K; Martin, Jeffrey S; Rector, R Scott; Davis, J Wade; Laughlin, M Harold

    2014-11-15

    We used next-generation RNA sequencing (RNA-Seq) technology on the whole transcriptome to identify genes whose expression is consistently affected by obesity across multiple arteries. Specifically, we examined transcriptional profiles of the iliac artery as well as the feed artery, first, second, and third branch order arterioles in the soleus, gastrocnemius, and diaphragm muscles from obese Otsuka Long-Evans Tokushima Fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats. Within the gastrocnemius and soleus muscles, the number of genes differentially expressed with obesity tended to increase with increasing branch order arteriole number (i.e., decreasing size of the artery). This trend was opposite in the diaphragm. We found a total of 15 genes that were consistently upregulated with obesity (MIS18A, CTRB1, FAM151B, FOLR2, PXMP4, OAS1B, SREBF2, KLRA17, SLC25A44, SNX10, SLFN3, MEF2BNB, IRF7, RAD23A, LGALS3BP) and five genes that were consistently downregulated with obesity (C2, GOLGA7, RIN3, PCP4, CYP2E1). A small fraction (∼9%) of the genes affected by obesity was modulated across all arteries examined. In conclusion, the present study identifies a select number of genes (i.e., 20 genes) whose expression is consistently altered throughout the arterial network in response to obesity and provides further insight into the heterogeneous vascular effects of obesity. Although there is no known direct function of the majority of 20 genes related to vascular health, the obesity-associated upregulation of SREBF2, LGALS3BP, IRF7, and FOLR2 across all arteries is suggestive of an unfavorable vascular phenotypic alteration with obesity. These data may serve as an important resource for identifying novel therapeutic targets against obesity-related vascular complications.

  3. Development of resistant tomato population with bacterial canker resistance genes from interspecific hybrids by the support of embryo rescue

    OpenAIRE

    Aylin KABAŞ; Esin ARI; Sinan ZENGİN; Hülya İLBİ; AYSAN, Yeşim; Asu OĞUZ

    2016-01-01

    Bacterial canker is one of the most important diseases causing economic yield loss in tomato production areas in the world. The best way to control for this disease is to use resistant varieties. However, there are few studies on variety breeding studies of this disease compared with other disease resistant breeding studies. In this study we aimed to improve inbred lines carrying bacterial canker resistance genes to use in the breeding of resistant varieties. Susceptible inbred line AK1 (S. e...

  4. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    Science.gov (United States)

    Sotero-Martins, Adriana; de Jesus, Michele Silva; Lacerda, Michele; Moreira, Josino Costa; Filgueiras, Ana Luzia Lauria; Barrocas, Paulo Rubens Guimarães

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  5. Altered Levels of Aroma and Volatiles by Metabolic Engineering of Shikimate Pathway Genes in Tomato Fruits

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    Vered Tzin

    2015-06-01

    Full Text Available The tomato (Solanum lycopersicum fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a “functional food”. Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG209-9 and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA12 were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG209 and PheA12 genes. Overexpression of the AroG209-9 gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA12 overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG209-9 and PheA12 genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG209-9 gene. However, the aroma ranking attributes of the tomato fruits from PheA12//AroG209-9 were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA12 gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of

  6. Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes

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    Olschwang Sylviane

    2008-10-01

    Full Text Available Abstract Background Chronic myelomonocytic leukemia (CMML is a hematological disease close to, but separate from both myeloproliferative disorders (MPD and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML or myelodysplastic (MD-CMML features. Not much is known about the molecular biology of this disease. Methods We studied a series of 30 CMML samples (13 MP- and 11 MD-CMMLs, and 6 acutely transformed cases from 29 patients by using Agilent high density array-comparative genomic hybridization (aCGH and sequencing of 12 candidate genes. Results Two-thirds of samples did not show any obvious alteration of aCGH profiles. In one-third we observed chromosome abnormalities (e.g. trisomy 8, del20q and gain or loss of genes (e.g. NF1, RB1 and CDK6. RAS mutations were detected in 4 cases (including an uncommon codon 146 mutation in KRAS and PTPN11 mutations in 3 cases. We detected 11 RUNX1 alterations (9 mutations and 2 rearrangements. The rearrangements were a new, cryptic inversion of chromosomal region 21q21-22 leading to break and fusion of RUNX1 to USP16. RAS and RUNX1 alterations were not mutually exclusive. RAS pathway mutations occurred in MP-CMMLs (~46% but not in MD-CMMLs. RUNX1 alterations (mutations and cryptic rearrangement occurred in both MP and MD classes (~38%. Conclusion We detected RAS pathway mutations and RUNX1 alterations. The latter included a new cryptic USP16-RUNX1 fusion. In some samples, two alterations coexisted already at this early chronic stage.

  7. Bacterial pathogen gene abundance and relation to recreational water quality at seven Great Lakes beaches.

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    Oster, Ryan J; Wijesinghe, Rasanthi U; Haack, Sheridan K; Fogarty, Lisa R; Tucker, Taaja R; Riley, Stephen C

    2014-12-16

    Quantitative assessment of bacterial pathogens, their geographic variability, and distribution in various matrices at Great Lakes beaches are limited. Quantitative PCR (qPCR) was used to test for genes from E. coli O157:H7 (eaeO157), shiga-toxin producing E. coli (stx2), Campylobacter jejuni (mapA), Shigella spp. (ipaH), and a Salmonella enterica-specific (SE) DNA sequence at seven Great Lakes beaches, in algae, water, and sediment. Overall, detection frequencies were mapA>stx2>ipaH>SE>eaeO157. Results were highly variable among beaches and matrices; some correlations with environmental conditions were observed for mapA, stx2, and ipaH detections. Beach seasonal mean mapA abundance in water was correlated with beach seasonal mean log10 E. coli concentration. At one beach, stx2 gene abundance was positively correlated with concurrent daily E. coli concentrations. Concentration distributions for stx2, ipaH, and mapA within algae, sediment, and water were statistically different (Non-Detect and Data Analysis in R). Assuming 10, 50, or 100% of gene copies represented viable and presumably infective cells, a quantitative microbial risk assessment tool developed by Michigan State University indicated a moderate probability of illness for Campylobacter jejuni at the study beaches, especially where recreational water quality criteria were exceeded. Pathogen gene quantification may be useful for beach water quality management.

  8. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

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    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  9. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    Science.gov (United States)

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. PMID:20805991

  10. Oxidative Stress Alters miRNA and Gene Expression Profiles in Villous First Trimester Trophoblasts

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    Courtney E. Cross

    2015-01-01

    Full Text Available The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE is not fully elucidated. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H2O2 at 12 different concentrations (0-1 mM for 0.5, 4, 24, and 48 h. Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2. RNA was extracted after 4 h exposure to H2O2 for miRNA and gene expression profiling. H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50 significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. Tool for annotations of microRNAs (TAM analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Exposure to H2O2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile and expression of genes implicated in placental development.

  11. Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations.

    Science.gov (United States)

    Bueno, Raphael; Stawiski, Eric W; Goldstein, Leonard D; Durinck, Steffen; De Rienzo, Assunta; Modrusan, Zora; Gnad, Florian; Nguyen, Thong T; Jaiswal, Bijay S; Chirieac, Lucian R; Sciaranghella, Daniele; Dao, Nhien; Gustafson, Corinne E; Munir, Kiara J; Hackney, Jason A; Chaudhuri, Amitabha; Gupta, Ravi; Guillory, Joseph; Toy, Karen; Ha, Connie; Chen, Ying-Jiun; Stinson, Jeremy; Chaudhuri, Subhra; Zhang, Na; Wu, Thomas D; Sugarbaker, David J; de Sauvage, Frederic J; Richards, William G; Seshagiri, Somasekar

    2016-04-01

    We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score ≥ 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (∼2%; 4/216) and TRAF7 (∼2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

  12. Terminal restriction fragment length polymorphism (T-RFLP) profiling of bacterial 16S rRNA genes.

    Science.gov (United States)

    Osborne, Catherine A

    2014-01-01

    T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

  13. Ex vivo proteomics of Campylobacter jejuni 81-176 reveal that FabG affects fatty acid composition to alter bacterial growth fitness in the chicken gut.

    Science.gov (United States)

    Asakura, Hiroshi; Kawamoto, Keiko; Murakami, Satoshi; Tachibana, Masato; Kurazono, Hisao; Makino, Sou-Ichi; Yamamoto, Shigeki; Igimi, Shizunobu

    2016-01-01

    Campylobacter jejuni is one of the leading causes of foodborne gastrointestinal illness worldwide. Here we performed ex vivo proteomic analysis of C. jejuni 81-176 in chicken, a main reservoir for human infection. At 0, 1 and 4 weeks post-infection (p.i.) with the GFP-expressing 81-176 strain, inocula were recovered from chicken ceca by cell sorting using flow cytometry. iTRAQ-coupled 2D-LC-MS/MS analyses that detected 55 C. jejuni proteins, among which either 3 (FabG, HydB, CJJ81176_0876) or 7 (MscS, CetB, FlhF, PurH, PglJ, LpxC, Icd) proteins exhibited >1.4-fold-increased expression at 1 or 4 week(s) p.i. compared with those at 0 weeks p.i., respectively. Deletion of the fabG gene clearly decreased the proportion of bacterial unsaturated fatty acids (UFAs) and chicken colonization. The UFA proportion of the parental strain was not altered when grown at 42 °C. These findings suggest that FabG might play a pivotal role in UFA production, linked to bacterial adaptation in the poultry host. To our knowledge, this is the first example of ex vivo C. jejuni proteomics, in which fatty acid metabolism might affect bacterial adaptation to the chicken host.

  14. Involvement of the cell-specific pigment genes pks and sult in bacterial defense response of sea urchins Strongylocentrotus intermedius.

    Science.gov (United States)

    Kiselev, Konstantin V; Ageenko, Natalya V; Kurilenko, Valeria V

    2013-03-26

    Bacterial infections are one of the most important problems in mass aquaculture, causing the loss of millions of juvenile organisms. We isolated 22 bacterial strains from the cavity fluid of the sea urchin Strongylocentrotus pallidus and used phylogenetic analysis based on 16S rRNA gene sequences to separate the bacterial strains into 9 genera (Aliivibrio, Bizionia, Colwellia, Olleya, Paenibacillus, Photobacterium, Pseudoalteromonas, Shewanella, and Vibrio). Incubating Strongylocentrotus intermedius larvae with a strain from each of the 9 bacterial genera, we investigated the viability of the larvae, the amount of pigment cells, and the level of polyketide synthase (pks) and sulfotransferase (sult) gene expression. Results of the assay on sea urchin development showed that all bacterial strains, except Pseudoalteromonas and Bizionia, suppressed sea urchin development (resulting in retardation of the embryos' development with cellular disorders) and reduced cell viability. We found that pks expression in the sea urchin larvae after incubation with the bacteria of 9 tested genera was significantly increased, while the sult expression was increased only after the treatment with Pseudoalteromonas and Shewanella. Shikimic acid, which is known to activate the biosynthesis of naphthoquinone pigments, increased the tolerance of the sea urchin embryos to the bacteria. In conclusion, we show that the cell-specific pigment genes pks and sult are involved in the bacterial defense response of sea urchins.

  15. Rescue of Metabolic Alterations in AR113Q Skeletal Muscle by Peripheral Androgen Receptor Gene Silencing

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    Elisa Giorgetti

    2016-09-01

    Full Text Available Spinal and bulbar muscular atrophy (SBMA, a progressive degenerative disorder, is caused by a CAG/glutamine expansion in the androgen receptor (polyQ AR. Recent studies demonstrate that skeletal muscle is an important site of toxicity that contributes to the SBMA phenotype. Here, we sought to identify critical pathways altered in muscle that underlie disease manifestations in AR113Q mice. This led to the unanticipated identification of gene expression changes affecting regulators of carbohydrate metabolism, similar to those triggered by denervation. AR113Q muscle exhibits diminished glycolysis, altered mitochondria, and an impaired response to exercise. Strikingly, the expression of genes regulating muscle energy metabolism is rescued following peripheral polyQ AR gene silencing by antisense oligonucleotides (ASO, a therapeutic strategy that alleviates disease. Our data establish the occurrence of a metabolic imbalance in SBMA muscle triggered by peripheral expression of the polyQ AR and indicate that alterations in energy utilization contribute to non-neuronal disease manifestations.

  16. Altered activities of transcription factors and their related gene expression in cardiac tissues of diabetic rats.

    Science.gov (United States)

    Nishio, Y; Kashiwagi, A; Taki, H; Shinozaki, K; Maeno, Y; Kojima, H; Maegawa, H; Haneda, M; Hidaka, H; Yasuda, H; Horiike, K; Kikkawa, R

    1998-08-01

    Gene regulation in the cardiovascular tissues of diabetic subjects has been reported to be altered. To examine abnormal activities in transcription factors as a possible cause of this altered gene regulation, we studied the activity of two redox-sensitive transcription factors--nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1)--and the change in the mRNA content of heme oxygenase-1, which is regulated by these transcription factors in the cardiac tissues of rats with streptozotocin-induced diabetes. Increased activity of NF-kappaB and AP-1 but not nuclear transcription-activating factor, as determined by an electrophoretic mobility shift assay, was found in the hearts of 4-week diabetic rats. Glycemic control by a subcutaneous injection of insulin prevented these diabetes-induced changes in transcription factor activity. In accordance with these changes, the mRNA content of heme oxygenase-1 was increased fourfold in 4-week diabetic rats and threefold in 24-week diabetic rats as compared with control rats (P oxidative stress is involved in the activation of the transcription factors NF-kappaB and AP-1 in the cardiac tissues of diabetic rats, and that these abnormal activities of transcription factors could be associated with the altered gene regulation observed in the cardiovascular tissues of diabetic rats.

  17. Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

    Science.gov (United States)

    Twomey, Kate B; Alston, Mark; An, Shi-Qi; O'Connell, Oisin J; McCarthy, Yvonne; Swarbreck, David; Febrer, Melanie; Dow, J Maxwell; Plant, Barry J; Ryan, Robert P

    2013-01-01

    Chronic polymicrobial infections of the lung are the foremost cause of morbidity and mortality in cystic fibrosis (CF) patients. The composition of the microbial flora of the airway alters considerably during infection, particularly during patient exacerbation. An understanding of which organisms are growing, their environment and their behaviour in the airway is of importance for designing antibiotic treatment regimes and for patient prognosis. To this end, we have analysed sputum samples taken from separate cohorts of CF and non-CF subjects for metabolites and in parallel, and we have examined both isolated DNA and RNA for the presence of 16S rRNA genes and transcripts by high-throughput sequencing of amplicon or cDNA libraries. This analysis revealed that although the population size of all dominant orders of bacteria as measured by DNA- and RNA- based methods are similar, greater discrepancies are seen with less prevalent organisms, some of which we associated with CF for the first time. Additionally, we identified a strong relationship between the abundance of specific anaerobes and fluctuations in several metabolites including lactate and putrescine during patient exacerbation. This study has hence identified organisms whose occurrence within the CF microbiome has been hitherto unreported and has revealed potential metabolic biomarkers for exacerbation.

  18. Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

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    Kate B Twomey

    Full Text Available Chronic polymicrobial infections of the lung are the foremost cause of morbidity and mortality in cystic fibrosis (CF patients. The composition of the microbial flora of the airway alters considerably during infection, particularly during patient exacerbation. An understanding of which organisms are growing, their environment and their behaviour in the airway is of importance for designing antibiotic treatment regimes and for patient prognosis. To this end, we have analysed sputum samples taken from separate cohorts of CF and non-CF subjects for metabolites and in parallel, and we have examined both isolated DNA and RNA for the presence of 16S rRNA genes and transcripts by high-throughput sequencing of amplicon or cDNA libraries. This analysis revealed that although the population size of all dominant orders of bacteria as measured by DNA- and RNA- based methods are similar, greater discrepancies are seen with less prevalent organisms, some of which we associated with CF for the first time. Additionally, we identified a strong relationship between the abundance of specific anaerobes and fluctuations in several metabolites including lactate and putrescine during patient exacerbation. This study has hence identified organisms whose occurrence within the CF microbiome has been hitherto unreported and has revealed potential metabolic biomarkers for exacerbation.

  19. Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

    Science.gov (United States)

    Ochi, Kozo; Tanaka, Yukinori; Tojo, Shigeo

    2014-02-01

    Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed.

  20. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    Science.gov (United States)

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  1. PROGNOSTIC VALUE OF VHL GENE ALTERATION IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

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    D. A. Nosov

    2011-01-01

    Full Text Available Objective: to estimate the rate, predictive and prognostic value of VHL gene alterations in the population of patients with sporadic metastatic renal cell carcinoma (mRCC.Subjects and methods. Paraffin embedded tumor tissue blocks were available from 88 patients with mRCC who had undergone antitumor therapy in 1994- 2010. Of them, 53 patients received only immunotherapy regimens with interferon (IFN-α and 35 patients had targeted therapy with VEGFR inhibitors. VHL mutations were detected by polymerase chain reaction (PCR for exons of 1-3, single-strand conformation polymorphism analysis of PCR products, and further sequencing. VHL gene methylation was determined by methyl-sensitive PCR.Results. Somatic mutations and/or promoter hypermethylation of the VHL gene were found in 23 (26% patients; Of them, VHL gene mutations and promoter hypermethylation were found in 15 patients and 7 patients respectively. Mutation and promoter methylation VHL were simultaneously observed in one case. VHL gene mutations were detected only in patients with clear cell RCC while aberrant promoter methylation was seen in both clear cell and papillary RCC. With a median follow-up of 34 months (range, 2-127 months, the median time to progression (TTP and median overall survival (OS for the entire group of patients were 5.8 and 26.7 months, respectively. In patients with and without VHL gene alterations, the median TTP was 5.5 and 6.9 months, respectively (p = 0.15 and the median overall survival time was 22.0 and 34.5 months, respectively (p = 0.98. Moreover, the subgroup analysis revealed that VHL gene inactivation events had no impact on the objective response rate (ORR, TTP and OS in the subgroup of patients who received immunotherapy (n = 53 or antiangiogenic targeted therapy (n = 35 (p > 0.05.Conclusion. VHL gene mutations and/or promotor hypermethylation observed in 26% of patients with mRCC. These VHL gene alterations were neither prognostic nor predictive factors

  2. PROGNOSTIC VALUE OF VHL GENE ALTERATION IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

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    D. A. Nosov

    2014-08-01

    Full Text Available Objective: to estimate the rate, predictive and prognostic value of VHL gene alterations in the population of patients with sporadic metastatic renal cell carcinoma (mRCC.Subjects and methods. Paraffin embedded tumor tissue blocks were available from 88 patients with mRCC who had undergone antitumor therapy in 1994- 2010. Of them, 53 patients received only immunotherapy regimens with interferon (IFN-α and 35 patients had targeted therapy with VEGFR inhibitors. VHL mutations were detected by polymerase chain reaction (PCR for exons of 1-3, single-strand conformation polymorphism analysis of PCR products, and further sequencing. VHL gene methylation was determined by methyl-sensitive PCR.Results. Somatic mutations and/or promoter hypermethylation of the VHL gene were found in 23 (26% patients; Of them, VHL gene mutations and promoter hypermethylation were found in 15 patients and 7 patients respectively. Mutation and promoter methylation VHL were simultaneously observed in one case. VHL gene mutations were detected only in patients with clear cell RCC while aberrant promoter methylation was seen in both clear cell and papillary RCC. With a median follow-up of 34 months (range, 2-127 months, the median time to progression (TTP and median overall survival (OS for the entire group of patients were 5.8 and 26.7 months, respectively. In patients with and without VHL gene alterations, the median TTP was 5.5 and 6.9 months, respectively (p = 0.15 and the median overall survival time was 22.0 and 34.5 months, respectively (p = 0.98. Moreover, the subgroup analysis revealed that VHL gene inactivation events had no impact on the objective response rate (ORR, TTP and OS in the subgroup of patients who received immunotherapy (n = 53 or antiangiogenic targeted therapy (n = 35 (p > 0.05.Conclusion. VHL gene mutations and/or promotor hypermethylation observed in 26% of patients with mRCC. These VHL gene alterations were neither prognostic nor predictive factors

  3. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

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    Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2013-02-15

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  4. Altered gene expression in schizophrenia: findings from transcriptional signatures in fibroblasts and blood.

    Directory of Open Access Journals (Sweden)

    Nadia Cattane

    Full Text Available Whole-genome expression studies in the peripheral tissues of patients affected by schizophrenia (SCZ can provide new insight into the molecular basis of the disorder and innovative biomarkers that may be of great utility in clinical practice. Recent evidence suggests that skin fibroblasts could represent a non-neural peripheral model useful for investigating molecular alterations in psychiatric disorders.A microarray expression study was conducted comparing skin fibroblast transcriptomic profiles from 20 SCZ patients and 20 controls. All genes strongly differentially expressed were validated by real-time quantitative PCR (RT-qPCR in fibroblasts and analyzed in a sample of peripheral blood cell (PBC RNA from patients (n = 25 and controls (n = 22. To evaluate the specificity for SCZ, alterations in gene expression were tested in additional samples of fibroblasts and PBCs RNA from Major Depressive Disorder (MDD (n = 16; n = 21, respectively and Bipolar Disorder (BD patients (n = 15; n = 20, respectively.Six genes (JUN, HIST2H2BE, FOSB, FOS, EGR1, TCF4 were significantly upregulated in SCZ compared to control fibroblasts. In blood, an increase in expression levels was confirmed only for EGR1, whereas JUN was downregulated; no significant differences were observed for the other genes. EGR1 upregulation was specific for SCZ compared to MDD and BD.Our study reports the upregulation of JUN, HIST2H2BE, FOSB, FOS, EGR1 and TCF4 in the fibroblasts of SCZ patients. A significant alteration in EGR1 expression is also present in SCZ PBCs compared to controls and to MDD and BD patients, suggesting that this gene could be a specific biomarker helpful in the differential diagnosis of major psychoses.

  5. Altered Gene Expression in Schizophrenia: Findings from Transcriptional Signatures in Fibroblasts and Blood

    Science.gov (United States)

    Cattane, Nadia; Minelli, Alessandra; Milanesi, Elena; Maj, Carlo; Bignotti, Stefano; Bortolomasi, Marco; Chiavetto, Luisella Bocchio; Gennarelli, Massimo

    2015-01-01

    Background Whole-genome expression studies in the peripheral tissues of patients affected by schizophrenia (SCZ) can provide new insight into the molecular basis of the disorder and innovative biomarkers that may be of great utility in clinical practice. Recent evidence suggests that skin fibroblasts could represent a non-neural peripheral model useful for investigating molecular alterations in psychiatric disorders. Methods A microarray expression study was conducted comparing skin fibroblast transcriptomic profiles from 20 SCZ patients and 20 controls. All genes strongly differentially expressed were validated by real-time quantitative PCR (RT-qPCR) in fibroblasts and analyzed in a sample of peripheral blood cell (PBC) RNA from patients (n = 25) and controls (n = 22). To evaluate the specificity for SCZ, alterations in gene expression were tested in additional samples of fibroblasts and PBCs RNA from Major Depressive Disorder (MDD) (n = 16; n = 21, respectively) and Bipolar Disorder (BD) patients (n = 15; n = 20, respectively). Results Six genes (JUN, HIST2H2BE, FOSB, FOS, EGR1, TCF4) were significantly upregulated in SCZ compared to control fibroblasts. In blood, an increase in expression levels was confirmed only for EGR1, whereas JUN was downregulated; no significant differences were observed for the other genes. EGR1 upregulation was specific for SCZ compared to MDD and BD. Conclusions Our study reports the upregulation of JUN, HIST2H2BE, FOSB, FOS, EGR1 and TCF4 in the fibroblasts of SCZ patients. A significant alteration in EGR1 expression is also present in SCZ PBCs compared to controls and to MDD and BD patients, suggesting that this gene could be a specific biomarker helpful in the differential diagnosis of major psychoses. PMID:25658856

  6. iGC-an integrated analysis package of gene expression and copy number alteration.

    Science.gov (United States)

    Lai, Yi-Pin; Wang, Liang-Bo; Wang, Wei-An; Lai, Liang-Chuan; Tsai, Mong-Hsun; Lu, Tzu-Pin; Chuang, Eric Y

    2017-01-14

    With the advancement in high-throughput technologies, researchers can simultaneously investigate gene expression and copy number alteration (CNA) data from individual patients at a lower cost. Traditional analysis methods analyze each type of data individually and integrate their results using Venn diagrams. Challenges arise, however, when the results are irreproducible and inconsistent across multiple platforms. To address these issues, one possible approach is to concurrently analyze both gene expression profiling and CNAs in the same individual. We have developed an open-source R/Bioconductor package (iGC). Multiple input formats are supported and users can define their own criteria for identifying differentially expressed genes driven by CNAs. The analysis of two real microarray datasets demonstrated that the CNA-driven genes identified by the iGC package showed significantly higher Pearson correlation coefficients with their gene expression levels and copy numbers than those genes located in a genomic region with CNA. Compared with the Venn diagram approach, the iGC package showed better performance. The iGC package is effective and useful for identifying CNA-driven genes. By simultaneously considering both comparative genomic and transcriptomic data, it can provide better understanding of biological and medical questions. The iGC package's source code and manual are freely available at https://www.bioconductor.org/packages/release/bioc/html/iGC.html .

  7. Preliminary evidence of phenytoin-induced alterations in embryonic gene expression in a mouse model.

    Science.gov (United States)

    Musselman, A C; Bennett, G D; Greer, K A; Eberwine, J H; Finnell, R H

    1994-01-01

    SWV mouse embryos collected on gestational days (GD) 9:12 and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both Wnt-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:12 embryos, including the growth factors TGF-beta and NT3, the proto-oncogene Wnt-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.

  8. FGFR gene alterations in lung squamous cell carcinoma are potential targets for the multikinase inhibitor nintedanib.

    Science.gov (United States)

    Hibi, Masaaki; Kaneda, Hiroyasu; Tanizaki, Junko; Sakai, Kazuko; Togashi, Yosuke; Terashima, Masato; De Velasco, Marco Antonio; Fujita, Yoshihiko; Banno, Eri; Nakamura, Yu; Takeda, Masayuki; Ito, Akihiko; Mitsudomi, Tetsuya; Nakagawa, Kazuhiko; Okamoto, Isamu; Nishio, Kazuto

    2016-11-01

    Fibroblast growth factor receptor (FGFR) gene alterations are relatively frequent in lung squamous cell carcinoma (LSCC) and are a potential targets for therapy with FGFR inhibitors. However, little is known regarding the clinicopathologic features associated with FGFR alterations. The angiokinase inhibitor nintedanib has shown promising activity in clinical trials for non-small cell lung cancer. We have now applied next-generation sequencing (NGS) to characterize FGFR alterations in LSCC patients as well as examined the antitumor activity of nintedanib in LSCC cell lines positive for FGFR1 copy number gain (CNG). The effects of nintedanib on the proliferation of and FGFR signaling in LSCC cell lines were examined in vitro, and its effects on tumor formation were examined in vivo. A total of 75 clinical LSCC specimens were screened for FGFR alterations by NGS. Nintedanib inhibited the proliferation of FGFR1 CNG-positive LSCC cell lines in association with attenuation of the FGFR1-ERK signaling pathway in vitro and in vivo. FGFR1 CNG (10.7%), FGFR1 mutation (2.7%), FGFR2 mutation (2.7%), FGFR4 mutation (5.3%), and FGFR3 fusion (1.3%) were detected in LSCC specimens by NGS. Clinicopathologic features did not differ between LSCC patients positive or negative for FGFR alterations. However, among the 36 patients with disease recurrence after surgery, prognosis was significantly worse for those harboring FGFR alterations. Screening for FGFR alterations by NGS warrants further study as a means to identify patients with LSCC recurrence after surgery who might benefit from nintedanib therapy.

  9. The expression of petunia strigolactone pathway genes is altered as part of the endogenous developmental program

    Directory of Open Access Journals (Sweden)

    Revel S M Drummond

    2012-01-01

    Full Text Available Analysis of mutants with increased branching has revealed the strigolactone synthesis/perception pathway which regulates branching in plants. However, whether variation in this well conserved developmental signalling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologues of the Arabidopsis MAX1 and MAX2 genes to characterise their role in petunia architecture. A single orthologue of MAX1, PhMAX1 which encodes a cytochrome P450, was identified and was able to complement the max1 mutant of Arabidopsis. Petunia has two copies of the MAX2 gene, PhMAX2A and PhMAX2B which encode F-Box proteins. Differences in the transcript levels of these two MAX2-like genes suggest diverging functions. Unlike PhMAX2B, PhMAX2A mRNA levels increase as leaves age. Nonetheless, this gene functionally complements the Arabidopsis max2 mutant indicating that the biochemical activity of the PhMAX2A protein is not significantly different from MAX2. The expression of the petunia strigolactone pathway genes (PhCCD7, PhCCD8, PhMAX1, PhMAX2A, and PhMAX2B was then further investigated throughout the development of wild-type petunia plants. Three of these genes showed changes in mRNA levels over the development series. Alterations to the expression of these genes over time, or in different regions of the plant, may influence the branching growth habit of the plant. Alterations to strigolactone production and/or sensitivity could allow both subtle and dramatic changes to branching within and between species.

  10. Comparative and Experimental Studies on the Genes Altered by Chronic Hypoxia in Human Brain Microendothelial Cells

    Directory of Open Access Journals (Sweden)

    Eugenia Mata-Greenwood

    2017-05-01

    Full Text Available Background : Hypoxia inducible factor 1 alpha (HIF1A is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia.Results : We examined the effect of chronic hypoxia (3 days; 0.5% oxygen on human brain micro endothelial cells (HBMEC viability and apoptosis. Hypoxia caused a significant reduction in cell viability and an increase in apoptosis. Next, we examined chronic hypoxia associated changes in transcriptome and genome-wide promoter methylation. The data obtained was compared with 16 other microarray studies on chronic hypoxia. Nine genes were altered in response to chronic hypoxia in all 17 studies. Interestingly, HIF1A was not altered with chronic hypoxia in any of the studies. Furthermore, we compared our data to three other studies that identified HIF-responsive genes by various approaches. Only two genes were found to be HIF dependent. We silenced each of these 9 genes using CRISPR/Cas9 system. Downregulation of EGLN3 significantly increased the cell death under chronic hypoxia, whereas downregulation of ERO1L, ENO2, adrenomedullin, and spag4 reduced the cell death under hypoxia.Conclusions : We provide a core group of genes that regulates cellular acclimatization under chronic hypoxic stress, and most of them are HIF independent.

  11. Sulfonamide and tetracycline resistance genes in total- and culturable-bacterial assemblages in South African aquatic environments

    Directory of Open Access Journals (Sweden)

    Satoru eSuzuki

    2015-08-01

    Full Text Available Antibiotic resistant bacteria (ARB are ubiquitous in the natural environment. The introduction of effluent derived antibiotic resistance genes (ARGs into aquatic environments is of concern in the spreading of genetic risk. This study showed the prevalence of sulfonamide and tetracycline resistance genes, sul1, sul2, sul3 and tet(M, in the total bacterial assemblage and colony forming bacterial assemblage in river and estuarine water and sewage treatment plants (STP in South Africa. There was no correlation between antibiotic concentrations and ARGs, suggesting the targeted ARGs are spread in a wide area without connection to selection pressure. Among sul genes, sul1 and sul2 were major genes in the total (over 10-2 copies/16S and colony forming bacteria assemblages (approx 10-1 copies/16S. In urban waters, the sul3 gene was mostly not detectable in total and culturable assemblages, suggesting sul3 is not abundant. tet(M was found in natural assemblages with 10-3 copies/16S level in STP, but was not detected in colony forming bacteria, suggesting the non-culturable (yet-to-be cultured bacterial community in urban surface waters and STP effluent possess the tet(M gene. Sulfamethoxazole resistant (SMXr and oxytetracycline resistant (OTCr bacterial communities in urban waters possessed not only sul1 and sul2 but also sul3 and tet(M genes. These genes are widely distributed in SMXr and OTCr bacteria. In conclusion, urban river and estuarine water and STP effluent in the Durban area were highly contaminated with ARGs, and the yet-to-be cultured bacterial community may act as a non-visible ARG reservoir in certain situations.

  12. HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

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    Lehto Kirsi

    2011-04-01

    Full Text Available Abstract Background RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs. These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter the helper component-proteinase (HC-Pro derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent. Results Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1 were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S

  13. HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers.

    Science.gov (United States)

    Soitamo, Arto J; Jada, Balaji; Lehto, Kirsi

    2011-04-20

    RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs). These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA) mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter) the helper component-proteinase (HC-Pro) derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent). Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1) were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S-adenosyl-L-methionine (SAM) were also decreased in

  14. Testing the infinitely many genes model for the evolution of the bacterial core genome and pangenome.

    Science.gov (United States)

    Collins, R Eric; Higgs, Paul G

    2012-11-01

    When groups of related bacterial genomes are compared, the number of core genes found in all genomes is usually much less than the mean genome size, whereas the size of the pangenome (the set of genes found on at least one of the genomes) is much larger than the mean size of one genome. We analyze 172 complete genomes of Bacilli and compare the properties of the pangenomes and core genomes of monophyletic subsets taken from this group. We then assess the capabilities of several evolutionary models to predict these properties. The infinitely many genes (IMG) model is based on the assumption that each new gene can arise only once. The predictions of the model depend on the shape of the evolutionary tree that underlies the divergence of the genomes. We calculate results for coalescent trees, star trees, and arbitrary phylogenetic trees of predefined fixed branch length. On a star tree, the pangenome size increases linearly with the number of genomes, as has been suggested in some previous studies, whereas on a coalescent tree, it increases logarithmically. The coalescent tree gives a better fit to the data, for all the examples we consider. In some cases, a fixed phylogenetic tree proved better than the coalescent tree at reproducing structure in the gene frequency spectrum, but little improvement was gained in predictions of the core and pangenome sizes. Most of the data are well explained by a model with three classes of gene: an essential class that is found in all genomes, a slow class whose rate of origination and deletion is slow compared with the time of divergence of the genomes, and a fast class showing rapid origination and deletion. Although the majority of genes originating in a genome are in the fast class, these genes are not retained for long periods, and the majority of genes present in a genome are in the slow or essential classes. In general, we show that the IMG model is useful for comparison with experimental genome data both for species level and

  15. Concerning RNA-guided gene drives for the alteration of wild populations

    Science.gov (United States)

    Esvelt, Kevin M; Smidler, Andrea L; Catteruccia, Flaminia; Church, George M

    2014-01-01

    Gene drives may be capable of addressing ecological problems by altering entire populations of wild organisms, but their use has remained largely theoretical due to technical constraints. Here we consider the potential for RNA-guided gene drives based on the CRISPR nuclease Cas9 to serve as a general method for spreading altered traits through wild populations over many generations. We detail likely capabilities, discuss limitations, and provide novel precautionary strategies to control the spread of gene drives and reverse genomic changes. The ability to edit populations of sexual species would offer substantial benefits to humanity and the environment. For example, RNA-guided gene drives could potentially prevent the spread of disease, support agriculture by reversing pesticide and herbicide resistance in insects and weeds, and control damaging invasive species. However, the possibility of unwanted ecological effects and near-certainty of spread across political borders demand careful assessment of each potential application. We call for thoughtful, inclusive, and well-informed public discussions to explore the responsible use of this currently theoretical technology. DOI: http://dx.doi.org/10.7554/eLife.03401.001 PMID:25035423

  16. The enteric bacterial metabolite propionic acid alters brain and plasma phospholipid molecular species: further development of a rodent model of autism spectrum disorders

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    Thomas Raymond H

    2012-07-01

    Full Text Available Abstract Gastrointestinal symptoms and altered blood phospholipid profiles have been reported in patients with autism spectrum disorders (ASD. Most of the phospholipid analyses have been conducted on the fatty acid composition of isolated phospholipid classes following hydrolysis. A paucity of information exists on how the intact phospholipid molecular species are altered in ASD. We applied ESI/MS to determine how brain and blood intact phospholipid species were altered during the induction of ASD-like behaviors in rats following intraventricular infusions with the enteric bacterial metabolite propionic acid. Animals were infused daily for 8 days, locomotor activity assessed, and animals killed during the induced behaviors. Propionic acid infusions increased locomotor activity. Lipid analysis revealed treatment altered 21 brain and 30 blood phospholipid molecular species. Notable alterations were observed in the composition of brain SM, diacyl mono and polyunsaturated PC, PI, PS, PE, and plasmalogen PC and PE molecular species. These alterations suggest that the propionic acid rat model is a useful tool to study aberrations in lipid metabolism known to affect membrane fluidity, peroxisomal function, gap junction coupling capacity, signaling, and neuroinflammation, all of which may be associated with the pathogenesis of ASD.

  17. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  18. Contemporary human-altered landscapes and oceanic barriers reduce bumble bee gene flow.

    Science.gov (United States)

    Jha, S

    2015-03-01

    Much of the world's terrestrial landscapes are being altered by humans in the form of agriculture, urbanization and pastoral systems, with major implications for biodiversity. Bumble bees are one of the most effective pollinators in both natural and cultivated landscapes, but are often the first to be extirpated in human-altered habitats. Yet, little is known about the role of natural and human-altered habitats in promoting or limiting bumble bee gene flow. In this study, I closely examine the genetic structure of the yellow-faced bumble bee, Bombus vosnesenskii, across the southwestern US coast and find strong evidence that natural oceanic barriers, as well as contemporary human-altered habitats, limit bee gene flow. Heterozygosity and allelic richness were lower in island populations, while private allelic richness was higher in island populations compared to mainland populations. Genetic differentiation, measured for three indices across the 1000 km study region, was significantly greater than the null expectation (F(ST) = 0.041, F'(ST) = 0.044 and D(est) = 0.155) and correlated with geographic distance. Furthermore, genetic differentiation patterns were most strongly correlated with contemporary (2011) not past (2006, 2001) resistance maps calibrated for high dispersal limitation over oceans, impervious habitat and croplands. Despite the incorporation of dramatic elevation gradients, the analyses reveal that oceans and contemporary human land use, not mountains, are the primary dispersal barriers for B. vosnesenskii gene flow. These findings reinforce the importance of maintaining corridors of suitable habitat across the distribution range of native pollinators to promote their persistence and safeguard their ability to provide essential pollination services.

  19. Therapeutic strategies in male breast cancer: clinical implications of chromosome 17 gene alterations and molecular subtypes.

    Science.gov (United States)

    Schildhaus, Hans-Ulrich; Schroeder, Lars; Merkelbach-Bruse, Sabine; Binot, Elke; Büttner, Reinhard; Kuhn, Walther; Rudlowski, Christian

    2013-12-01

    Male breast cancer (MBC) is a rare disease. To date, therapy is mainly based on studies and clinical experiences with breast cancer in women. Only little is known about molecular typing of MBC, particularly with regard to potential biological predictors for adjuvant therapy. In female breast cancer tumors with chromosome 17 centromere (CEP17) duplication, HER2 and/or Topoisomerase II alpha (Topo II-α) gene alterations have been suggested to be associated with poor prognosis and increased sensitivity to anthracycline-containing regimens. In a well characterized cohort of 96 primary invasive MBC, we studied CEP17, HER2 and Topo II-α alterations by fluorescence in-situ hybridization (FISH), and expression of hormone receptors (HR), HER2 and Ki67 by immunohistochemistry to define molecular subtypes. Tumor characteristics and follow-up data were available and correlated with molecular findings. HER2 amplification and Topo II-α amplification/deletion were exceptionally rare in MBC (6.3% and 3.1%, respectively). CEP17 polysomy were found in 9.4% of tumors. HER2, Topo II-α and CEP17 gene alterations were not correlated to patients outcome. 96.9% of our cases were HR positive. Triple negative tumors were found in only 3.1% of the cases. In nodal negative tumors luminal A subtypes were significantly associated with better overall survival. Our results provide evidence for a predominant male breast cancer phenotype, characterized by HR expression and a lack of HER2/Topo II-α alterations and CEP17 duplicates. Therefore, the impact of anthracycline sensitivity linked to HER2/Topo II-α alterations as found in female breast cancer has low clinical significance for this specific male breast cancer phenotype. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

    Indian Academy of Sciences (India)

    Pornima Phatak; S Kalai Selvi; T Divya; A S Hegde; Sridevi Hegde; Kumaravel Somasundaram

    2002-12-01

    Alterations in the tumour suppressor p53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of the p53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5–9 of the p53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13.6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients.

  1. Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas.

    Science.gov (United States)

    Kaur, Simran; Norkina, Oxana; Ziemer, Donna; Samuelson, Linda C; De Lisle, Robert C

    2004-08-01

    The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.

  2. Whole blood gene expression profiling of neonates with confirmed bacterial sepsis

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    Paul Dickinson

    2015-03-01

    Full Text Available Neonatal infection remains a primary cause of infant morbidity and mortality worldwide and yet our understanding of how human neonates respond to infection remains incomplete. Changes in host gene expression in response to infection may occur in any part of the body, with the continuous interaction between blood and tissues allowing blood cells to act as biosensors for the changes. In this study we have used whole blood transcriptome profiling to systematically identify signatures and the pathway biology underlying the pathogenesis of neonatal infection. Blood samples were collected from neonates at the first clinical signs of suspected sepsis alongside age matched healthy control subjects. Here we report a detailed description of the study design, including clinical data collected, experimental methods used and data analysis workflows and which correspond with data in Gene Expression Omnibus (GEO data sets (GSE25504. Our data set has allowed identification of a patient invariant 52-gene classifier that predicts bacterial infection with high accuracy and lays the foundation for advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.

  3. Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene.

    Science.gov (United States)

    Vail, Andrew W; Wang, Ping; Uefuji, Hirotaka; Samac, Deborah A; Vance, Carroll P; Wackett, Lawrence P; Sadowsky, Michael J

    2015-06-01

    The widespread use of atrazine and other s-triazine herbicides to control weeds in agricultural production fields has impacted surface and groundwater in the United States and elsewhere. We previously reported the cloning, sequencing, and expression of six genes involved in the atrazine biodegradation pathway of Pseudomonas sp. strain ADP, which is initiated by atzA, encoding atrazine chlorohydrolase. Here we explored the use of enhanced expression of a modified bacterial atrazine chlorohydrolase, p-AtzA, in transgenic grasses (tall fescue, perennial ryegrass, and switchgrass) and the legume alfalfa for the biodegradation of atrazine. Enhanced expression of p-AtzA was obtained by using combinations of the badnavirus promoter, the maize alcohol dehydrogenase first intron, and the maize ubiquitin promoter. For alfalfa, we used the first intron of the 5'-untranslated region tobacco alcohol dehydrogenase gene and the cassava vein mosaic virus promoter. Resistance of plants to atrazine in agar-based and hydroponic growth assays was correlated with in vivo levels of gene expression and atrazine degradation. The in planta expression of p-atzA enabled transgenic tall fescue to transform atrazine into hydroxyatrazine and other metabolites. Results of our studies highlight the potential use of transgenic plants for bioremediating atrazine in the environment.

  4. Nucleotide diversity analysis of three major bacterial blight resistance genes in rice.

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    Waikhom Bimolata

    Full Text Available Nucleotide sequence polymorphisms among R gene alleles influence the process of co-evolutionary interaction between host and pathogen by shaping the response of host plants towards invading pathogens. Here, we present the DNA sequence polymorphisms and diversities present among natural alleles of three rice bacterial blight resistance genes, Xa21, Xa26 and xa5. The diversity was examined across different wild relatives and cultivars of Oryza species. Functional significance of selected alleles was evaluated through semi-quantitative reverse transcription polymerase chain reaction and real time PCR. The greatest nucleotide diversity and singleton variable sites (SVS were present in Xa26 (π = 0.01958; SVS = 182 followed by xa5 and Xa21 alleles. The highest frequency of single nucleotide polymorphisms were observed in Xa21 alleles and least in xa5. Transition bias was observed in all the genes and 'G' to 'A' transitions were more favored than other form of transitions. Neutrality tests failed to show the presence of selection at these loci, though negative Tajima's D values indicate the presence of a rare form of polymorphisms. At the interspecies level, O. nivara exhibited more diversity than O. sativa. We have also identified two nearly identical resistant alleles of xa5 and two sequentially identical alleles of Xa21. The alleles of xa5 showed basal levels of expression while Xa21 alleles were functionally not expressed.

  5. An acute dose of gamma-hydroxybutyric acid alters gene expression in multiple mouse brain regions.

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    Schnackenberg, B J; Saini, U T; Robinson, B L; Ali, S F; Patterson, T A

    2010-10-13

    Gamma-hydroxybutyric acid (GHB) is normally found in the brain in low concentrations and may function as a neurotransmitter, although the mechanism of action has not been completely elucidated. GHB has been used as a general anesthetic and is currently used to treat narcolepsy and alcoholism. Recreational use of GHB is primarily as a "club drug" and a "date rape drug," due to its amnesic effects. For this study, the hypothesis was that behavioral and neurochemical alterations may parallel gene expression changes in the brain after GHB administration. Adult male C57/B6N mice (n=5/group) were administered a single dose of 500 mg/kg GHB (i.p.) and were sacrificed 1, 2 and 4 h after treatment. Control mice were administered saline. Brains were removed and regionally dissected on ice. Total RNA from the hippocampus, cortex and striatum was extracted, amplified and labeled. Gene expression was evaluated using Agilent whole mouse genome 4x44K oligonucleotide microarrays. Microarray data were analyzed by ArrayTrack and differentially expressed genes (DEGs) were identified using P or = 1.7 as the criteria for significance. Principal component analysis (PCA) and Hierarchical Cluster Analysis (HCA) showed that samples from each time point clustered into distinct treatment groups with respect to sacrifice time. Ingenuity pathways analysis (IPA) was used to identify involved pathways. The results show that GHB induces gene expression alterations in hundreds of genes in the hippocampus, cortex and striatum, and the number of affected genes increases throughout a 4-h time course. Many of these DEGs are involved in neurological disease, apoptosis, and oxidative stress.

  6. Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows.

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    Loor, Juan J; Everts, Robin E; Bionaz, Massimo; Dann, Heather M; Morin, Dawn E; Oliveira, Rosane; Rodriguez-Zas, Sandra L; Drackley, James K; Lewin, Harris A

    2007-12-19

    Dairy cows are highly susceptible after parturition to developing liver lipidosis and ketosis, which are costly diseases to farmers. A bovine microarray platform consisting of 13,257-annotated oligonucleotides was used to study hepatic gene networks underlying nutrition-induced ketosis. On day 5 postpartum, 14 Holstein cows were randomly assigned to ketosis-induction (n = 7) or control (n = 7) groups. Cows in the ketosis-induction group were fed at 50% of day 4 intake until they developed signs of clinical ketosis, and cows in the control group were fed ad libitum throughout the treatment period. Liver was biopsied at 10-14 (ketosis) or 14 days postpartum (controls). Feed restriction increased blood concentrations of nonesterified fatty acids and beta-hydroxybutyrate, but decreased glucose. Liver triacylglycerol concentration also increased. A total of 2,415 genes were altered by ketosis (false discovery rate = 0.05). Ingenuity Pathway Analysis revealed downregulation of genes associated with oxidative phosphorylation, protein ubiquitination, and ubiquinone biosynthesis with ketosis. Other molecular adaptations included upregulation of genes and nuclear receptors associated with cytokine signaling, fatty acid uptake/transport, and fatty acid oxidation. Genes downregulated during ketosis included several associated with cholesterol metabolism, growth hormone signaling, proton transport, and fatty acid desaturation. Feed restriction and ketosis resulted in previously unrecognized alterations in gene network expression underlying key cellular functions and discrete metabolic events. These responses might help explain well-documented physiological adaptations to reduced feed intake in early postpartum cows and, thus, provide molecular targets that might be useful in prevention and treatment of liver lipidosis and ketosis.

  7. Addiction and Reward-related Genes Show Altered Expression in the Postpartum Nucleus Accumbens

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    Changjiu eZhao

    2014-11-01

    Full Text Available Motherhood involves a switch in natural rewards, whereby offspring become highly rewarding. Nucleus accumbens (NAC is a key CNS region for natural rewards and addictions, but to date no study has evaluated on a large scale the events in NAC that underlie the maternal change in natural rewards. In this study we utilized microarray and bioinformatics approaches to evaluate postpartum NAC gene expression changes in mice. Modular Single-set Enrichment Test (MSET indicated that postpartum (relative to virgin NAC gene expression profile was significantly enriched for genes related to addiction and reward in 5 of 5 independently curated databases (e.g., Malacards, Phenopedia. Over 100 addiction/reward related genes were identified and these included: Per1, Per2, Arc, Homer2, Creb1, Grm3, Fosb, Gabrb3, Adra2a, Ntrk2, Cry1, Penk, Cartpt, Adcy1, Npy1r, Htr1a, Drd1a, Gria1, and Pdyn. ToppCluster analysis found maternal NAC expression profile to be significantly enriched for genes related to the drug action of nicotine, ketamine, and dronabinol. Pathway analysis indicated postpartum NAC as enriched for RNA processing, CNS development/differentiation, and transcriptional regulation. Weighted Gene Coexpression Network Analysis identified possible networks for transcription factors, including Nr1d1, Per2, Fosb, Egr1, and Nr4a1. The postpartum state involves increased risk for mental health disorders and MSET analysis indicated postpartum NAC to be enriched for genes related to depression, bipolar disorder, and schizophrenia. Mental health related genes included: Fabp7, Grm3, Penk, and Nr1d1. We confirmed via quantitative PCR Nr1d1, Per2, Grm3, Penk, Drd1a, and Pdyn. This study indicates for the first time that postpartum NAC involves large scale gene expression alterations linked to addiction and reward. Because the postpartum state also involves decreased response to drugs, the findings could provide insights into how to mitigate addictions.

  8. Dehydration, rehydration, and overhydration alter patterns of gene expression in the Antarctic midge, Belgica antarctica.

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    Lopez-Martinez, Giancarlo; Benoit, Joshua B; Rinehart, Joseph P; Elnitsky, Michael A; Lee, Richard E; Denlinger, David L

    2009-05-01

    We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene's responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins (smHsp, Hsp70, Hsp90), antioxidants (superoxide dismutase, catalase), detoxification (metallothionein, cytochrome p450), genes involved in altering cell membranes (fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton (actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment.

  9. Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems.

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    Wang, H; Jones, R W

    1999-09-01

    Functional expression of a beta-D-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60 degrees C, whereas the enzyme activity was optimal at 40 degrees C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.

  10. Light without substrate amendment: the bacterial luciferase gene cassette as a mammalian bioreporter

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    Close, Dan M.; Xu, Tingting; Smartt, Abby E.; Jegier, Pat; Ripp, Steven A.; Sayler, Gary S.

    2011-06-01

    Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor development.

  11. NF-Y activates genes of metabolic pathways altered in cancer cells

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    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A.; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-01

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells. PMID:26646448

  12. Long-term oil contamination alters the molecular ecological networks of soil microbial functional genes

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    Yuting eLiang

    2016-02-01

    Full Text Available With knowledge on microbial composition and diversity, investigation of within-community interactions is a further step to elucidate microbial ecological functions, such as the biodegradation of hazardous contaminants. In this work, microbial functional molecular ecological networks were studied in both contaminated and uncontaminated soils to determine the possible influences of oil contamination on microbial interactions and potential functions. Soil samples were obtained from an oil-exploring site located in South China, and the microbial functional genes were analyzed with GeoChip, a high-throughput functional microarray. By building random networks based on null model, we demonstrated that overall network structures and properties were significantly different between contaminated and uncontaminated soils (P < 0.001. Network connectivity, module numbers, and modularity were all reduced with contamination. Moreover, the topological roles of the genes (module hub and connectors were altered with oil contamination. Subnetworks of genes involved in alkane and polycyclic aromatic hydrocarbon degradation were also constructed. Negative co-occurrence patterns prevailed among functional genes, thereby indicating probable competition relationships. The potential keystone genes, defined as either hubs or genes with highest connectivities in the network, were further identified. The network constructed in this study predicted the potential effects of anthropogenic contamination on microbial community co-occurrence interactions.

  13. Mechanical force alters morphogenetic movements and segmental gene expression patterns during Drosophila embryogenesis.

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    Abhishek Kumar

    Full Text Available The development of an organism is accompanied by various cellular morphogenetic movements, changes in cellular as well as nuclear morphology and transcription programs. Recent evidence suggests that intra and inter-cellular connections mediated by various adhesion proteins contribute to defining nuclear morphology. In addition, three dimensional organization of the cell nucleus regulate the transcription programs. However the link between cellular morphogenetic movements and its coupling to nuclear function in a developmental context is poorly understood. In this paper we use a point perturbation by tissue level laser ablation and sheet perturbation by application of force using magnetic tweezers to alter cellular morphogenetic movements and probe its impact on nuclear morphology and segmental gene expression patterns. Mechanical perturbations during blastoderm stage in a developing Drosophila embryo resulted in localized alterations in nuclear morphology and cellular movement. In addition, global defects in germ-band (GB extension and retraction are observed when external force is applied during morphogenetic movements, suggesting a long-range physical coupling within the GB layer of cells. Further local application of force resulted in redistribution of non muscle myosin-II in the GB layer. Finally these perturbations lead to altered segmental gene (engrailed expression patterns later during the development. Our observations suggest that there exists a tight regulation between nuclear morphology and cellular adhesive connections during morphogenetic movement of cells in the embryo. The observed spatial changes in patterning genes, with perturbation, highlight the importance of nuclear integrity to cellular movement in establishing gene expression program in a developmental system.

  14. Fetal and neonatal exposure to the endocrine disruptor methoxychlor causes epigenetic alterations in adult ovarian genes.

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    Zama, Aparna Mahakali; Uzumcu, Mehmet

    2009-10-01

    Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-beta, which was down-regulated, whereas ERalpha was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 microg/kgxd or 100 mg/kg.d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERalpha and ERbeta genes in postnatal d 50-60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERbeta promoter regions (P < 0.05), whereas the ERalpha promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

  15. Prevalence of antibiotic resistance genes in bacterial communities associated with Cladophora glomerata mats along the nearshore of Lake Ontario.

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    Ibsen, Michael; Fernando, Dinesh M; Kumar, Ayush; Kirkwood, Andrea E

    2017-05-01

    The alga Cladophora glomerata can erupt in nuisance blooms throughout the lower Great Lakes. Since bacterial abundance increases with the emergence and decay of Cladophora, we investigated the prevalence of antibiotic resistance (ABR) in Cladophora-associated bacterial communities up-gradient and down-gradient from a large sewage treatment plant (STP) on Lake Ontario. Although STPs are well-known sources of ABR, we also expected detectable ABR from up-gradient wetland communities, since they receive surface run-off from urban and agricultural sources. Statistically significant differences in aquatic bacterial abundance and ABR were found between down-gradient beach samples and up-gradient coastal wetland samples (ANOVA, Holm-Sidak test, p floating Cladophora sampled near the STP had the highest bacterial densities overall, including on ampicillin- and vancomycin-treated plates. However, quantitative polymerase chain reaction analysis of the ABR genes ampC, tetA, tetB, and vanA from environmental communities showed a different pattern. Some of the highest ABR gene levels occurred at the 2 coastal wetland sites (vanA). Overall, bacterial ABR profiles from environmental samples were distinguishable between living and decaying Cladophora, inferring that Cladophora may control bacterial ABR depending on its life-cycle stage. Our results also show how spatially and temporally dynamic ABR is in nearshore aquatic bacteria, which warrants further research.

  16. Genome wide transcriptome analysis of dendritic cells identifies genes with altered expression in psoriasis.

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    Kata Filkor

    Full Text Available Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS or peptidoglycan (PGN induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs upon PGN induced tolerance. Using SAGE-Seq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (Kegg analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified

  17. Alterations of c-Myc and c-erbB-2 genes in ovarian tumours

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    Pastor Tibor

    2009-01-01

    Full Text Available Introduction. According to clinical and epidemiological studies, ovarian cancer ranks fifth in cancer deaths among women. The causes of ovarian cancer remain largely unknown but various factors may increase the risk of developing it, such as age, family history of cancer, childbearing status etc. This cancer results from a succession of genetic alterations involving oncogenes and tumour suppressor genes, which have a critical role in normal cell growth regulation. Mutations and/or overexpression of three oncogenes, c-erbB-2, c-Myc and K-ras, and of the tumour suppressor gene p53, have been frequently observed in a sporadic ovarian cancer. Objective. The aim of the present study was to analyze c-Myc and c-erbB-2 oncogene alterations, specifically amplification, as one of main mechanisms of their activation in ovarian cancers and to establish a possible association with the pathogenic process. Methods. DNA was isolated from 15 samples of malignant and 5 benign ovarian tumours, using proteinase K digestion, followed by phenol-chloroform isoamyl extraction and ethanol precipitation. C-Myc and c-erbB-2 amplification were detected by differential PCR. The level of gene copy increase was measured using the Scion image software. Results. The amplification of both c-Myc and c-erbB-2 was detected in 26.7% of ovarian epithelial carcinoma specimens. Only one tumour specimen concomitantly showed increased gene copy number for both studied genes. Interestingly, besides amplification, gene deletion was also detected (26.7% for c-erbB-2. Most of the ovarian carcinomas with alterations in c-Myc and c-erbB-2 belonged to advanced FIGO stages. Conclusion. The amplification of c-Myc and c-erbB-2 oncogenes in ovarian epithelial carcinomas is most probably a late event in the pathogenesis conferring these tumours a more aggressive biological behaviour. Similarly, gene deletions point to genomic instability in epithelial carcinomas in higher clinical stages as the

  18. Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

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    Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

    2016-03-18

    A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

  19. Simulated microgravity alters the expression of key genes involved in fracture healing

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    McCabe, N. Patrick; Androjna, Caroline; Hill, Esther; Globus, Ruth K.; Midura, Ronald J.

    2013-11-01

    Fracture healing in animal models has been shown to be altered in both ground based analogs of spaceflight and in those exposed to actual spaceflight. The molecular mechanisms behind altered fracture healing as a result of chronic exposure to microgravity remain to be elucidated. This study investigates temporal gene expression of multiple factors involved in secondary fracture healing, specifically those integral to the development of a soft tissue callus and the transition to that of hard tissue. Skeletally mature female rats were subjected to a 4 week period of simulated microgravity and then underwent a closed femoral fracture procedure. Thereafter, they were reintroduced to the microgravity and allowed to heal for a 1 or 2 week period. A synchronous group of weight bearing rats was used as a normal fracture healing control. Utilizing Real-Time quantitative PCR on mRNA from fracture callus tissue, we found significant reductions in the levels of transcripts associated with angiogenesis, chondrogenesis, and osteogenesis. These data suggest an altered fracture healing process in a simulated microgravity environment, and these alterations begin early in the healing process. These findings may provide mechanistic insight towards developing countermeasure protocols to mitigate these adaptations.

  20. Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors

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    Hamm Christopher A

    2010-09-01

    Full Text Available Abstract Background Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established - namely, the Swarm Rat Chondrosarcoma (SRC - and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy. Methods To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC. Results The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-β4, c-fos, and CTGF may play in chondrosarcoma development and progression. Conclusion This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that

  1. Who Possesses Drug Resistance Genes in the Aquatic Environment? : Sulfamethoxazole (SMX Resistance Genes among the Bacterial Community in Water Environment of Metro-Manila, Philippines

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    Satoru eSuzuki

    2013-04-01

    Full Text Available Recent evidence has shown that antibiotic resistant bacteria (ARB and antibiotic resistance genes (ARG are ubiquitous in natural environments, including sites considered pristine. To understand the origin of ARGs and their dynamics, we must first define their actual presence in the natural bacterial assemblage. Here we found varying distribution profiles of sul genes in colony forming bacterial assemblages and natural bacterial assemblages. Our monitoring for antibiotic contamination revealed that sulfamethoxazole (SMX is a major contaminant in aquatic environments of Metro-Manila, which would have been derived from human and animal use, and subsequently decreased through the process of outflow from source to the sea. The SMX-resistant bacterial rate evaluated by the colony forming unit showed 10 to 86 % of the total colony numbers showed higher rates from freshwater sites compared to marine sites. When sul genes were quantified by qPCR, colony-forming bacteria conveyed sul1 and sul2 genes in freshwater and seawater (10-5-10-2 copy/16S but not sul3. Among the natural bacterial assemblage, all sul1, sul2 and sul3 were detected (10-5-10-3 copy/16S, whereas all sul genes were at an almost non-detectable level in the freshwater assemblage. This study suggests that sul1 and sul2 are main sul genes in culturable bacteria, whereas sul3 is conveyed by non-culturable bacteria in the sea. As a result marine bacteria possess sul1, sul2 and sul3 genes in the marine environment.

  2. Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure, with Concomitant Bacterial Population Changes

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    Wang, Hang; Li, Hongyi; Gilbert, Jack A.; Li, Haibo; Wu, Longhua; Liu, Meng; Wang, Liling; Zhou, Qiansheng; Yuan, Junxiang; Zhang, Zhijian

    2015-11-01

    Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems.

  3. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

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    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  4. Somatic Copy Number Alterations at Oncogenic Loci Show Diverse Correlations with Gene Expression

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    Roszik, Jason; Wu, Chang-Jiun; Siroy, Alan E.; Lazar, Alexander J.; Davies, Michael A.; Woodman, Scott E.; Kwong, Lawrence N.

    2016-01-01

    Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy.

  5. Altered glial gene expression, density, and architecture in the visual cortex upon retinal degeneration.

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    Cornett, Ashley; Sucic, Joseph F; Hillsburg, Dylan; Cyr, Lindsay; Johnson, Catherine; Polanco, Anthony; Figuereo, Joe; Cabine, Kenneth; Russo, Nickole; Sturtevant, Ann; Jarvinen, Michael K

    2011-11-08

    Genes encoding the proteins of cytoskeletal intermediate filaments (IF) are tightly regulated, and they are important for establishing neural connections. However, it remains uncertain to what extent neurological disease alters IF gene expression or impacts cells that express IFs. In this study, we determined the onset of visual deficits in a mouse model of progressive retinal degeneration (Pde6b(-) mice; Pde6b(+) mice have normal vision) by observing murine responses to a visual task throughout development, from postnatal day (PND) 21 to adult (N=174 reliable observations). Using Q-PCR, we evaluated whether expression of the genes encoding two Type III IF proteins, glial fibrillary acidic protein (GFAP) and vimentin was altered in the visual cortex before, during, and after the onset of visual deficits. Using immunohistochemical techniques, we investigated the impact of vision loss on the density and morphology of astrocytes that expressed GFAP and vimentin in the visual cortex. We found that Pde6b(-) mice displayed 1) evidence of blindness at PND 49, with visual deficits detected at PND 35, 2) reduced GFAP mRNA expression in the visual cortex between PND 28 and PND 49, and 3) an increased ratio of vimentin:GFAP-labeled astrocytes at PND 49 with reduced GFAP cell body area. Together, these findings demonstrate that retinal degeneration modifies cellular and molecular indices of glial plasticity in a visual system with drastically reduced visual input. The functional consequences of these structural changes remain uncertain.

  6. Altered expression of adipose differentiation-related protein gene in placental tissue of pre-eclampsia

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun-li; YAO Yuan-qing; LI Dong-hong; ZHANG Wei

    2006-01-01

    Objective: To investigate the altered expression of lipid metabolism-related gene adipose differentiation-related protein (ADRP) in pre-eclampsia. Methods: Semi-quantitative RT-PCR and Western blotting were used to validate the altered expression of ADRP gene between pre-eclamptic placentas (preeclampsia group) and normotensive placentas (control group) respectively. In situ hybridization (ISH)was used to localize ADRP mRNA in pre-eclamptic placentas. Results: There was a significant difference in the levels of placental ADRP mRNA between pre-eclampsia group and control group (1.98± 0. 50 vs 1. 09±0. 20, P<0.01). Western blotting showed that placentas both in pre-eclampsia group and control group expressed the special ADRP band at 48. 1 kD. The relative levels of ADRP protein in pre-eclampsia group were significantly higher than those of control group (0. 40 ±0. 19 vs 0. 19 ±0. 09, P< 0. 01).ADRP mRNA was diffusely distributed in pre-eclamptic placentas. Their positive staining existed in cytoplasm of trophoblast. Conclusion: Abnormal expression of ADRP gene in pre-eclamptic placenta may be associated with the pathogenesis of pre-eclampsia.

  7. Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling.

    Directory of Open Access Journals (Sweden)

    Chongwei Chen

    Full Text Available This paper aims to investigate whether equibiaxial tensile strain alters chondrocyte gene expression via controlling subcellular localization of histone deacetylase 4 (HDAC4.Murine chondrocytes transfected with GFP-HDAC4 were subjected to 3 h cyclic equibiaxial tensile strain (CTS, 6% strain at 0.25 Hz by a Flexcell® FX-5000™ Tension System. Fluorescence microscope and western blot were used to observe subcellular location of HDAC4. The gene expression was analyzed by real-time RT-PCR. The concentration of Glycosaminoglycans in culture medium was quantified by bimethylmethylene blue dye; Collagen II protein was evaluated by western blot. Cells phenotype was identified by immunohistochemistry. Cell viability was evaluated by live-dead cell detect kit. Okadaic acid, an inhibitor of HDAC4 nuclear relocation, was used to further validate whether HDAC4 nuclear relocation plays a role in gene expression in response to tension stimulation.87.5% of HDAC4 was located in the cytoplasm in chondrocytes under no loading condition, but it was relocated to the nucleus after CTS. RT-PCR analysis showed that levels of mRNA for aggrecan, collagen II, LK1 and SOX9 were all increased in chondrocytes subjected to CTS as compared to no loading control chondrocytes; in contrast, the levels of type X collagen, MMP-13, IHH and Runx2 gene expression were decreased in the chondrocytes subjected to CTS as compared to control chondrocytes. Meanwhile, CTS contributed to elevation of glycosaminoglycans and collagen II protein, but did not change collagen I production. When Okadaic acid blocked HDAC4 relocation from the cytoplasm to nucleus, the changes of the chondrocytes induced by CTS were abrogated. There was no chondrocyte dead detected in this study in response to CTS.CTS is able to induce HDAC4 relocation from cytoplasm to nucleus. Thus, CTS alters chondrocytes gene expression in association with the relocation of HDAC4 induced by CTS.

  8. Altered Protein Composition and Gene Expression in Strabismic Human Extraocular Muscles and Tendons

    Science.gov (United States)

    Agarwal, Andrea B.; Feng, Cheng-Yuan; Altick, Amy L.; Quilici, David R.; Wen, Dan; Johnson, L. Alan; von Bartheld, Christopher S.

    2016-01-01

    Purpose To determine whether structural protein composition and expression of key regulatory genes are altered in strabismic human extraocular muscles. Methods Samples from strabismic horizontal extraocular muscles were obtained during strabismus surgery and compared with normal muscles from organ donors. We used proteomics, standard and customized PCR arrays, and microarrays to identify changes in major structural proteins and changes in gene expression. We focused on muscle and connective tissue and its control by enzymes, growth factors, and cytokines. Results Strabismic muscles showed downregulation of myosins, tropomyosins, troponins, and titin. Expression of collagens and regulators of collagen synthesis and degradation, the collagenase matrix metalloproteinase (MMP)2 and its inhibitors, tissue inhibitor of metalloproteinase (TIMP)1 and TIMP2, was upregulated, along with tumor necrosis factor (TNF), TNF receptors, and connective tissue growth factor (CTGF), as well as proteoglycans. Growth factors controlling extracellular matrix (ECM) were also upregulated. Among 410 signaling genes examined by PCR arrays, molecules with downregulation in the strabismic phenotype included GDNF, NRG1, and PAX7; CTGF, CXCR4, NPY1R, TNF, NTRK1, and NTRK2 were upregulated. Signaling molecules known to control extraocular muscle plasticity were predominantly expressed in the tendon rather than the muscle component. The two horizontal muscles, medial and lateral rectus, displayed similar changes in protein and gene expression, and no obvious effect of age. Conclusions Quantification of proteins and gene expression showed significant differences in the composition of extraocular muscles of strabismic patients with respect to important motor proteins, elements of the ECM, and connective tissue. Therefore, our study supports the emerging view that the molecular composition of strabismic muscles is substantially altered. PMID:27768799

  9. Bisphenol A exposure alters developmental gene expression in the fetal rhesus macaque uterus.

    Directory of Open Access Journals (Sweden)

    Kathryn C Calhoun

    Full Text Available Bisphenol A (BPA exposure results in numerous developmental and functional abnormalities in reproductive organs in rodent models, but limited data are available regarding BPA effects in the primate uterus. To determine if maternal oral BPA exposure affects fetal uterine development in a non-human primate model, pregnant rhesus macaques carrying female fetuses were exposed orally to 400 µg/kg BPA or vehicle control daily from gestation day (GD 50-100 or GD100-165. Fetal uteri were collected at the completion of treatment (GD100 or GD165; tissue histology, cell proliferation, and expression of estrogen receptor alpha (ERα and progesterone receptor (PR were compared to that of controls. Gene expression analysis was conducted using rhesus macaque microarrays. There were no significant differences in histology or in the percentage of cells expressing the proliferation marker Ki-67, ERα, or PR in BPA-exposed uteri compared to controls at GD100 or GD165. Minimal differences in gene expression were observed between BPA-exposed and control GD100 uteri. However, at GD165, BPA-exposed uteri had significant differences in gene expression compared to controls. Several of the altered genes, including HOXA13, WNT4, and WNT5A, are critical for reproductive organ development and/or adult function. We conclude that second or third trimester BPA exposure does not significantly affect fetal uterus development based on morphological, proliferation, and steroid hormone receptor assessments. However, differences in expression of key developmental genes after third trimester exposure suggest that BPA could alter transcriptional signals influencing uterine function later in life.

  10. Silver nanoparticles mediated altered gene expression of melanin biosynthesis genes in Bipolaris sorokiniana.

    Science.gov (United States)

    Mishra, Sandhya; Singh, H B

    2015-03-01

    Melanin production in many fungal phytopathogens has been investigated to play direct or indirect role in pathogenesis. However, in Bipolaris sorokiniana, the spot blotch pathogen of wheat, much less is known about the role melanin play in pathogenesis. As an extension of our previous report, the present study aims to investigate the plausible association between melanin production and virulence factor in B. sorokiniana. In the previous study, we carried out analysis on the antifungal efficacy of biosynthesized silver nanoparticles (AgNPs) against B. sorokiniana. The present investigation revealed the gene expression analysis of melanin biosynthesis genes viz. polyketide synthase (PKS1) and scytalone dehydratase (SCD1) under the influence of AgNPs. The 0.05mg/ml concentration of AgNPs yielded noticeable inhibition of B. sorokiniana growth, while 0.1mg/ml concentration of AgNPs accounted for complete inhibition of pathogen growth. In addition, the semiquantitative RT-PCR analysis exhibited reduced expression of PKS1 and SCD1 under the influence of AgNPs treatment. Furthermore, the qRT-PCR demonstrated 6.47 and 1.808 fold significant decrease in the expression pattern of PKS1 and SCD1, respectively, in B. sorokiniana treated with AgNPs. The present study provides probable understanding of molecular events underlying the antifungal role of AgNPs against B. sorokiniana.

  11. Ginsenoside Rg1-induced alterations in gene expression in TNF-α stimulated endothelial cells

    Institute of Scientific and Technical Information of China (English)

    吕俊萍; 马增春; 杨静; 黄坚; 王树人; 王升启

    2004-01-01

    Background In China the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The authors investigated the effect of ginsenoside Rg1 on the spectrum of gene expression in the endothelial cells stimulated by TNF-α and further explored the potential molecular mechanism of endothelial protection by ginsenoside Rg1.Methods Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells(HUVECs) was measured by using an NO assay kit. A home-made oligonucleotide microarray containing approximately 400 cardiovascular disease-related genes was constructed. The alteration of the spectrum of gene expression induced by ginsenoside Rg1 in HUVECs which were activated by TNF-α were detected by oligonucleotide microarray analysis.Results NO production in HUVECs was decreased significantly after TNF-α treatment, while pretreatment with ginsenoside Rg1 enhanced NO production in TNF-αstimulated HUVECs. Ginsenoside Rg1 affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-αstimulated HUVECs.Conclusions Ginsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-α stimulated HUVECs. Ginsenoside Rg1 regulated sets of genes in endothelial cells and protected endothelial cells from TNF-αactivation. Microarray analysis provided us with valuable insights into the atheroprotective mechanism by gingsenoside Rg1.

  12. Recreational Music-Making alters gene expression pathways in patients with coronary heart disease.

    Science.gov (United States)

    Bittman, Barry; Croft, Daniel T; Brinker, Jeannie; van Laar, Ryan; Vernalis, Marina N; Ellsworth, Darrell L

    2013-02-25

    Psychosocial stress profoundly impacts long-term cardiovascular health through adverse effects on sympathetic nervous system activity, endothelial dysfunction, and atherosclerotic development. Recreational Music Making (RMM) is a unique stress amelioration strategy encompassing group music-based activities that has great therapeutic potential for treating patients with stress-related cardiovascular disease. Participants (n=34) with a history of ischemic heart disease were subjected to an acute time-limited stressor, then randomized to RMM or quiet reading for one hour. Peripheral blood gene expression using GeneChip® Human Genome U133A 2.0 arrays was assessed at baseline, following stress, and after the relaxation session. Full gene set enrichment analysis identified 16 molecular pathways differentially regulated (Pstress that function in immune response, cell mobility, and transcription. During relaxation, two pathways showed a significant change in expression in the control group, while 12 pathways governing immune function and gene expression were modulated among RMM participants. Only 13% (2/16) of pathways showed differential expression during stress and relaxation. Human stress and relaxation responses may be controlled by different molecular pathways. Relaxation through active engagement in Recreational Music Making may be more effective than quiet reading at altering gene expression and thus more clinically useful for stress amelioration.

  13. Phylogeny Inference of Closely Related Bacterial Genomes: Combining the Features of Both Overlapping Genes and Collinear Genomic Regions

    Science.gov (United States)

    Zhang, Yan-Cong; Lin, Kui

    2015-01-01

    Overlapping genes (OGs) represent one type of widespread genomic feature in bacterial genomes and have been used as rare genomic markers in phylogeny inference of closely related bacterial species. However, the inference may experience a decrease in performance for phylogenomic analysis of too closely or too distantly related genomes. Another drawback of OGs as phylogenetic markers is that they usually take little account of the effects of genomic rearrangement on the similarity estimation, such as intra-chromosome/genome translocations, horizontal gene transfer, and gene losses. To explore such effects on the accuracy of phylogeny reconstruction, we combine phylogenetic signals of OGs with collinear genomic regions, here called locally collinear blocks (LCBs). By putting these together, we refine our previous metric of pairwise similarity between two closely related bacterial genomes. As a case study, we used this new method to reconstruct the phylogenies of 88 Enterobacteriale genomes of the class Gammaproteobacteria. Our results demonstrated that the topological accuracy of the inferred phylogeny was improved when both OGs and LCBs were simultaneously considered, suggesting that combining these two phylogenetic markers may reduce, to some extent, the influence of gene loss on phylogeny inference. Such phylogenomic studies, we believe, will help us to explore a more effective approach to increasing the robustness of phylogeny reconstruction of closely related bacterial organisms. PMID:26715828

  14. Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Dalton, Helen M.; Angels, Mark;

    1997-01-01

    A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells: a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of Fluorescently labelled oligonucleotide probes to rRNA. The method a...

  15. Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium.

    Science.gov (United States)

    Shippy, Daniel C; Heintz, Joseph A; Albrecht, Ralph M; Eakley, Nicholas M; Chopra, Ashok K; Fadl, Amin A

    2012-06-01

    Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.

  16. Overexpression of bacterial mtlD gene in peanut improves drought tolerance through accumulation of mannitol.

    Science.gov (United States)

    Bhauso, Tengale Dipak; Radhakrishnan, Thankappan; Kumar, Abhay; Mishra, Gyan Prakash; Dobaria, Jentilal Ramjibhai; Patel, Kirankumar; Rajam, Manchikatla Venkat

    2014-01-01

    In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

  17. Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure, with Concomitant Bacterial Population Changes

    Science.gov (United States)

    Wang, Hang; Li, Hongyi; Gilbert, Jack A.; Li, Haibo; Wu, Longhua; Liu, Meng; Wang, Liling; Zhou, Qiansheng; Yuan, Junxiang

    2015-01-01

    Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems. PMID:26296728

  18. Identification of molecular markers linked to rice bacterial blight resistance genes from Oryza meyeriana

    Directory of Open Access Journals (Sweden)

    Jing WANG,Chen CHENG,Yanru ZHOU,Yong YANG,Qiong MEI,Junmin LI,Ye CHENG,Chengqi YAN,Jianping CHEN

    2015-09-01

    Full Text Available Y73 is a progeny of asymmetric somatic hybridization between Oryza sativa cv. Dalixiang and the wild rice species Oryza meyeriana. Inoculation with a range of strains of Xanthomonas oryzae pv. oryzae showed that Y73 had inherited a high level of resistance to rice bacterial blight (BB from its wild parent. An F2 population of 7125 individuals was constructed from the cross between Y73 and a BB-susceptible cultivar IR24. After testing 615 SSR and STS markers covering the 12 rice chromosomes, 186 markers were selected that showed polymorphism between Y73 and IR24. Molecular markers linked to the BB resistance genes in Y73 were scanned using the F2 population and the polymorphic markers. The SSR marker RM128 on chromosome 1, the STS marker R03D159 on chromosome 3 and the STS marker R05D104 on chromosome 5 were found to be linked to the rice BB resistance genes in Y73.

  19. Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    Directory of Open Access Journals (Sweden)

    Tengale Dipak Bhauso

    2014-01-01

    Full Text Available In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L. crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated.

  20. Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

    Science.gov (United States)

    Öztürk, Başak; Ghequire, Maarten; Nguyen, Thi Phi Oanh; De Mot, René; Wattiez, Ruddy; Springael, Dirk

    2016-12-01

    Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low KM towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    Science.gov (United States)

    Bhauso, Tengale Dipak; Radhakrishnan, Thankappan; Kumar, Abhay; Mishra, Gyan Prakash; Dobaria, Jentilal Ramjibhai; Patel, Kirankumar; Rajam, Manchikatla Venkat

    2014-01-01

    In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated. PMID:25436223

  2. A profile-based method for identifying functional divergence of orthologous genes in bacterial genomes.

    Science.gov (United States)

    Wheeler, Nicole E; Barquist, Lars; Kingsley, Robert A; Gardner, Paul P

    2016-12-01

    Next generation sequencing technologies have provided us with a wealth of information on genetic variation, but predicting the functional significance of this variation is a difficult task. While many comparative genomics studies have focused on gene flux and large scale changes, relatively little attention has been paid to quantifying the effects of single nucleotide polymorphisms and indels on protein function, particularly in bacterial genomics. We present a hidden Markov model based approach we call delta-bitscore (DBS) for identifying orthologous proteins that have diverged at the amino acid sequence level in a way that is likely to impact biological function. We benchmark this approach with several widely used datasets and apply it to a proof-of-concept study of orthologous proteomes in an investigation of host adaptation in Salmonella enterica We highlight the value of the method in identifying functional divergence of genes, and suggest that this tool may be a better approach than the commonly used dN/dS metric for identifying functionally significant genetic changes occurring in recently diverged organisms. A program implementing DBS for pairwise genome comparisons is freely available at: https://github.com/UCanCompBio/deltaBS CONTACT: nicole.wheeler@pg.canterbury.ac.nz or lars.barquist@uni-wuerzburg.deSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  3. Altered Pathway Analyzer: A gene expression dataset analysis tool for identification and prioritization of differentially regulated and network rewired pathways

    Science.gov (United States)

    Kaushik, Abhinav; Ali, Shakir; Gupta, Dinesh

    2017-01-01

    Gene connection rewiring is an essential feature of gene network dynamics. Apart from its normal functional role, it may also lead to dysregulated functional states by disturbing pathway homeostasis. Very few computational tools measure rewiring within gene co-expression and its corresponding regulatory networks in order to identify and prioritize altered pathways which may or may not be differentially regulated. We have developed Altered Pathway Analyzer (APA), a microarray dataset analysis tool for identification and prioritization of altered pathways, including those which are differentially regulated by TFs, by quantifying rewired sub-network topology. Moreover, APA also helps in re-prioritization of APA shortlisted altered pathways enriched with context-specific genes. We performed APA analysis of simulated datasets and p53 status NCI-60 cell line microarray data to demonstrate potential of APA for identification of several case-specific altered pathways. APA analysis reveals several altered pathways not detected by other tools evaluated by us. APA analysis of unrelated prostate cancer datasets identifies sample-specific as well as conserved altered biological processes, mainly associated with lipid metabolism, cellular differentiation and proliferation. APA is designed as a cross platform tool which may be transparently customized to perform pathway analysis in different gene expression datasets. APA is freely available at http://bioinfo.icgeb.res.in/APA. PMID:28084397

  4. Iron deficiency alters expression of genes implicated in Alzheimer disease pathogenesis.

    Science.gov (United States)

    Carlson, Erik S; Magid, Rhamy; Petryk, Anna; Georgieff, Michael K

    2008-10-27

    Neonatal brain iron deficiency occurs after insufficient maternal dietary iron intake, maternal hypertension, and maternal diabetes mellitus and results in short and long-term neurologic and behavioral deficits. Early iron deficiency affects the genomic profile of the developing hippocampus that persists despite iron repletion. The purpose of the present study was threefold: 1) quantitative PCR confirmation of our previous microarray results, demonstrating upregulation of a network of genes leading to beta-amyloid production and implicated in Alzheimer disease etiology in iron-deficient anemic rat pups at the time of hippocampal differentiation; 2) investigation of the potential contributions of iron deficiency anemia and iron treatment to this differential gene expression in the hippocampus; and 3) investigation of these genes over a developmental time course in a mouse model where iron deficiency is limited to hippocampus, is not accompanied by anemia and is not repletable. Quantitative PCR confirmed altered regulation in 6 of 7 Alzheimer-related genes (Apbb1, C1qa, Clu, App, Cst3, Fn1, Htatip) in iron-deficient rats relative to iron-sufficient controls at P15. Comparison of untreated to treated iron-deficient animals at this age suggested the strong role of iron deficiency, not treatment, in the upregulation of this gene network. The non-anemic hippocampal iron-deficient mouse demonstrated upregulation of all 7 genes in this pathway from P5 to P25. Our results suggest a role for neonatal iron deficiency in dysregulation of genes that may set the stage for long-term neurodegenerative disease and that this may occur through a histone modification mechanism.

  5. Variance of gene expression identifies altered network constraints in neurological disease.

    Directory of Open Access Journals (Sweden)

    Jessica C Mar

    2011-08-01

    Full Text Available Gene expression analysis has become a ubiquitous tool for studying a wide range of human diseases. In a typical analysis we compare distinct phenotypic groups and attempt to identify genes that are, on average, significantly different between them. Here we describe an innovative approach to the analysis of gene expression data, one that identifies differences in expression variance between groups as an informative metric of the group phenotype. We find that genes with different expression variance profiles are not randomly distributed across cell signaling networks. Genes with low-expression variance, or higher constraint, are significantly more connected to other network members and tend to function as core members of signal transduction pathways. Genes with higher expression variance have fewer network connections and also tend to sit on the periphery of the cell. Using neural stem cells derived from patients suffering from Schizophrenia (SZ, Parkinson's disease (PD, and a healthy control group, we find marked differences in expression variance in cell signaling pathways that shed new light on potential mechanisms associated with these diverse neurological disorders. In particular, we find that expression variance of core networks in the SZ patient group was considerably constrained, while in contrast the PD patient group demonstrated much greater variance than expected. One hypothesis is that diminished variance in SZ patients corresponds to an increased degree of constraint in these pathways and a corresponding reduction in robustness of the stem cell networks. These results underscore the role that variation plays in biological systems and suggest that analysis of expression variance is far more important in disease than previously recognized. Furthermore, modeling patterns of variability in gene expression could fundamentally alter the way in which we think about how cellular networks are affected by disease processes.

  6. Is gene transcription in mussel gills altered after exposure to Ag nanoparticles?

    Science.gov (United States)

    Bebianno, M J; Gonzalez-Rey, M; Gomes, T; Mattos, J J; Flores-Nunes, F; Bainy, A C D

    2015-11-01

    Nanotechnology is a rapid field of development with the enhancement of the production of different types of nanoparticles (NPs) applied in several industrial and commercial applications which increase the risk of their presence in the aquatic environment. Ag NPs have a wide application in everyday life products. However, there is concern about the exposure effects on aquatic organisms to these NPs. Therefore, this study aims to assess gene transcription alterations in mussels Mytilus galloprovincialis gills exposed for 2 weeks to Ag NPs (42 ± 10 nm, 10 μg.L(-1)). The genes were selected based on previous biomarkers and proteomic results and included superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), caspase 3/7-1 (CAS), cathepsin L (CATH), heat-shock protein 70 (HSP 70), cytochrome P450 4YA (CYP 4YA), the elongation factor (EF1), actin and α- tubulin. No significant changes in gene transcription profiles were observed after exposure of M. galloprovincialis to Ag NPs for 15 days. The lack of significant gene transcription responses is in light with previous results obtained for mussels exposed to these NPs and may be related to the fact that enzyme kinetics and relative abundance of proteins (increase of antioxidant enzymes and metalllothioneins (MTs) with the time of exposure) do not always directly reflect their relative mRNA levels. Nevertheless, their overall expression maintenance may signify that, at end of the exposure period (15 days), the transcription of the respective genes is no longer required, pointing out to a possible adaptation effect to nanoparticles or due to the levels of Ag NPs accumulated in this tissue at this exposure time. This study highlights that gene transcription application and role as an additional and/or alternative end point approach is important to understand the mode of action of these emergent contaminants in aquatic organisms. However, in future studies, the time window needs to be adjusted, as

  7. Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

    Science.gov (United States)

    Bandara, H M H N; K Cheung, B P; Watt, R M; Jin, L J; Samaranayake, L P

    2013-02-01

    Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

  8. Zinc source and concentration altered physiological responses of beef heifers during a combined viral-bacterial respiratory challenge

    Science.gov (United States)

    Three treatments were evaluated in feedlot heifers to determine the effects of zinc supplementation on the immune response to a combined viral-bacterial respiratory disease challenge. Thirty-two beef heifers (255+/-15 kg) were subjected to a 30d period of Zn depletion, then randomly assigned to one ...

  9. Increases in mean annual temperature do not alter soil bacterial community structure in tropical montane wet forests

    Science.gov (United States)

    Paul C. Selmants; Karen L. Adair; Creighton M. Litton; Christian P. Giardina; Egbert Schwartz

    2016-01-01

    Soil bacteria play a key role in regulating terrestrial biogeochemical cycling and greenhouse gas fluxes across the soil-atmosphere continuum. Despite their importance to ecosystem functioning, we lack a general understanding of how bacterial communities respond to climate change, especially in relatively understudied ecosystems like tropical montane wet...

  10. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions.

    Science.gov (United States)

    Silver, Simon; Phung, Le T

    2005-12-01

    Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.

  11. Chromosomal antioxidant genes have metal ion-specific roles as determinants of bacterial metal tolerance.

    Science.gov (United States)

    Harrison, Joe J; Tremaroli, Valentina; Stan, Michelle A; Chan, Catherine S; Vacchi-Suzzi, Caterina; Heyne, Belinda J; Parsek, Matthew R; Ceri, Howard; Turner, Raymond J

    2009-10-01

    Microbiological metal toxicity involves redox reactions between metal species and cellular molecules, and therefore, we hypothesized that antioxidant systems might be chromosomal determinants affecting the susceptibility of bacteria to metal toxicity. Here, survival was quantified in metal ion-exposed planktonic cultures of several Escherichia coli strains, each bearing a mutation in a gene important for redox homeostasis. This characterized approximately 250 gene-metal combinations and identified that sodA, sodB, gor, trxA, gshA, grxA and marR have distinct roles in safeguarding or sensitizing cells to different toxic metal ions (Cr(2)O(7)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), AsO(2)(-), SeO(3)(2-) or TeO(3)(2-)). To shed light on these observations, fluorescent sensors for reactive oxygen species (ROS) and reduced thiol (RSH) quantification were used to ascertain that different metal ions exert oxidative toxicity through disparate modes-of-action. These oxidative mechanisms of metal toxicity were categorized as involving ROS and thiol-disulfide chemistry together (AsO(2)(-), SeO(3)(2-)), ROS predominantly (Cu(2+), Cr(2)O(7)(2-)) or thiol-disulfide chemistry predominantly (Ag(+), Co(2+), Zn(2+), TeO(3)(2-)). Corresponding to this, promoter-luxCDABE fusions showed that toxic doses of different metal ions up- or downregulate the transcription of gene sets marking distinct pathways of cellular oxidative stress. Altogether, our findings suggest that different metal ions are lethal to cells through discrete pathways of oxidative biochemistry, and moreover, indicate that chromosomally encoded antioxidant systems may have metal ion-specific physiological roles as determinants of bacterial metal tolerance.

  12. The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter.

    Science.gov (United States)

    Close, Dan; Xu, Tingting; Smartt, Abby; Rogers, Alexandra; Crossley, Robert; Price, Sarah; Ripp, Steven; Sayler, Gary

    2012-01-01

    The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

  13. The Evolution of the Bacterial Luciferase Gene Cassette (lux) as a Real-Time Bioreporter

    Science.gov (United States)

    Close, Dan; Xu, Tingting; Smartt, Abby; Rogers, Alexandra; Crossley, Robert; Price, Sarah; Ripp, Steven; Sayler, Gary

    2012-01-01

    The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted. PMID:22368493

  14. The Evolution of the Bacterial Luciferase Gene Cassette (lux as a Real-Time Bioreporter

    Directory of Open Access Journals (Sweden)

    Gary Sayler

    2012-01-01

    Full Text Available The bacterial luciferase gene cassette (lux is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

  15. A plant natriuretic peptide-like gene in the bacterial pathogen Xanthomonas axonopodis may induce hyper-hydration in the plant host: a hypothesis of molecular mimicry

    Directory of Open Access Journals (Sweden)

    Sayed Muhammed

    2004-03-01

    Full Text Available Abstract Background Plant natriuretic peptides (PNPs are systemically mobile molecules that regulate homeostasis at nanomolar concentrations. PNPs are up-regulated under conditions of osmotic stress and PNP-dependent processes include changes in ion transport and increases of H2O uptake into protoplasts and whole tissue. Presentation of the hypothesis The bacterial citrus pathogen Xanthomonas axonopodis pv. Citri str. 306 contains a gene encoding a PNP-like protein. We hypothesise that this bacterial protein can alter plant cell homeostasis and thus is likely to represent an example of molecular mimicry that enables the pathogen to manipulate plant responses in order to bring about conditions favourable to the pathogen such as the induced plant tissue hyper-hydration seen in the wet edged lesions associated with Xanthomonas axonopodis infection. Testing the hypothesis We found a Xanthomonas axonopodis PNP-like protein that shares significant sequence similarity and identical domain organisation with PNPs. We also observed a significant excess of conserved residues between the two proteins within the domain previously identified as being sufficient to induce biological activity. Structural modelling predicts identical six stranded double-psi β barrel folds for both proteins thus supporting the hypothesis of similar modes of action. No significant similarity between the Xanthomonas axonopodis protein and other bacterial proteins from GenBank was found. Sequence similarity of the Xanthomonas axonopodis PNP-like protein with the Arabidopsis thaliana PNP (AtPNP-A, shared domain organisation and incongruent phylogeny suggest that the PNP-gene may have been acquired by the bacteria in an ancient lateral gene transfer event. Finally, activity of a recombinant Xanthomonas axonopodis protein in plant tissue and changes in symptoms induced by a Xanthomonas axonopodis mutant with a knocked-out PNP-like gene will be experimental proof of molecular mimicry

  16. Sludge as a potential important source of antibiotic resistance genes in both the bacterial and bacteriophage fractions.

    Science.gov (United States)

    Calero-Cáceres, William; Melgarejo, Ana; Colomer-Lluch, Marta; Stoll, Claudia; Lucena, Francisco; Jofre, Juan; Muniesa, Maite

    2014-07-01

    The emergence and prevalence of antibiotic resistance genes (ARGs) in the environment is a serious global health concern. ARGs found in bacteria can become mobilized in bacteriophage particles in the environment. Sludge derived from secondary treatment in wastewater treatment plants (WWTPs) constitutes a concentrated pool of bacteria and phages that are removed during the treatment process. This study evaluates the prevalence of ARGs in the bacterial and phage fractions of anaerobic digested sludge; five ARGs (blaTEM, blaCTX-M, qnrA, qnrS, and sul1) are quantified by qPCR. Comparison between the wastewater and sludge revealed a shift in the prevalence of ARGs (blaTEM and sul1 became more prevalent in sludge), suggesting there is a change in the bacterial and phage populations from wastewater to those selected during the secondary treatment and the later anaerobic mesophilic digestion of the sludge. ARGs densities were higher in the bacterial than in the phage fraction, with high densities in both fractions; particularly for blaTEM and sul1 (5 and 8 log10 gene copies (GC)/g, respectively, in bacterial DNA; 5.5 and 4.4 log10 GC/g, respectively, in phage DNA). These results question the potential agricultural uses of treated sludge, as it could contribute to the spread of ARGs in the environment and have an impact on the bacterial communities of the receiving ecosystem.

  17. Paralysis and killing of Caenorhabditis elegans by enteropathogenic Escherichia coli requires the bacterial tryptophanase gene.

    Science.gov (United States)

    Anyanful, Akwasi; Dolan-Livengood, Jennifer M; Lewis, Taiesha; Sheth, Seema; Dezalia, Mark N; Sherman, Melanie A; Kalman, Lisa V; Benian, Guy M; Kalman, Daniel

    2005-08-01

    Pathogenic Escherichia coli, including enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) are major causes of food and water-borne disease. We have developed a genetically tractable model of pathogenic E. coli virulence based on our observation that these bacteria paralyse and kill the nematode Caenorhabditis elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting that a secreted toxin mediates the effect. Virulence against C. elegans required tryptophan and bacterial tryptophanase, the enzyme catalysing the production of indole and other molecules from tryptophan. Thus, lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyse or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a cocultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal non-pathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyse and kill nematodes. Furthermore, tryptophanase activity was required for full activation of the LEE1 promoter, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes including sek-1, mek-1, mev-1, pgp-1,3 and vhl-1, rendered C. elegans more

  18. Multiple insulin degrading enzyme variants alter in vitro reporter gene expression.

    Directory of Open Access Journals (Sweden)

    Olivia Belbin

    Full Text Available The insulin degrading enzyme (IDE variant, v311 (rs6583817, is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ, decreased risk for Alzheimer's disease (AD and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957, for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2 successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04 but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5' to the promoter (1.18 fold-change, p = 0.006 and 3' to the reporter gene (1.29 fold change, p = 0.003, an effect contributed to by four variants (v10, v315, v154 and v311. Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.

  19. Protein and Amino Acid Supplementation Does Not Alter Proteolytic Gene Expression following Immobilization

    Directory of Open Access Journals (Sweden)

    Jennifer A. Bunn

    2011-01-01

    Full Text Available Objective. To determine if supplementation of protein and amino acids (PAA decreases skeletal muscle expression of atrophy-related genes, muscle mass, and strength during immobilization in humans. Methods. Twenty males wore a lower-limb immobilization boot for 28 days and consumed either a PAA supplement (28 g protein or carbohydrate placebo (28 g maltodextrose, while consuming their normal daily diet. Testing sessions included dietary analysis, lower-leg girth and body composition measurements, strength testing, and gastrocnemius muscle biopsies. Muscle was analyzed for mRNA expression of markers in the ubiquitin and calpain systems, myostatin, TNF-α, and NF-κB. Results. All genes of interest increased over time (P<.05, but there was no difference between groups. Lower-leg girth decreased over time (P=0.02; however, there were no significant changes in body composition or strength. Conclusion. Short-term lower-limb disuse, despite the absence of significant muscle atrophy, is associated with increases in skeletal muscle gene expression of several proteolysis-related genes. These changes do not appear to be altered by oral PAA supplementation.

  20. Epigenetic alteration of imprinted genes during neural differentiation of germline-derived pluripotent stem cells.

    Science.gov (United States)

    Lee, Hye Jeong; Choi, Na Young; Lee, Seung-Won; Ko, Kisung; Hwang, Tae Sook; Han, Dong Wook; Lim, Jisun; Schöler, Hans R; Ko, Kinarm

    2016-03-01

    Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.

  1. Stress alters the expression of cancer-related genes in the prostate.

    Science.gov (United States)

    Flores, Ivan E; Sierra-Fonseca, Jorge A; Davalos, Olinamyr; Saenz, Luis A; Castellanos, Maria M; Zavala, Jaidee K; Gosselink, Kristin L

    2017-09-05

    Prostate cancer is a major contributor to mortality worldwide, and significant efforts are being undertaken to decipher specific cellular and molecular pathways underlying the disease. Chronic stress is known to suppress reproductive function and promote tumor progression in several cancer models, but our understanding of the mechanisms through which stress contributes to cancer development and progression is incomplete. We therefore examined the relationship between stress, modulation of the gonadotropin-releasing hormone (GnRH) system, and changes in the expression of cancer-related genes in the rat prostate. Adult male rats were acutely or repeatedly exposed to restraint stress, and compared to unstressed controls and groups that were allowed 14 days of recovery from the stress. Prostate tissue was collected and frozen for gene expression analyses by PCR array before the rats were transcardially perfused; and brain tissues harvested and immunohistochemically stained for Fos to determine neuronal activation. Acute stress elevated Fos expression in the paraventricular nucleus of the hypothalamus (PVH), an effect that habituated with repeated stress exposure. Data from the PCR arrays showed that repeated stress significantly increases the transcript levels of several genes associated with cellular proliferation, including proto-oncogenes. Data from another array platform showed that both acute and repeated stress can induce significant changes in metastatic gene expression. The functional diversity of genes with altered expression, which includes transcription factors, growth factor receptors, apoptotic genes, and extracellular matrix components, suggests that stress is able to induce aberrant changes in pathways that are deregulated in prostate cancer. Our findings further support the notion that stress can affect cancer outcomes, perhaps by interfering with neuroendocrine mechanisms involved in the control of reproduction.

  2. Obesity is associated with depot-specific alterations in adipocyte DNA methylation and gene expression.

    Science.gov (United States)

    Sonne, Si Brask; Yadav, Rachita; Yin, Guangliang; Dalgaard, Marlene Danner; Myrmel, Lene Secher; Gupta, Ramneek; Wang, Jun; Madsen, Lise; Kajimura, Shingo; Kristiansen, Karsten

    2017-04-03

    The present study aimed to identify genes exhibiting concomitant obesity-dependent changes in DNA methylation and gene expression in adipose tissues in the mouse using diet-induced obese (DIO) C57BL/6J and genetically obese ob/ob mice as models. Mature adipocytes were isolated from epididymal and inguinal adipose tissues of ob/ob and DIO C57BL/6J mice. DNA methylation was analyzed by MeDIP-sequencing and gene expression by microarray analysis. The majority of differentially methylated regions (DMRs) were hypomethylated in obese mice. Global methylation of long interspersed elements indicated that hypomethylation did not reflect methyl donor deficiency. In both DIO and ob/ob mice, we observed more obesity-associated methylation changes in epididymal than in inguinal adipocytes. Assignment of DMRs to promoter, exon, intron and intergenic regions demonstrated that DIO-induced changes in DNA methylation in C57BL/6J mice occurred primarily in exons, whereas inguinal adipocytes of ob/ob mice exhibited a higher enrichment of DMRs in promoter regions than in other regions of the genome, suggesting an influence of leptin on DNA methylation in inguinal adipocytes. We observed altered methylation and expression of 9 genes in epididymal adipocytes, including the known obesity-associated genes, Ehd2 and Kctd15, and a novel candidate gene, Irf8, possibly involved in immune type 1/type2 balance. The use of 2 obesity models enabled us to dissociate changes associated with high fat feeding from those associated with obesity per se. This information will be of value in future studies on the mechanisms governing the development of obesity and changes in adipocyte function associated with obesity.

  3. Finding immune gene expression differences induced by marine bacterial pathogens in the Deep-sea hydrothermal vent mussel Bathymodiolus azoricus

    Science.gov (United States)

    Martins, E.; Queiroz, A.; Serrão Santos, R.; Bettencourt, R.

    2013-11-01

    The deep-sea hydrothermal vent mussel Bathymodiolus azoricus lives in a natural environment characterised by extreme conditions of hydrostatic pressure, temperature, pH, high concentrations of heavy metals, methane and hydrogen sulphide. The deep-sea vent biological systems represent thus the opportunity to study and provide new insights into the basic physiological principles that govern the defense mechanisms in vent animals and to understand how they cope with microbial infections. Hence, the importance of understanding this animal's innate defense mechanisms, by examining its differential immune gene expressions toward different pathogenic agents. In the present study, B. azoricus mussels were infected with single suspensions of marine bacterial pathogens, consisting of Vibrio splendidus, Vibrio alginolyticus, or Vibrio anguillarum, and a pool of these Vibrio bacteria. Flavobacterium suspensions were also used as a non-pathogenic bacterium. Gene expression analyses were carried out using gill samples from infected animals by means of quantitative-Polymerase Chain Reaction aimed at targeting several immune genes. We also performed SDS-PAGE protein analyses from the same gill tissues. We concluded that there are different levels of immune gene expression between the 12 h to 24 h exposure times to various bacterial suspensions. Our results from qPCR demonstrated a general pattern of gene expression, decreasing from 12 h over 24 h post-infection. Among the bacteria tested, Flavobacterium is the bacterium inducing the highest gene expression level in 12 h post-infections animals. The 24 h infected animals revealed, however, greater gene expression levels, using V. splendidus as the infectious agent. The SDS-PAGE analysis also pointed at protein profile differences between 12 h and 24 h, particularly evident for proteins of 18-20 KDa molecular mass, where most dissimilarity was found. Multivariate analyses demonstrated that immune genes, as well as experimental

  4. Alterations in Gene Expression in Depression: Prospects for Personalize Patient Treatment.

    Science.gov (United States)

    Donev, Rossen; Alawam, Khaled

    2015-01-01

    The number of people around the world suffering from depression has dramatically increased in last few decades. It has been predicted that by 2020 depression will become the second most common cause of disability. Furthermore, depression is often misdiagnosed and confused with other psychiatric disorders showing similar symptoms, i.e., anxiety and bipolar disorder, due to the fact that diagnosing is often carried out by medical workers who are not psychiatrically trained. These facts prompt us to prepare this review which focuses on alterations in gene expression in depression. We believe that an in-depth knowledge of molecular bases of behavior in depression and other mood disorders would be of a great benefit for the correct diagnosing of these disorders, as well as for prescribing a treatment that best suits each individual depending on expression alterations in depression-related genes. Therefore, the main aim of this review is to promote further translational research on the biochemistry of mood disorders and take the results further for the design of new targeted therapeutics that can be used for personalized treatment with minimal adverse effects.

  5. Responses of plasmid-mediated quinolone resistance genes and bacterial taxa to (fluoro)quinolones-containing manure in arable soil.

    Science.gov (United States)

    Xiong, Wenguang; Sun, Yongxue; Ding, Xueyao; Zhang, Yiming; Zhong, Xiaoxia; Liang, Wenfei; Zeng, Zhenling

    2015-01-01

    The aim of the present study was to investigate the fate of plasmid-mediated quinolone resistance (PMQR) genes and the disturbance of soil bacterial communities posed by (fluoro)quinolones (FQNs)-containing manure in arable soil. Representative FQNs (enrofloxacin (ENR), ciprofloxacin (CIP) and norfloxacin (NOR)), PMQR genes (qepA, oqxA, oqxB, aac(6')-Ib-cr and qnrS) and bacterial communities in untreated soil, +manure and +manure+FQNs groups were analyzed using culture independent methods. The significantly higher abundance of oqxA, oqxB and aac(6')-Ib-cr, and significantly higher abundance of qnrS in +manure group than those in untreated soil disappeared at day 30 and day 60, respectively. All PMQR genes (oqxA, oqxB, aac(6')-Ib-cr and qnrS) dissipated 1.5-1.7 times faster in +manure group than those in +manure+FQNs group. The disturbance of soil bacterial communities posed by FQNs-containing manure was also found. The results indicated that significant effects of PMQR genes (oqxA, oqxB, aac(6')-Ib and qnrS) on arable soils introduced by manure disappeared 2 month after manure application. FQNs introduced by manure slowed down the dissipation of PMQR genes. The presence of high FQNs provided a selective advantage for species affiliated to the phylum including Acidobacteria, Verrucomicrobia and Planctomycetes while suppressing Proteobacteria and Actinobacteria.

  6. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens;

    2008-01-01

    of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...

  7. Altered cortical expression of GABA-related genes in schizophrenia: illness progression vs developmental disturbance.

    Science.gov (United States)

    Hoftman, Gil D; Volk, David W; Bazmi, H Holly; Li, Siyu; Sampson, Allan R; Lewis, David A

    2015-01-01

    Schizophrenia is a neurodevelopmental disorder with altered expression of GABA-related genes in the prefrontal cortex (PFC). However, whether these gene expression abnormalities reflect disturbances in postnatal developmental processes before clinical onset or arise as a consequence of clinical illness remains unclear. Expression levels for 7 GABA-related transcripts (vesicular GABA transporter [vGAT], GABA membrane transporter [GAT1], GABAA receptor subunit α1 [GABRA1] [novel in human and monkey cohorts], glutamic acid decarboxylase 67 [GAD67], parvalbumin, calretinin, and somatostatin [previously reported in human cohort, but not in monkey cohort]) were quantified in the PFC from 42 matched pairs of schizophrenia and comparison subjects and from 49 rhesus monkeys ranging in age from 1 week postnatal to adulthood. Levels of vGAT and GABRA1, but not of GAT1, messenger RNAs (mRNAs) were lower in the PFC of the schizophrenia subjects. As previously reported, levels of GAD67, parvalbumin, and somatostatin, but not of calretinin, mRNAs were also lower in these subjects. Neither illness duration nor age accounted for the levels of the transcripts with altered expression in schizophrenia. In monkey PFC, developmental changes in expression levels of many of these transcripts were in the opposite direction of the changes observed in schizophrenia. For example, mRNA levels for vGAT, GABRA1, GAD67, and parvalbumin all increased with age. Together with published reports, these findings support the interpretation that the altered expression of GABA-related transcripts in schizophrenia reflects a blunting of normal postnatal development changes, but they cannot exclude a decline during the early stages of clinical illness. © The Author 2013. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Genetic alterations in mesiodens as revealed by targeted next-generation sequencing and gene co-occurrence network analysis.

    Science.gov (United States)

    Kim, Y Y; Hwang, J; Kim, H-S; Kwon, H J; Kim, S; Lee, J H; Lee, J H

    2017-04-17

    Mesiodens is the most common type of supernumerary tooth which includes a population prevalence of 0.15%-1.9%. Alongside evidence that the condition is heritable, mutations in single genes have been reported in few human supernumerary tooth cases. Gene sequencing methods in tradition way are time-consuming and labor-intensive, whereas next-generation sequencing and bioinformatics are cost-effective for large samples and target sizes. We describe the application of a targeted next-generation sequencing (NGS) and bioinformatics approach to samples from 17 mesiodens patients. Subjects were diagnosed on the basis of panoramic radiograph. A total of 101 candidate genes which were captured custom genes were sequenced on the Illumina HiSeq 2500. Multistep bioinformatics processing was performed including variant identification, base calling, and in silico analysis of putative disease-causing variants. Targeted capture identified 88 non-synonymous, rare, exonic variants involving 42 of the 101 candidate genes. Moreover, we investigated gene co-occurrence relationships between the genomic alterations and identified 88 significant relationships among 18 most recurrent driver alterations. Our search for co-occurring genetic alterations revealed that such alterations interact cooperatively to drive mesiodens. We discovered a gene co-occurrence network in mesiodens patients with functionally enriched gene groups in the sonic hedgehog (SHH), bone morphogenetic proteins (BMP), and wingless integrated (WNT) signaling pathways. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

  9. Alteration of gene expression in human cells treated with the agricultural chemical diazinon: possible interaction in fetal development.

    Science.gov (United States)

    Mankame, T; Hokanson, R; Fudge, R; Chowdhary, R; Busbee, D

    2006-05-01

    Agricultural chemicals frequently alter human health or development, typically because they have endocrine agonist or antagonist activities and alter hormone-regulation of gene expression. The insecticide, diazinon, was evaluated for gene expression disrupting activity using MCF-7 cells, an estrogen-dependent human cell line, to examine the capacity of the insecticide to disrupt gene expression essential for morphological development, immune system development or function, and/or central nervous system development and function. MCF-7 cells were treated with 30, 50 or 67 ppm diazinon, and gene expression was measured in treated cells compared to expression in untreated or estrogen-treated cells. DNA microarray analysis of diazinon-treated cells showed significant up- or down-regulation of a large number of genes compared to untreated cells. Of the 600 human genes on the Phase 1 chip utilized for these studies, two specific genes--calreticulin and TGF-beta3--were selected for corroboration using quantitative real time PCR (qrtPCR). qrtPCR, completed to assess gene expression levels for calreticulin and TGFbeta3, confirmed results showing significant up-regulation of these two genes obtained from the microarray data. These studies were designed to provide baseline data on the gene expression-altering capacity of a specific chemical, diazinon, and allow a partial assessment of the potentially deleterious effects associated with exposure of human cells to this chemical. Currently, it is not known whether results from cells in vitro can be extrapolated to human health consequences of chemical exposure.

  10. NF-κB Inhibition after Cecal Ligation and Puncture Reduces Sepsis-Associated Lung Injury without Altering Bacterial Host Defense

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    Hui Li

    2013-01-01

    Full Text Available Introduction. Since the NF-κB pathway regulates both inflammation and host defense, it is uncertain whether interventions targeting NF-κB would be beneficial in sepsis. Based on the kinetics of the innate immune response, we postulated that selective NF-κB inhibition during a defined time period after the onset of sepsis would reduce acute lung injury without compromising bacterial host defense. Methods. Mice underwent cecal ligation and puncture (CLP. An NF-κB inhibitor, BMS-345541 (50 µg/g mice, was administered by peroral gavage beginning 2 hours after CLP and repeated at 6 hour intervals for 2 additional doses. Results. Mice treated with BMS-345541 after CLP showed reduced neutrophilic alveolitis and lower levels of KC in bronchoalveolar lavage fluid compared to mice treated with CLP+vehicle. In addition, mice treated with CLP+BMS had minimal histological evidence of lung injury and normal wet-dry ratios, indicating protection from acute lung injury. Treatment with the NF-κB inhibitor did not affect the ability of cultured macrophages to phagocytose bacteria and did not alter bacterial colony counts in blood, lung tissue, or peritoneal fluid at 24 hours after CLP. While BMS-345541 treatment did not alter mortality after CLP, our results showed a trend towards improved survival. Conclusion. Transiently blocking NF-κB activity after the onset of CLP-induced sepsis can effectively reduce acute lung injury in mice without compromising bacterial host defense or survival after CLP.

  11. GESTATIONAL DIABETES MELLITUS ALTERS APOPTOTIC AND INFLAMMATORY GENE EXPRESSION OF TROPHOBASTS FROM HUMAN TERM PLACENTA

    Science.gov (United States)

    MAGEE, Thomas R.; ROSS, Michael G.; WEDEKIND, Lauren; DESAI, Mina; KJOS, Siri; BELKACEMI, Louiza

    2014-01-01

    AIM Increased placental growth secondary to reduced apoptosis may contribute to the development of macrosomia in GDM pregnancies. We hypothesize that reduced apoptosis in GDM placentas is caused by dysregulation of apoptosis related genes from death receptors or mitochondrial pathway or both to enhance placental growth in GDM pregnancies. METHODS Newborn and placental weights from women with no pregnancy complications (controls; N=5), or with GDM (N=5) were recorded. Placental villi from both groups were either fixed for TUNEL assay, or snap frozen for gene expression analysis by apoptosis PCR microarrays and qPCR. RESULTS Maternal, placental and newborn weights were significantly higher in the GDM group vs. Controls. Apoptotic index of placentas from the GDM group was markedly lower than the Controls. At a significant threshold of 1.5, seven genes (BCL10, BIRC6, BIRC7, CASP5, CASP8P2, CFLAR, and FAS) were down regulated, and 13 genes (BCL2, BCL2L1, BCL2L11, CASP4, DAPK1, IκBκE, MCL1, NFκBIZ, NOD1, PEA15, TNF, TNFRSF25, and XIAP) were unregulated in the GDM placentas. qPCR confirmed the consistency of the PCR microarray. Using Western blotting we found significantly decreased placental pro-apoptotic FAS receptor and FAS ligand (FASL), and increased mitochondrial anti-apoptotic BCL2 post GDM insult. Notably, caspase-3, which plays a central role in the execution-phase of apoptosis, and its substrate poly (ADP-ribose) polymerase (PARP) were significantly down regulated in GDM placentas, as compared to non-diabetic Control placentas. CONCLUSION . Women with gestational diabetes (GDM) are at increased risk for having macrosomic newborns, and larger placentas with reduced apoptosis. Decreased apoptosis subsequent to alterations in apoptotic and inflammatory genes may promote elevated weight in the GDM placentas. PMID:24768206

  12. Dietary Phenethyl Isothiocyanate Alters Gene Expression in Human Breast Cancer Cells

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    Young Jin Moon

    2011-01-01

    Full Text Available Phenethyl isothiocyanate (PEITC, a component in cruciferous vegetables, can block chemical carcinogenesis in animal models. Our objective was to determine the effect of treatment with PEITC on gene expression changes in MCF-7 human breast cancer cells in order to evaluate potential mechanisms involved in its chemopreventive effects. MCF-7 cells were treated for 48 hours with either PEITC (3 μM or the vehicle. Total RNA was extracted from cell membrane preparations, and labeled cDNA's representing the mRNA pool were reverse-transcribed directly from total RNA isolated for use in the microarray hybridizations. Two specific human GE Array Kits (Superarray Inc. that both contain 23 marker genes, related to signal transduction pathways or cancer/tumor suppression, plus 2 housekeeping genes (β-actin and GAPDH, were utilized. Arrays from treated and control cells (n=4 per group were evaluated using a Student's t-test. Gene expression was significantly induced for tumor protein p53 (p53, cyclin-dependent kinase inhibitor 1C (p57 Kip2, breast cancer Type 2 early onset (BRCA2, cAMP responsive element binding protein 2 (ATF-2, interleukin 2 (IL-2, heat shock 27 KD protein (hsp27, and CYP19 (aromatase. Induction of p57 Kip2, p53, BRCA2, IL-2, and ATF-2 would be expected to decrease cellular proliferation and increase tumor suppression and/or apoptosis. PEITC treatment produced significant alterations in some genes involved in tumor suppression and cellular proliferation/apoptosis that may be important in explaining the chemopreventive effects of PEITC.

  13. Effect of different biochars on antibiotic resistance genes and bacterial community during chicken manure composting.

    Science.gov (United States)

    Cui, Erping; Wu, Ying; Zuo, Yiru; Chen, Hong

    2016-03-01

    Rice straw biochar (RSB) and mushroom biochar (MB) were added to lab-scale chicken manure composting to evaluate their effects on the behaviors of antibiotic resistance genes (ARGs) and on total and bio-available heavy metals (Cu, Zn and As). The associated bacterial community was characterized by 16SrRNA high-throughput sequencing. The abundance of pathogenic bacteria was also calculated. At the end of the control composting experiment, the average removal rate of ARGs was 0.86 log units and the removal rate of pathogenic bacteria was 57.1%. MB addition resulted in a higher removal rate than that in the control composting experiment. However, RSB addition yielded opposite results, which may be due to the higher abundance of Erysipelotrichaceae, Lactobacillaceae, Family_XI_Incertae_Sedis (belonging to Firmicutes carrying and disseminating ARGs) and pathogenic bacteria carrying ARGs. Furthermore, the correlations between bio-available heavy metals and ARGs were more obvious than those between total heavy metals and ARGs.

  14. Sequence Alterations of I(Ks Potassium Channel Genes in Kazakhstani Patients with Atrial Fibrillation

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    Ainur Akilzhanova

    2014-12-01

    Full Text Available Introduction. Atrial fibrillation (AF is the most common sustained arrhythmia, and it results in significant morbidity and mortality. However, the pathogenesis of AF remains unclear to date. Recently, more pieces of evidence indicated that AF is a multifactorial disease resulting from the interaction between environmental factors and genetics. Recent studies suggest that genetic mutation of the slow delayed rectifier potassium channel (I(Ks may underlie AF.Objective. To investigate sequence alterations of I(Ks potassium channel genes KCNQ1, KCNE1 and KCNE2 in Kazakhstani patients with atrial fibrillation.Methods. Genomic DNA of 69 cases with atrial fibrillation and 27 relatives were analyzed for mutations in all protein-coding exons and their flanking splice site regions of the genes KCNQ1 (NM_000218.2 and NM_181798.1, KCNE1 (NM_000219.2, and KCNE2 (NM_172201.1 using bidirectional sequencing on the ABI 3730xL DNA Analyzer (Applied Biosystems, Foster City, CA, USA.Results. In total, a disease-causing mutation was identified in 39 of the 69 (56.5% index cases. Of these, altered sequence variants in the KCNQ1 gene accounted for 14.5% of the mutations, whereas a KCNE1 mutation accounted for 43.5% of the mutations and KCNE2 mutation accounted for 1.4% of the mutations. The majority of the distinct mutations were found in a single case (80%, whereas 20% of the mutations were observed more than once. We found two sequence variants in KCNQ1 exon 13 (S546S G1638A and exon 16 (Y662Y, C1986T in ten patients (14.5%. In KCNE1 gene in exon 3 mutation, S59G A280G was observed in 30 of 69 patients (43.5% and KCNE2 exon 2 T10K C29A in 1 patient (1.4%. Genetic cascade screening of 27 relatives to the 69 index cases with an identified mutation revealed 26.9% mutation carriers  who were at risk of cardiac events such as syncope or sudden unexpected death.Conclusion. In this cohort of Kazakhstani index cases with AF, a disease-causing mutation was identified in

  15. Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis.

    Science.gov (United States)

    Thiesen, H-J; Steinbeck, F; Maruschke, M; Koczan, D; Ziems, B; Hakenberg, O W

    2017-01-01

    Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value.

  16. Electroporation of proviral RCAS DNA alters gene expression in the embryonic chick hindbrain.

    Science.gov (United States)

    Hermann, Petra M; Logan, C Cairine

    2003-11-01

    Gene transfer by means of electroporation is an effective method for delivering DNA into cells. Expression vectors encoding green fluorescent protein (GFP) are routinely used as a control for this technique and are also regularly used to indirectly or directly monitor the expression of introduced transgenes. However, recent studies suggest that GFP may have nonspecific and/or cytotoxic side effects. In this study, we investigated the effects of enhanced GFP (EGFP) expression delivered by means of electroporation of proviral RCASBP(B)-EGFP DNA on gene expression in the hindbrain of chick embryos. We examined, via whole-mount in situ hybridization, the expression of a number of transcription factors. We found that Tlx-1 was ectopically expressed following electroporation of proviral RCASBP(B)-EGFP DNA. In contrast, the number of cells expressing Tlx-3, Phox2a, and Phox2b were reduced. Intriguingly, these effects could be mimicked by electroporation of wild-type proviral RCASBP(B) DNA (i.e., lacking the GFP insert). However, neither delivery of the EGFP transgene by means of viral infection nor electroporation alone yielded aberrant expression patterns. Together our data indicate that alterations of gene expression patterns are not directly due to the expression of EGFP but instead reflect a confounding effect of electroporating proviral DNA.

  17. Androgen receptor regulation of the seladin-1/DHCR24 gene: altered expression in prostate cancer.

    Science.gov (United States)

    Bonaccorsi, Lorella; Luciani, Paola; Nesi, Gabriella; Mannucci, Edoardo; Deledda, Cristiana; Dichiara, Francesca; Paglierani, Milena; Rosati, Fabiana; Masieri, Lorenzo; Serni, Sergio; Carini, Marco; Proietti-Pannunzi, Laura; Monti, Salvatore; Forti, Gianni; Danza, Giovanna; Serio, Mario; Peri, Alessandro

    2008-10-01

    Prostate cancer (CaP) represents a major leading cause of morbidity and mortality in the Western world. Elevated cholesterol levels, resulting from altered cholesterol metabolism, have been found in CaP cells. Seladin-1 (SELective Alzheimer Disease INdicator-1)/DHCR24 is a recently described gene involved in cholesterol biosynthesis. Here, we demonstrated the androgen regulation of seladin-1/DHCR24 expression, due to the presence of androgen responsive element sequences in its promoter region. In metastatic androgen receptor-negative CaP cells seladin-1/DHCR24 expression and cholesterol amount were reduced compared to androgen receptor-positive cells. In tumor samples from 61 patients who underwent radical prostatectomy the expression of seladin-1/DHCR24 was significantly higher with respect to normal tissues. In addition, in cancer tissues mRNA levels were positively related to T stage. In tumor specimens from 23 patients who received androgen ablation treatment for 3 months before surgery seladin-1/DHCR24 expression was significantly lower with respect to patients treated by surgery only. In conclusion, our study demonstrated for the first time the androgen regulation of the seladin-1/DHCR24 gene and the presence of a higher level of expression in CaP tissues, compared to the normal prostate. These findings, together with the results previously obtained in metastatic disease, suggest an involvement of this gene in CaP.

  18. Altered expression of mitochondrial and extracellular matrix genes in the heart of human fetuses with chromosome 21 trisomy

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    Olla Carlo

    2007-08-01

    Full Text Available Abstract Background The Down syndrome phenotype has been attributed to overexpression of chromosome 21 (Hsa21 genes. However, the expression profile of Hsa21 genes in trisomic human subjects as well as their effects on genes located on different chromosomes are largely unknown. Using oligonucleotide microarrays we compared the gene expression profiles of hearts of human fetuses with and without Hsa21 trisomy. Results Approximately half of the 15,000 genes examined (87 of the 168 genes on Hsa21 were expressed in the heart at 18–22 weeks of gestation. Hsa21 gene expression was globally upregulated 1.5 fold in trisomic samples. However, not all genes were equally dysregulated and 25 genes were not upregulated at all. Genes located on other chromosomes were also significantly dysregulated. Functional class scoring and gene set enrichment analyses of 473 genes, differentially expressed between trisomic and non-trisomic hearts, revealed downregulation of genes encoding mitochondrial enzymes and upregulation of genes encoding extracellular matrix proteins. There were no significant differences between trisomic fetuses with and without heart defects. Conclusion We conclude that dosage-dependent upregulation of Hsa21 genes causes dysregulation of the genes responsible for mitochondrial function and for the extracellular matrix organization in the fetal heart of trisomic subjects. These alterations might be harbingers of the heart defects associated with Hsa21 trisomy, which could be based on elusive mechanisms involving genetic variability, environmental factors and/or stochastic events.

  19. Using bacterial extract along with differential gene expression in Acropora millepora larvae to decouple the processes of attachment and metamorphosis.

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    Nachshon Siboni

    Full Text Available Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP, isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA Neogoniolithon fosliei, induced coral larval metamorphosis (100% with little or no attachment (0-2%. To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR and employed 47 genes of interest (GOI, selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p<0.05 in gene expression profiles of 24 GOI, 12 hours post exposure. Fourteen of those GOI also presented differences in expression (p<0.05 following exposure to the threshold concentration of bacterial TBP-containing extract. The specificity of the bacterial TBP-containing extract to induce the metamorphic stage in A. millepora larvae without attachment, using a robust, low cost, accurate, ecologically relevant and highly reproducible RT-qPCR assay, allowed partially decoupling of the transcriptomic processes of attachment and metamorphosis. The bacterial TBP-containing extract provided a unique opportunity to monitor the regulation of genes exclusively involved in the process of metamorphosis, contrasting previous gene expression studies that

  20. Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression

    Science.gov (United States)

    Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan

    2012-07-01

    unloading in spaceflight, we conducted genome wide microarray analysis of total RNA isolated from the mouse pelvis. Specifically, 16 week old mice were subjected to 15 days spaceflight onboard NASA's STS-131 space shuttle mission. The pelvis of the mice was dissected, the bone marrow was flushed and the bones were briefly stored in RNAlater. The pelvii were then homogenized, and RNA was isolated using TRIzol. RNA concentration and quality was measured using a Nanodrop spectrometer, and 0.8% agarose gel electrophoresis. Samples of cDNA were analyzed using an Affymetrix GeneChip\\S Gene 1.0 ST (Sense Target) Array System for Mouse and GenePattern Software. We normalized the ST gene arrays using Robust Multichip Average (RMA) normalization, which summarizes perfectly matched spots on the array through the median polish algorithm, rather than normalizing according to mismatched spots. We also used Limma for statistical analysis, using the BioConductor Limma Library by Gordon Smyth, and differential expression analysis to identify genes with significant changes in expression between the two experimental conditions. Finally we used GSEApreRanked for Gene Set Enrichment Analysis (GSEA), with Kolmogorov-Smirnov style statistics to identify groups of genes that are regulated together using the t-statistics derived from Limma. Preliminary results show that 6,603 genes expressed in pelvic bone had statistically significant alterations in spaceflight compared to ground controls. These prominently included cell cycle arrest molecules p21, and p18, cell survival molecule Crbp1, and cell cycle molecules cyclin D1, and Cdk1. Additionally, GSEA results indicated alterations in molecular targets of cyclin D1 and Cdk4, senescence pathways resulting from abnormal laminin maturation, cell-cell contacts via E-cadherin, and several pathways relating to protein translation and metabolism. In total 111 gene sets out of 2,488, about 4%, showed statistically significant set alterations. These

  1. Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

    Science.gov (United States)

    Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

    2011-01-01

    To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

  2. Multidirectional chemical signalling between Mammalian hosts, resident microbiota, and invasive pathogens: neuroendocrine hormone-induced changes in bacterial gene expression.

    Science.gov (United States)

    Karavolos, Michail H; Khan, C M Anjam

    2014-01-01

    Host-pathogen communication appears to be crucial in establishing the outcome of bacterial infections. There is increasing evidence to suggest that this communication can take place by bacterial pathogens sensing and subsequently responding to host neuroendocrine (NE) stress hormones. Bacterial pathogens have developed mechanisms allowing them to eavesdrop on these communication pathways within their hosts. These pathogens can use intercepted communication signals to adjust their fitness to persist and cause disease in their hosts. Recently, there have been numerous studies highlighting the ability of NE hormones to act as an environmental cue for pathogens, helping to steer their responses during host infection. Host NE hormone sensing can take place indirectly or directly via bacterial adrenergic receptors (BARs). The resulting changes in bacterial gene expression can be of strategic benefit to the pathogen. Furthermore, it is intriguing that not only can bacteria sense NE stress hormones but they are also able to produce key signalling molecules known as autoinducers. The rapid advances in our knowledge of the human microbiome, and its impact on health and disease highlights the potential importance of communication between the microbiota, pathogens and the host. It is indeed likely that the microbiota input significantly in the neuroendocrinological homeostasis of the host by catabolic, anabolic, and signalling processes. The arrival of unwanted guests, such as bacterial pathogens, clearly has a major impact on these delicately balanced interactions. Unravelling the pathways involved in interkingdom communication between invading bacterial pathogens, the resident microbiota, and hosts, may provide novel targets in our continuous search for new antimicrobials to control disease.

  3. Ubiquity and diversity of heterotrophic bacterial nasA genes in diverse marine environments.

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    Xuexia Jiang

    Full Text Available Nitrate uptake by heterotrophic bacteria plays an important role in marine N cycling. However, few studies have investigated the diversity of environmental nitrate assimilating bacteria (NAB. In this study, the diversity and biogeographical distribution of NAB in several global oceans and particularly in the western Pacific marginal seas were investigated using both cultivation and culture-independent molecular approaches. Phylogenetic analyses based on 16S rRNA and nasA (encoding the large subunit of the assimilatory nitrate reductase gene sequences indicated that the cultivable NAB in South China Sea belonged to the α-Proteobacteria, γ-Proteobacteria and CFB (Cytophaga-Flavobacteria-Bacteroides bacterial groups. In all the environmental samples of the present study, α-Proteobacteria, γ-Proteobacteria and Bacteroidetes were found to be the dominant nasA-harboring bacteria. Almost all of the α-Proteobacteria OTUs were classified into three Roseobacter-like groups (I to III. Clone library analysis revealed previously underestimated nasA diversity; e.g. the nasA gene sequences affiliated with β-Proteobacteria, ε-Proteobacteria and Lentisphaerae were observed in the field investigation for the first time, to the best of our knowledge. The geographical and vertical distributions of seawater nasA-harboring bacteria indicated that NAB were highly diverse and ubiquitously distributed in the studied marginal seas and world oceans. Niche adaptation and separation and/or limited dispersal might mediate the NAB composition and community structure in different water bodies. In the shallow-water Kueishantao hydrothermal vent environment, chemolithoautotrophic sulfur-oxidizing bacteria were the primary NAB, indicating a unique nitrate-assimilating community in this extreme environment. In the coastal water of the East China Sea, the relative abundance of Alteromonas and Roseobacter-like nasA gene sequences responded closely to algal blooms, indicating

  4. Ubiquity and diversity of heterotrophic bacterial nasA genes in diverse marine environments.

    Science.gov (United States)

    Jiang, Xuexia; Dang, Hongyue; Jiao, Nianzhi

    2015-01-01

    Nitrate uptake by heterotrophic bacteria plays an important role in marine N cycling. However, few studies have investigated the diversity of environmental nitrate assimilating bacteria (NAB). In this study, the diversity and biogeographical distribution of NAB in several global oceans and particularly in the western Pacific marginal seas were investigated using both cultivation and culture-independent molecular approaches. Phylogenetic analyses based on 16S rRNA and nasA (encoding the large subunit of the assimilatory nitrate reductase) gene sequences indicated that the cultivable NAB in South China Sea belonged to the α-Proteobacteria, γ-Proteobacteria and CFB (Cytophaga-Flavobacteria-Bacteroides) bacterial groups. In all the environmental samples of the present study, α-Proteobacteria, γ-Proteobacteria and Bacteroidetes were found to be the dominant nasA-harboring bacteria. Almost all of the α-Proteobacteria OTUs were classified into three Roseobacter-like groups (I to III). Clone library analysis revealed previously underestimated nasA diversity; e.g. the nasA gene sequences affiliated with β-Proteobacteria, ε-Proteobacteria and Lentisphaerae were observed in the field investigation for the first time, to the best of our knowledge. The geographical and vertical distributions of seawater nasA-harboring bacteria indicated that NAB were highly diverse and ubiquitously distributed in the studied marginal seas and world oceans. Niche adaptation and separation and/or limited dispersal might mediate the NAB composition and community structure in different water bodies. In the shallow-water Kueishantao hydrothermal vent environment, chemolithoautotrophic sulfur-oxidizing bacteria were the primary NAB, indicating a unique nitrate-assimilating community in this extreme environment. In the coastal water of the East China Sea, the relative abundance of Alteromonas and Roseobacter-like nasA gene sequences responded closely to algal blooms, indicating that NAB may be

  5. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    Energy Technology Data Exchange (ETDEWEB)

    Walters, William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada, Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, J. Gregory; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob; Bik, Holly

    2015-12-22

    ABSTRACT

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection ofThaumarchaeotaand clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

    ImportanceWe continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to

  6. Metagenomic analysis of bacterial community composition and antibiotic resistance genes in a wastewater treatment plant and its receiving surface water.

    Science.gov (United States)

    Tang, Junying; Bu, Yuanqing; Zhang, Xu-Xiang; Huang, Kailong; He, Xiwei; Ye, Lin; Shan, Zhengjun; Ren, Hongqiang

    2016-10-01

    The presence of pathogenic bacteria and the dissemination of antibiotic resistance genes (ARGs) may pose big risks to the rivers that receive the effluent from municipal wastewater treatment plants (WWTPs). In this study, we investigated the changes of bacterial community and ARGs along treatment processes of one WWTP, and examined the effects of the effluent discharge on the bacterial community and ARGs in the receiving river. Pyrosequencing was applied to reveal bacterial community composition including potential bacterial pathogen, and Illumina high-throughput sequencing was used for profiling ARGs. The results showed that the WWTP had good removal efficiency on potential pathogenic bacteria (especially Arcobacter butzleri) and ARGs. Moreover, the bacterial communities of downstream and upstream of the river showed no significant difference. However, the increase in the abundance of potential pathogens and ARGs at effluent outfall was observed, indicating that WWTP effluent might contribute to the dissemination of potential pathogenic bacteria and ARGs in the receiving river. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Salinity altered root distribution and increased diversity of bacterial communities in the rhizosphere soil of Jerusalem artichoke

    Science.gov (United States)

    Yang, Hui; Hu, Jinxiang; Long, Xiaohua; Liu, Zhaopu; Rengel, Zed

    2016-02-01

    The interaction between roots and bacterial communities in halophytic species is poorly understood. Here, we used Jerusalem artichoke cultivar Nanyu 1 (NY-1) to characterise root distribution patterns and determine diversity and abundance of bacteria in the rhizosphere soil under variable salinity. Root growth was not inhibited within the salinity range 1.2 to 1.9 g salt/kg, but roots were mainly confined to 0–20 cm soil layer vertically and 0–30 cm horizontally from the plant centre. Root concentrations of K+, Na+, Mg2+ and particularly Ca2+ were relatively high under salinity stress. High salinity stress decreased soil invertase and catalase activity. Using a next-generation, Illumina-based sequencing approach, we determined higher diversity of bacteria in the rhizosphere soil at high than low salinity. More than 15,500 valid reads were obtained, and Proteobacteria, Acidobacteria, Bacteroidetes and Actinobacteria predominated in all samples, accounting for >80% of the reads. On a genus level, 636 genera were common to the low and high salinity treatments at 0–5 cm and 5–10 cm depth. The abundance of Steroidobacter and Sphingomonas was significantly decreased by increasing salinity. Higher Shannon and Chao 1 indices with increasing severity of salt stress indicated that high salt stress increased diversity in the bacterial communities.

  8. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions

    Institute of Scientific and Technical Information of China (English)

    Carol L Fischer; Katherine S Walters; David R Drake; Deborah V Dawson; Derek R Blanchette; Kim A Brogden; Philip W Wertz

    2013-01-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria;however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

  9. Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

    Science.gov (United States)

    Fischer, Carol L; Walters, Katherine S; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2013-09-01

    Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

  10. Genetic Alterations within the DENND1A Gene in Patients with Polycystic Ovary Syndrome (PCOS)

    DEFF Research Database (Denmark)

    Eriksen, Mette B; Nielsen, Michael F B; Brusgaard, Klaus

    2013-01-01

    sequencing. SNP genotyping was tested by allelic discrimination in real-time PCR in the additional patients and controls. Sequencing of the DENND1A gene identified eight SNPs; seven were not known to be associated with any diseases. One missense SNP was detected (rs189947178, A/C), potentially altering...... with PCOS. Additional patients (n = 251) and healthy female controls (n = 248) were included for SNP genotyping. Patients underwent clinical examination including Ferriman-Gallwey score (FG-score), biochemical analyses and transvaginal ultrasound. Mutation analysis was carried out by bidirectional...... the structural conformation of the DENND1A protein. SNP genotyping of rs189947178 showed significantly more carriers among patients with PCOS and moderate hirsutism compared to controls. However, due to small sample size and lack of multiple regression analysis supporting an association between rs189947178...

  11. Methamphetamine and HIV-Tat Alter Murine Cardiac DNA Methylation and Gene Expression

    Science.gov (United States)

    Koczor, Christopher A.; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

    2015-01-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change>1.5, p<0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. PMID:26307267

  12. Chronic LSD alters gene expression profiles in the mPFC relevant to schizophrenia.

    Science.gov (United States)

    Martin, David A; Marona-Lewicka, Danuta; Nichols, David E; Nichols, Charles D

    2014-08-01

    Chronic administration of lysergic acid diethylamide (LSD) every other day to rats results in a variety of abnormal behaviors. These build over the 90 day course of treatment and can persist at full strength for at least several months after cessation of treatment. The behaviors are consistent with those observed in animal models of schizophrenia and include hyperactivity, reduced sucrose-preference, and decreased social interaction. In order to elucidate molecular changes that underlie these aberrant behaviors, we chronically treated rats with LSD and performed RNA-sequencing on the medial prefrontal cortex (mPFC), an area highly associated with both the actions of LSD and the pathophysiology of schizophrenia and other psychiatric illnesses. We observed widespread changes in the neurogenetic state of treated animals four weeks after cessation of LSD treatment. QPCR was used to validate a subset of gene expression changes observed with RNA-Seq, and confirmed a significant correlation between the two methods. Functional clustering analysis indicates differentially expressed genes are enriched in pathways involving neurotransmission (Drd2, Gabrb1), synaptic plasticity (Nr2a, Krox20), energy metabolism (Atp5d, Ndufa1) and neuropeptide signaling (Npy, Bdnf), among others. Many processes identified as altered by chronic LSD are also implicated in the pathogenesis of schizophrenia, and genes affected by LSD are enriched with putative schizophrenia genes. Our results provide a relatively comprehensive analysis of mPFC transcriptional regulation in response to chronic LSD, and indicate that the long-term effects of LSD may bear relevance to psychiatric illnesses, including schizophrenia. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Laminin gene LAMB4 is somatically mutated and expressionally altered in gastric and colorectal cancers.

    Science.gov (United States)

    Choi, Mi Ryoung; An, Chang Hyeok; Yoo, Nam Jin; Lee, Sug Hyung

    2015-01-01

    Laminins are important in tumor invasion and metastasis as well as in maintenance of normal epithelial cell structures. However, mutation status of laminin chain-encoding genes remains unknown in cancers. Aim of this study was to explore whether laminin chain genes are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that laminin chain genes LAMA1, LAMA3, LAMB1 and LAMB4 had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with microsatellite instability (MSI). We analyzed the genes in 88 GC and 139 CRC [high MSI (MSI-H) or stable MSI/low MSI (MSS/MSI-L)] by single strand conformation polymorphism analysis and DNA sequencing. In the present study, we found LAMB4 (11.8% of GC and 7.6% of CRC with MSI-H), LAMA3 (2.9% of GC and 2.5 of CRC with MSI-H), LAMA1 (5.9% of GC with MSI-H) and LAMB1 frameshift mutations (1.3% of CRC with MSI-H). These mutations were not found in MSS/MSI-L (0/114). We also analyzed LAMB4 expression in GC and CRC by immunohistochemistry. Loss of LAMB4 expression was identified in 17-32% of the GC and CRC. Of note, the loss expression was more common in the cancers with LAMB4 mutation or those with MSI-H. Our data show that frameshift mutations of LAMA1, LAMA3, LAMB1 and LAMB4, and loss of LAMB4 may be features of GC and CRC with MSI-H.

  14. The alteration of zinc transporter gene expression is associated with inflammatory markers in obese women.

    Science.gov (United States)

    Noh, Hwayoung; Paik, Hee Young; Kim, Jihye; Chung, Jayong

    2014-04-01

    Obesity, a chronic inflammatory state, is associated with altered zinc metabolism. ZnT and Zip transporters are involved in the regulation of zinc metabolism. This study examined the relationships among obesity, zinc transporter gene expression, and inflammatory markers in young Korean women. The messenger RNA (mRNA) levels of leukocyte zinc transporters between obese (BMI = 28.3 ± 0.5 kg/m(2), n = 35) and nonobese (BMI = 20.7 ± 0.2 kg/m(2), n = 20) women aged 18-28 years were examined using quantitative real-time polymerase chain reaction. Inflammatory markers, such as C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6, were measured in serum by enzyme immunoassay. ZnT1 and Zip1 were the most abundantly expressed zinc transporters in leukocytes. The mRNA levels of many zinc transporters (ZnT4, ZnT5, ZnT9, Zip1, Zip4, and Zip6) were significantly lower in obese women, and expression of these genes was inversely correlated with BMI and body fat percentage. In addition, inflammatory markers (CRP and TNF-α) were significantly higher in obese women. The mRNA levels of ZnT4, Zip1, and Zip6 were inversely correlated with CRP (P zinc transporters such as ZnT4, ZnT5, Zip1, and Zip6 (P zinc transporters may be altered in obese individuals. Changes in zinc transporters may also be related to the inflammatory state associated with obesity.

  15. A Casparian strip domain-like gene, CASPL, negatively alters growth and cold tolerance.

    Science.gov (United States)

    Yang, Jinghua; Ding, Changqing; Xu, Baochen; Chen, Cuiting; Narsai, Reena; Whelan, Jim; Hu, Zhongyuan; Zhang, Mingfang

    2015-09-24

    A cold-induced transcript encoding a Casparian strip membrane domain (CASP)-like protein (ClCASPL) was identified in watermelon (Citrullus lanatus). Fluorescence microscopy analysis showed that ClCASPL-GFP is localized in the plasma membrane. The orthologous gene in Arabidopsis thaliana (AtCASPL4C1) was also found to play an important role in cold tolerance. Expression analysis using a β-glucuronidase (GUS) reporter reveals that AtCASPL4C1 is widely expressed in a variety of organs and is cold inducible. Analysis of AtCASPL4C1 T-DNA knock-out plants showed altered growth dynamics, faster growth, increased biomass (dry weight) and earlier flowering compared to wild type (Col-0) and ClCASPL overexpressing plants. AtCASPL4C1 knock-out plants showed elevated tolerance to cold stress, while overexpressing CICASPL resulted in increased sensitivity to cold stress in Arabidopsis. Interestingly, AtCASPL4C1 knock-out plants did not display significant alterations in the Casparian strip formation in roots. Thus, the combination of these results suggests a role for CICASPL and AtCASPL4C1 beyond Casparian strip formation in roots, possibly indicating a more fundamental role in vascular tissue.

  16. Salmonella plasmid virulence gene spvB enhances bacterial virulence by inhibiting autophagy in a zebrafish infection model.

    Science.gov (United States)

    Li, Yuan-Yuan; Wang, Ting; Gao, Song; Xu, Guang-Mei; Niu, Hua; Huang, Rui; Wu, Shu-Yan

    2016-02-01

    Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative intracellular pathogen that can cause gastroenteritis and systemic infection in a wide range of hosts. Salmonella plasmid virulence gene spvB is closely related to bacterial virulence in different cells and animal models, and the encoded protein acts as an intracellular toxin required for ADP-ribosyl transferase activity. However, until now there is no report about the pathogenecity of spvB gene on zebrafish. Due to the outstanding advantages of zebrafish in analyzing bacteria-host interactions, a S. typhimurium infected zebrafish model was set up here to study the effect of spvB on autophagy and intestinal pathogenesis in vivo. We found that spvB gene could decrease the LD50 of S. typhimurium, and the strain carrying spvB promoted bacterial proliferation and aggravated the intestinal damage manifested by the narrowed intestines, fallen microvilli, blurred epithelium cell structure and infiltration of inflammatory cells. Results demonstrated the enhanced virulence induced by spvB in zebrafish. In spvB-mutant strain infected zebrafish, the levels of Lc3 turnover and Beclin1 expression increased, and the double-membraned autophagosome structures were observed, suggesting that spvB can inhibit autophagy activity. In summary, our results indicate that S. typhimurium strain containing spvB displays more virulence, triggering an increase in bacterial survival and intestine injuries by suppressing autophagy for the first time. This model provides novel insights into the role of Salmonella plasmid virulence gene in bacterial pathogenesis, and can help to further elucidate the relationship between bacteria and host immune response.

  17. Colon cancer and gene alterations: their immunological implications and suggestions for prognostic indices and improvements in biotherapy.

    Science.gov (United States)

    Contasta, Ida; Pellegrini, Patrizia; Berghella, Anna Maria; Del Beato, Tiziana; Adorno, Domenico

    2006-10-01

    Studies have shown that changes occur in c-Ki-ras, p53, and Bcl2 gene structure and function during the various stages of human colon carcinogenesis. Alterations of these genes are responsible for the establishment of a state of continuous stimulus for cell division and apoptotic inhibition at physiological and pharmacological levels. This paper focuses on the results of our research aimed at investigating how these gene alterations influence tumoral mechanisms on an immunological level and how immunological parameters can be used as prognostic markers for the passage of normal tissue to adenoma and adenoma to carcinoma. Overall, our data suggest that an alteration in the c-Ki-ras gene results in a switch to a suppressive type of immune response, determining an impairment of immune cell activation at both antigen- presenting-cell and T-cell levels. c-Ki-ras gene mutations, p53 deletions, and Bc12 expression, on the other hand, can be used as prognostic markers for the passage of normal tissue to adenoma and adenoma to carcinoma. The p53 oncogene does not appear to impair patients' immunological response further. In conclusion, an evaluation of c-Ki-ras, rather than p53 gene alterations, would seem to be more relevant in colon cancer prevention programs and biotherapy improvement.

  18. Interactions among genes influencing bacterial recognition increase IBD risk in a population-based New Zealand cohort.

    Science.gov (United States)

    Petermann, Ivonne; Huebner, Claudia; Browning, Brian L; Gearry, Richard B; Barclay, Murray L; Kennedy, Martin; Roberts, Rebecca; Shelling, Andrew N; Philpott, Martin; Han, Dug Yeo; Ferguson, Lynnette R

    2009-06-01

    Bacterial sensing is crucial for appropriate response by the innate and adaptive immune system against invading microorganisms. Single nucleotide polymorphisms (SNPs) in genes involved in bacterial recognition, CARD15 and TLR4, increased the risk of inflammatory bowel disease (IBD) in a New Zealand Caucasian case-control cohort. We now consider the effects of SNPs in CD14, TLR9, and BPI, analyzed individually, in association with one another, and with SNPs in CARD15 or TLR4 in this same population group. SNPs in CD14 (c.-159 C>T), TLR9 (c.-1237T>C) and BPI (c.645A>G) showed no significant allele or genotype frequency differences between IBD cases and controls. Genotype-phenotype mapping reveals an association with BPI and ileocolonic Crohn's disease (CD) as well as an association with CD14 and early-onset ulcerative colitis (UC). Genotype interaction analyses using three different statistical approaches provided significant evidence of interaction for the following combinations: CARD15/TLR4 (CD and UC), CARD15/CD14 (CD and UC), CD14/TLR4 (UC only), and CD14/BPI (UC only). A trend for an association between BPI and TLR4 was observed in UC patients, but failed to reach statistical significance. Our findings support the idea of gene-gene interactions for genes involved in closely related pathways (i.e. bacterial detection). There is evidence that carrying two SNPs in genes may lead to statistical significance for genes and SNPs that do not otherwise confirm as risk alleles for disease aetiology when analysed alone.

  19. Bacterial genetics in meningitis: Associating meningococcal and pneumococcal genes with clinical outcome

    NARCIS (Netherlands)

    Piet, J.R.

    2016-01-01

    The objective of this thesis is to provide more insight in the association of bacterial genetics with clinical characteristics of patients with bacterial meningitis. In a genetic association study using a cohort of 258 meningococcal meningitis patients, we show that specific meningococcal clonal

  20. Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase

    Directory of Open Access Journals (Sweden)

    Dugas Sandra L

    2003-07-01

    Full Text Available Abstract Background The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects. Results Phylogenetic analysis of aligned arsC sequences shows broad similarities to the established 16S rRNA phylogeny, with separation of bacterial, archaeal, and subsequently eukaryotic arsC genes. However, inconsistencies between arsC and 16S rRNA are apparent for some taxa. Cyanobacteria and some of the γ-Proteobacteria appear to possess arsC genes that are similar to those of Low GC Gram-positive Bacteria, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relationships. There is no clear separation of plasmid-borne and chromosomal arsC genes, although a number of the Enterobacteriales (γ-Proteobacteria possess similar plasmid-encoded arsC sequences. Conclusion The overall phylogeny of the arsenate reductases suggests a single, early origin of the arsC gene and subsequent sequence divergence to give the distinct arsC classes that exist today. Discrepancies between 16S rRNA and arsC phylogenies support the role of horizontal gene transfer (HGT in the evolution of arsenate reductases, with a number of instances of HGT early in bacterial arsC evolution. Plasmid-borne arsC genes are not monophyletic suggesting multiple cases of chromosomal-plasmid exchange and subsequent HGT. Overall, arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms.

  1. Identification and gene prediction of a 24 kb region containing xa5, a recessive bacterial blight resistance gene in rice (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    ZHONG Yiming; JIANG Guanghuai; CHEN Xuewei; XIA Zhihui; LI Xiaobing; ZHU Lihuang; ZHAI Wenxue

    2003-01-01

    Rice xa5 gene provides recessive, race-specific resistance to bacterial blight disease caused by the pathogen Xanthomonas oryzae pv. oryzae and has great value for research and breeding. In an effort to clone xa5, an F2 population of 4892 individuals was developed from the xa5 near isogenic lines, IR24 and IRBB5. A fine mapping procedure was conducted and tightly linked RFLP markers were used to screen a BAC library of IRBB56, a resistant rice line containing the xa5 gene. A 213 kb contig covering the xa5 locus was constructed. According to the sequences from the International Rice Genome Sequening Project (IRGSP), the Chinese Superhybrid Rice Genome Project (SRGP) and some sub-clones of the contig, twelve SSLP and CAPS markers were developed for fine mapping. The xa5 gene was mapped to a 0.3 cM interval between markers K5 and T4, which spanned an interval of approximately 24 kb, co-segregating with marker T2. Sequence analysis of the 24 kb region revealed that an ABC transporter and a basal transcription factor (TFIIa) were potential candidates for the xa5 resistance gene product. The molecular mechanism by which the xa5 gene provides recessive, race-specific resistance to bacterial blight will be elucidated by the functional tests of the 24 kb DNA and the candidate genes.

  2. In vivo alteration of the keratin 17 gene in hair follicles by oligonucleotide-directed gene targeting.

    Science.gov (United States)

    Fan, W; Yoon, K

    2003-12-01

    Using intradermal injection of a chimeric RNA-DNA oligonucleotide (RDO) or a single-stranded oligonucleotide (ssODN) into murine skin, we attempted to make a dominant mutation (R94p) in the conserve alpha-helical domain of keratin 17 (K17), the same mutation found in pachyononychia congenichia type 2 (PC-2) patients with phenotypes ranging from twisted hair and multiple pilosebaceous cysts. Both K17A-RDO and -ssODN contained a single base mismatch (CGC to CCC) to alter the normal K17 sequence to cause an amino acid substitution (R94P). The complexes consisting of oligonucleotides and cationic liposomes were injected to C57B1/6 murine skin at 2 and 5 day after birth. Histological examination of skin biopsies at postnatal day 8 from several mice showed consistent twisted hair shafts or broken hair follicles at the sebaceous gland level and occasional rupture of the hair bulb or epidermal cyst-like changes. In the injected area, the number of full anagen hair follicles decrease by 50%. Injection of the control oligonucleotide, identical to K17A-RDO but containing no mismatch to the normal sequence, did not result in any detectable abnormality. The frequency of gene alteration was lower than 3%, according to the restriction fragment length polymorphism (RFLP) analysis of the genomic DNA isolated by dissection of hair follicles from slides. Although intradermal injection of K17A-RDO or K17-ssODN caused a dominant mutation in K17 affecting hair growth and morphology, these phenotypic changes were transient either due to the compensation of K17 by other keratins or the replacement of the mutated cells by normal surrounding cells during hair growth.

  3. [Effect of plasmid pKM101 on the expression of bacterial genes not related to DNa metabolism].

    Science.gov (United States)

    Skavronskaya, A G; Tiganova, I G; Andreeva, I V; Rusina, O Iu

    1999-02-01

    An experimental system ensuring fusion of bacterial genes to the lac operon of the Mu dl(Aplac) phage was used. Fusion operons in which the lac operon was under the control of promoters of the elt gene, responsible for synthesis of the LT toxin, of the tetracyclin-resistance tet gene, and sfiA gene encoding filament production, was studied. Using this experimental system, plasmid pKM101 was shown to be capable of activating the expression of the above Escherichia coli and Salmonella typhimurium genes, which is manifested as the activation of beta-galactosidase synthesis. The activation of the elt gene expression by the pKM101 plasmid was also confirmed in experiments on detecting the LT toxin synthesized by bacteria carrying this plasmid. Effect of the plasmid on the activation of elt operon expression, unlike the effect of this plasmid on mutability, does not depend on the functioning of the lexA and recA genes, i.e., this is not a SOS-regulated process. The mutant plasmid pGW12, a derivative of pKM101, deficient in the mucAB genes responsible for mutagenesis, causes a more pronounced activation of the elt gene than plasmid pKM101.

  4. Reduction of rainbow trout spleen size by splenectomy does not alter resistance against bacterial cold water disease.

    Science.gov (United States)

    Wiens, Gregory D; Marancik, David P; Zwollo, Patty; Kaattari, Stephen L

    2015-03-01

    In lower vertebrates, the contribution of the spleen to anti-bacterial immunity is poorly understood. We have previously reported a phenotypic and genetic correlation between resistance to Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) and spleen somatic index (spleen weight normalized to body weight, SI). Fish families with larger pre-challenge SI values were found to have greater BCWD survival (resistance) following intraperitoneal injection of a lethal dose of F. psychrophilum. Since the mammalian spleen is known to be crucial for capture and destruction of encapsulated bacteria, we tested the hypothesis that reduction of spleen size, by surgical splenectomy, should reduce the survival advantage of the larger-spleen, disease-resistant fish. Experiments were performed using two separate lines of fish that had previously been selected either based on BCWD survival (resistant and susceptible), or selected based on spleen size (high and low SI). Following 65 to 81 days post-surgical recovery, fish were challenged with F. psychrophilum and mortality monitored for a minimum of 21 days. No significant difference in the relative survival was detected between splenectomized or sham-operated groups, while SI of splenectomized fish was reduced to an average of 8-12% of control animals. A positive correlation was observed between the SI, measured at the time of splenectomy, and time-to-death post-challenge. In summary, these experiments argue that larger spleen size alone is not sufficient for greater BCWD resistance, but rather it is an indirect indicator of immunological status.

  5. CLUSEAN: a computer-based framework for the automated analysis of bacterial secondary metabolite biosynthetic gene clusters.

    Science.gov (United States)

    Weber, T; Rausch, C; Lopez, P; Hoof, I; Gaykova, V; Huson, D H; Wohlleben, W

    2009-03-10

    Bacterial secondary metabolites are an important source of antimicrobial and cytostatic drugs. These molecules are often synthesized in a stepwise fashion by multimodular megaenzymes that are encoded in clusters of genes encoding enzymes for precursor supply and modification. In this work,we present an open source software pipeline, CLUSEAN (CLUster SEquence ANalyzer) that helps to annotate and analyze such gene clusters. CLUSEAN integrates standard analysis tools, like BLAST and HMMer, with specific tools for the identification of the functional domains and motifs in nonribosomal peptide synthetases (NRPS)/type I polyketide synthases (PKS) and the prediction of specificities of NRPS.

  6. Ketamine influences CLOCK:BMAL1 function leading to altered circadian gene expression.

    Directory of Open Access Journals (Sweden)

    Marina M Bellet

    Full Text Available Major mood disorders have been linked to abnormalities in circadian rhythms, leading to disturbances in sleep, mood, temperature, and hormonal levels. We provide evidence that ketamine, a drug with rapid antidepressant effects, influences the function of the circadian molecular machinery. Ketamine modulates CLOCK:BMAL1-mediated transcriptional activation when these regulators are ectopically expressed in NG108-15 neuronal cells. Inhibition occurs in a dose-dependent manner and is attenuated after treatment with the GSK3β antagonist SB21673. We analyzed the effect of ketamine on circadian gene expression and observed a dose-dependent reduction in the amplitude of circadian transcription of the Bmal1, Per2, and Cry1 genes. Finally, chromatin-immunoprecipitation analyses revealed that ketamine altered the recruitment of the CLOCK:BMAL1 complex on circadian promoters in a time-dependent manner. Our results reveal a yet unsuspected molecular mode of action of ketamine and thereby may suggest possible pharmacological antidepressant strategies.

  7. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    Directory of Open Access Journals (Sweden)

    Junko Kimura-Kuroda

    2016-10-01

    Full Text Available Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of longterm (14 days and low dose (1 μM exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  8. Alterations in seed development gene expression affect size and oil content of Arabidopsis seeds.

    Science.gov (United States)

    Fatihi, Abdelhak; Zbierzak, Anna Maria; Dörmann, Peter

    2013-10-01

    Seed endosperm development in Arabidopsis (Arabidopsis thaliana) is under control of the polycomb group complex, which includes Fertilization Independent Endosperm (FIE). The polycomb group complex regulates downstream factors, e.g. Pheres1 (PHE1), by genomic imprinting. In heterozygous fie mutants, an endosperm develops in ovules carrying a maternal fie allele without fertilization, finally leading to abortion. Another endosperm development pathway depends on MINISEED3 (a WRKY10 transcription factor) and HAIKU2 (a leucine-rich repeat kinase). While the role of seed development genes in the embryo and endosperm establishment has been studied in detail, their impact on metabolism and oil accumulation remained unclear. Analysis of oil, protein, and sucrose accumulation in mutants and overexpression plants of the four seed development genes revealed that (1) seeds carrying a maternal fie allele accumulate low oil with an altered composition of triacylglycerol molecular species; (2) homozygous mutant seeds of phe1, mini3, and iku2, which are smaller, accumulate less oil and slightly less protein, and starch, which accumulates early during seed development, remains elevated in mutant seeds; (3) embryo-specific overexpression of FIE, PHE1, and MINI3 has no influence on seed size and weight, nor on oil, protein, or sucrose content; and (4) overexpression of IKU2 results in seeds with increased size and weight, and oil content of overexpressed IKU2 seeds is increased by 35%. Thus, IKU2 overexpression represents a novel strategy for the genetic manipulation of the oil content in seeds.

  9. Vitamin D receptor gene is epigenetically altered and transcriptionally up-regulated in multiple sclerosis

    Science.gov (United States)

    Soriano, Luis; Olaskoaga, Ander; Roldán, Miren; Otano, María; Ajuria, Iratxe; Soriano, Gerardo; Lacruz, Francisco

    2017-01-01

    Objective Vitamin D deficiency has been linked to increased risk of multiple sclerosis (MS) and poor outcome. However, the specific role that vitamin D plays in MS still remains unknown. In order to identify potential mechanisms underlying vitamin D effects in MS, we profiled epigenetic changes in vitamin D receptor (VDR) gene to identify genomic regulatory elements relevant to MS pathogenesis. Methods Human T cells derived from whole blood by negative selection were isolated in a set of 23 relapsing-remitting MS (RRMS) patients and 12 controls matched by age and gender. DNA methylation levels were assessed by bisulfite cloning sequencing in two regulatory elements of VDR. mRNA levels were measured by RT-qPCR to assess changes in VDR expression between patients and controls. Results An alternative VDR promoter placed at exon 1c showed increased DNA methylation levels in RRMS patients (median 30.08%, interquartile range 19.2%) compared to controls (18.75%, 9.5%), p-value<0.05. Moreover, a 6.5-fold increase in VDR mRNA levels was found in RRMS patients compared to controls (p-value<0.001). Conclusions An alternative promoter of the VDR gene shows altered DNA methylation levels in patients with multiple sclerosis, and it is associated with VDR mRNA upregulation. This locus may represent a candidate regulatory element in the genome relevant to MS pathogenesis. PMID:28355272

  10. Rarity of DNA sequence alterations in the promoter region of the human androgen receptor gene

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    D.F. Cabral

    2004-12-01

    Full Text Available The human androgen receptor (AR gene promoter lies in a GC-rich region containing two principal sites of transcription initiation and a putative Sp1 protein-binding site, without typical "TATA" and "CAAT" boxes. It has been suggested that mutations within the 5'untranslated region (5'UTR may contribute to the development of prostate cancer by changing the rates of gene transcription and/or translation. In order to investigate this question, the aim of the present study was to search for the presence of mutations or polymorphisms at the AR-5'UTR in 92 prostate cancer patients, where histological diagnosis of adenocarcinoma was established in specimens obtained from transurethral resection or after prostatectomy. The AR-5'UTR was amplified by PCR from genomic DNA samples of the patients and of 100 healthy male blood donors, included as controls. Conformation-sensitive gel electrophoresis was used for DNA sequence alteration screening. Only one band shift was detected in one individual from the blood donor group. Sequencing revealed a new single nucleotide deletion (T in the most conserved portion of the promoter region at position +36 downstream from the transcription initiation site I. Although the effect of this specific mutation remains unknown, its rarity reveals the high degree of sequence conservation of the human androgen promoter region. Moreover, the absence of detectable variation within the critical 5'UTR in prostate cancer patients indicates a low probability of its involvement in prostate cancer etiology.

  11. Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression.

    Science.gov (United States)

    Jia, Hong-Mei; Li, Qi; Zhou, Chao; Yu, Meng; Yang, Yong; Zhang, Hong-Wu; Ding, Gang; Shang, Hai; Zou, Zhong-Mei

    2016-03-23

    Depression is a complex disease characterized by a series of pathological changes. Research on depression is mainly focused on the changes in brain, but not on liver. Therefore, we initially explored the metabolic profiles of hepatic extracts from rats treated with chronic unpredictive mild stress (CUMS) by UPLC-Q-TOF/MS. Using multivariate statistical analysis, a total of 26 altered metabolites distinguishing CUMS-induced depression from normal control were identified. Using two-stage receiver operating characteristic (ROC) analysis, 18 metabolites were recognized as potential biomarkers related to CUMS-induced depression via 12 metabolic pathways. Subsequently, we detected the mRNA expressions levels of apoptosis-associated genes such as Bax and Bcl-2 and four key enzymes including Pla2g15, Pnpla6, Baat and Gad1 involved in phospholipid and primary bile acid biosynthesis in liver tissues of CUMS rats by real-time qRT-PCR assay. The expression levels of Bax, Bcl-2, Pla2g15, Pnpla6 and Gad1 mRNA were 1.43,1.68, 1.74, 1.67 and 1.42-fold higher, and those of Baat, Bax/Bcl-2 ratio mRNA were 0.83, 0.85-fold lower in CUMS rats compared with normal control. Results of liver-targeted metabonomics and mRNA expression demonstrated that CUMS-induced depression leads to variations in hepatic metabolic profile and gene expression, and ultimately results in liver injury.

  12. Evaluation of the gene encoding the enzyme βHPMEH for the bacterial wilt inhibition caused by Ralstonia solanacearum

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    Elizabeth Fernandez

    2015-10-01

    Full Text Available Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease that attacks important agricultural crops such as potato, tomato, banana, among others, causing serious yield losses. Control of R. solanacearum is difficult because of its wide range of alternate hosts, its long survival in soil, its biological and genetic variation, the lack of natural resistance sources and the insufficiency of the appropriate chemical control measures. Quorum sensing is the term that describes the phenomenon whereby the accumulation of molecules allows bacteria to know the number of bacteria found in the environment (population density. R. solanacearum has a quorum sensing system for the regulation of the expression of virulence genes; the molecule 3-OH-PAME is the self-regulatory signal. The molecule ΒHPMEH hydrolyzes 3-OH-PAME nullifying the signal of virulence, and thus, the quorum sensing communication in R. solanacearum. In order to evaluate the βhpmeh gene we designed two vectors that express this gene under the control of two different promoters. Both vectors were verified by restriction analysis and sequencing. Agroinfiltration assays were used to analyze gene expression and the effect against R. solanacearum in potato (Solanum tuberosum leaves. The results of the transient expression experiments showed that the expression of gene βhpmeh caused a delay in the appearance of symptoms of bacterial wilt and thus is a good candidate for whole genetic plant transformation.

  13. Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library.

    Science.gov (United States)

    Jacquiod, Samuel; Demanèche, Sandrine; Franqueville, Laure; Ausec, Luka; Xu, Zhuofei; Delmont, Tom O; Dunon, Vincent; Cagnon, Christine; Mandic-Mulec, Ines; Vogel, Timothy M; Simonet, Pascal

    2014-11-20

    A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events.

  14. Changes in mitochondrial DNA alter expression of nuclear encoded genes associated with tumorigenesis

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    Jandova, Jana; Janda, Jaroslav [Southern Arizona VA Healthcare System, Department of Medicine, Dermatology Division and Arizona Cancer Center, University of Arizona, 1515 N Campbell Avenue, Tucson, AZ 857 24 (United States); Sligh, James E, E-mail: jsligh@azcc.arizona.edu [Southern Arizona VA Healthcare System, Department of Medicine, Dermatology Division and Arizona Cancer Center, University of Arizona, 1515 N Campbell Avenue, Tucson, AZ 857 24 (United States)

    2012-10-15

    We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-{kappa}B (NF-{kappa}B) is a key transcription factor for production of MMPs. An inhibitor of NF-{kappa}B activation, Bay 11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-{kappa}B in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids. -- Highlights: Black-Right-Pointing-Pointer Cybrids are useful models to study the role of mtDNA changes in cancer development. Black-Right-Pointing-Pointer mtDNA changes affect the expression of nuclear

  15. Altered surfactant protein A gene expression and protein homeostasis in rats with emphysematous changes

    Institute of Scientific and Technical Information of China (English)

    HU Qiong-jie; XIONG Sheng-dao; ZHANG Hui-lan; SHI Xue-mei; XU Yong-jian; ZHANG Zhen-xiang; ZHEN Guo-hua; ZHAO Jian-ping

    2008-01-01

    Background The decrease of suffactant protein(SP)secreted by the alveolar type Ⅱ cell is one of the important causes of limiting air of pulmonary emphysema.However,the SP-A gene and protein changes in this disease are rarely studied.This study was undertaken to investigate alterations in SP-A gene activity and protein,and to explore their roles in the pathogenesis of emphysematous changes.Methods Twenty Wistar rats were divided randomly into a normal control group(n=10)and a cigarette smoking(CS)+lipopolysaccharide(LPS)group(n=10).Ultra-structural changes were obsewed under an electron microscope.The number of cells positive for SP-A was measured by immunohistochemistry.The mRNA expression and protein Ievel of SP-A in the lung tissues were determined by quantitative polymerase chain reaction(qPCR)and Western blot separately.The protein level of SP-A in lavage fluid was determined by Western blot.Results The number of cells positive for SP-A of the CS+LPS group(0.35±0.03)was lower than that of the blank control group(0.72±0.06,P<0.05).The level of SP-A in the lung tissues of rats in the CS+LPS group(0.2765±0.0890)was lower than that in the blank controI group(0.6875±0.1578,P<0.05).The level of SP-A in the lavage fluid of rats in the CS+LPS group(0.8567±0.1458)was lower than that in the blank controI group(1.3541±0.2475,P<0.05).The lung tissues of rats in the CS+LPS group showed an approximate increase(0.4-fold)in SP-A mRNA levels relative to β-actin mRNA (P<0.05).Conclusions The changes of SP-A may be related to emphysematous changes in the lung.And cigarette smoke and LPS alter lung SP-A gene activity and protein homeostasis.

  16. Altered expression of mitochondrial related genes in the native Tibetan placents by mitochondrial cDNA array analysis

    Institute of Scientific and Technical Information of China (English)

    Luo Yongjun; Gao Wenxiang; Zhao Xiuxin; Suo Lang; Chen Li; Liu Fuyu; Song Tonglin; Chen Jian; Gao Yuqi

    2009-01-01

    Objective: To explore the mechanism of native Tibetan fetuses adaptation to hypoxia, we tried to find the different expression genes about mitochondrial function in the native Tibetan placents. Methods: In this study, the placents of native Tibetan and the high-altitude Han (ha-Han) were collected. After the total RNA extraction, the finally synthesized cDNAs were hybridized to mitochondrial array to find the altered expression genes between them. Then, the cytochrome c oxidase 17 (Coxl7), dynactin 2 (DCTN2, also known as p50), and vascular endothelial growth factor receptor (VEGFR, also known as KDR) were chosen from the altered expression genes to further verify the array results using the SYBR Green real-time PCR. Because the altered expression genes (such as Cybb and Coxl 7) in the array results related to the activities of COXI and COXIV, the placental mitochondria activities of COXI and COXIV were measured to find their changes in the hypoxia. Results: By a standard of >1.5 or <0.67, there were 24 different expressed genes between the native Tibetan and the ha-Han placents, including 3 up-regulated genes and 21 down-regulated genes. These genes were related to energy metabolism, signal transduction, cell proliferation, electron transport, cell adhesion, nucleotide-excision repair. The array results of Coxl7, DCTN2 and KDR were further verified by the real-time RT-PCR. Through the mitochondria respiration measurements, the activity of COXI in the native Tibetan placents were higher than that of ha-Han, there was no difference in COXIV activity between them. Conclusion: The altered mitochondrial related genes in the native Tibetan placents may have a role in the high altitude adaptation for fetuses through changing the activity of mitochondrial COX.

  17. PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development

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    Damjanovski Sashko

    2011-04-01

    Full Text Available Abstract Background Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis. Results Microinjecting haemagglutinin (HA tagged Pex11β in early embryos resulted in increased RNA levels for peroxisome related genes PMP70 and catalase at developmental stages 10 and 20, versus uninjected embryos. Catalase and PMP70 proteins were found in punctate structures at stage 20 in control embryos, whereas the injection of ectopic HA-Pex11β induced their earlier localization in punctate structures at stage 10. Furthermore, the peroxisomal marker GFP-SKL, which was found localized as peroxisome-like structures at stage 20, was similarly found at stage 10 when co-microinjected with HA-Pex11β. Conclusions Overexpressed Pex11β altered peroxisomal gene levels and induced the early formation of peroxisomes-like structures during development, both of which demonstrate that Pex11β may be a key regulator of peroxisome number in early Xenopus embryos.

  18. Altered gene expression and ecological divergence in sibling allopolyploids of Dactylorhiza (Orchidaceae

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    Moat Justin

    2011-04-01

    Full Text Available Abstract Background Hybridization and polyploidy are potent forces that have regularly stimulated plant evolution and adaptation. Dactylorhiza majalis s.s., D. traunsteineri s.l. and D. ebudensis are three allopolyploid species of a polyploid complex formed through unidirectional (and, in the first two cases, recurrent hybridization between the widespread diploids D. fuchsii and D. incarnata. Differing considerably in geographical extent and ecological tolerance, the three allopolyploids together provide a useful system to explore genomic responses to allopolyploidization and reveal their role in adaptation to contrasting environments. Results Analyses of cDNA-AFLPs show a significant increase in the range of gene expression of these allopolyploid lineages, demonstrating higher potential for phenotypic plasticity than is shown by either parent. Moreover, allopolyploid individuals express significantly more gene variants (including novel alleles than their parents, providing clear evidence of increased biological complexity following allopolyploidization. More genetic mutations seem to have accumulated in the older D. majalis compared with the younger D. traunsteineri since their respective formation. Conclusions Multiple origins of the polyploids contribute to differential patterns of gene expression with a distinct geographic structure. However, several transcripts conserved within each allopolyploid taxon differ between taxa, indicating that habitat preferences shape similar expression patterns in these independently formed tetraploids. Statistical signals separate several transcripts - some of them novel in allopolyploids - that appear correlated with adaptive traits and seem to play a role favouring the persistence of individuals in their native environments. In addition to stabilizing the allopolyploid genome, genetic and epigenetic alterations are key determinants of adaptive success of the new polyploid species after recurrent

  19. Relationship between Microsatellite Alterations of RASSF1A Gene and Development of Cervical Carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHAO Fu-xi; YAN Jie; LIU Run-hua; WANG Xi-ying; CUI Ke

    2007-01-01

    Objective: To explore the relationship between microsatellite alterations of RASSF1A gene and the development of cervical carcinoma, and its relationship with HPV16 infection. Methods: Two sites of microsatellite polymorphism of RASSF1A gene were selected. Polymerase chain reaction (PCR) technique was used to detect LOH and MSI in 50 cases of cervical carcinoma and 40 cases of cervical intraepithelial neoplasia (CIN), and to detect the infection state of HPV16. Results: At D3S1478 and D3S4604, the LOH rates of cervical carcinomas were 32.6% (14/43) and 48.9% (23/47), the MSI rates were 14% (6/43) and 19.1% (9/47), respectively. The LOH rates of CINs were 31.4% (11/35) and 39.5% (15/38), the MSI rates were 11.4% (4/35) and 15.8% (6/38), respectively. There were no significant differences between cervical carcinomas and CINs in respect to their positive rates of LOH and MSI at D3S1478 and D3S4604 (P>0.05). There were significant differences in LOH rates at D3S1478 and D3S4604 between the stage Ⅰ-Ⅱ and Ⅲ-Ⅳ cervical carcinomas and between the well/moderately differentiated cervical carcinomas and the poorly differentiated cervical carcinomas (P<0.05). The positive rates of LOH and MSI for CIN Ⅲ and noninvasive cervical carcinomas were higher than those in CIN Ⅰ-Ⅱ. The rates of the infection of HPV16 in cervical cancer was obviously higher than that in CIN and in normal cervical tissues (P<0.05), and the incidence of LOH of RASSF1A gene was higher in HPV16(+) than that in HPV16(-) (P<0.05). Conclusion: The RASSF1A gene change is a relatively late event in cervical carcinomas. The detection of LOH and MSI of RASSF1A gene might be helpful to the early diagnosis and the screening of cervical carcinoma. It might also be useful for predicting the prognosis of cervical carcinoma.

  20. Functional alterations due to amino acid changes and evolutionary comparative analysis of ARPKD and ADPKD genes

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    Burhan M. Edrees

    2016-12-01

    Full Text Available A targeted customized sequencing of genes implicated in autosomal recessive polycystic kidney disease (ARPKD phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified four potential pathogenic variants in PKHD1 gene [c.4870C>T, p.(Arg1624Trp, c.5725C>T, p.(Arg1909Trp, c.1736C>T, p.(Thr579Met and c.10628T>G, p.(Leu3543Trp] among 12 out of 18 samples. However, one variant c.4870C>T, p.(Arg1624Trp was common among eight patients. Some patient samples also showed few variants in autosomal dominant polycystic kidney disease (ADPKD disease causing genes PKD1 and PKD2 such as c.12433G>A, p.(Val4145Ile and c.1445T>G, p.(Phe482Cys, respectively. All causative variants were validated by capillary sequencing and confirmed the presence of a novel homozygous variant c.10628T>G, p.(Leu3543Trp in a male proband. We have recently published the results of these studies (Edrees et al., 2016. Here we report for the first time the effect of the common mutation p.(Arg1624Trp found in eight samples on the protein structure and function due to the specific amino acid changes of PKHD1 protein using molecular dynamics simulations. The computational approaches provide tool predict the phenotypic effect of variant on the structure and function of the altered protein. The structural analysis with the common mutation p.(Arg1624Trp in the native and mutant modeled protein were also studied for solvent accessibility, secondary structure and stabilizing residues to find out the stability of the protein between wild type and mutant forms. Furthermore, comparative genomics and evolutionary analyses of variants observed in PKHD1, PKD1, and PKD2 genes were also performed in some mammalian species including human to understand the complexity of genomes among closely related mammalian species. Taken together, the results revealed that the evolutionary comparative analyses and characterization of PKHD1, PKD1

  1. Sulfamethoxazole and COD increase abundance of sulfonamide resistance genes and change bacterial community structures within sequencing batch reactors.

    Science.gov (United States)

    Guo, Xueping; Pang, Weihai; Dou, Chunling; Yin, Daqiang

    2017-05-01

    The abundant microbial community in biological treatment processes in wastewater treatment plants (WWTPs) may potentially enhance the horizontal gene transfer of antibiotic resistance genes with the presence of antibiotics. A lab-scale sequencing batch reactor was designed to investigate response of sulfonamide resistance genes (sulI, sulII) and bacterial communities to various concentrations of sulfamethoxazole (SMX) and chemical oxygen demand (COD) of wastewater. The SMX concentrations (0.001 mg/L, 0.1 mg/L and 10 mg/L) decreased with treatment time and higher SMX level was more difficult to remove. The presence of SMX also significantly reduced the removal efficiency of ammonia nitrogen, affecting the normal function of WWTPs. All three concentrations of SMX raised both sulI and sulII genes with higher concentrations exhibiting greater increases. The abundance of sul genes was positive correlated with treatment time and followed the second-order reaction kinetic model. Interestingly, these two genes have rather similar activity. SulI and sulII gene abundance also performed similar response to COD. Simpson index and Shannon-Weiner index did not show changes in the microbial community diversity. However, the 16S rRNA gene cloning and sequencing results showed the bacterial community structures varied during different stages. The results demonstrated that influent antibiotics into WWTPs may facilitate selection of ARGs and affect the wastewater conventional treatment as well as the bacteria community structures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies.

    Science.gov (United States)

    Mašlaňová, Ivana; Doškař, Jiří; Varga, Marian; Kuntová, Lucie; Mužík, Jan; Malúšková, Denisa; Růžičková, Vladislava; Pantůček, Roman

    2013-02-01

    Staphylococcus aureus is a serious human and veterinary pathogen in which new strains with increasing virulence and antimicrobial resistance occur due to acquiring new genes by horizontal transfer. It is generally accepted that temperate bacteriophages play a major role in gene transfer. In this study, we proved the presence of various bacterial genes of the S. aureus COL strain directly within the phage particles via qPCR and quantified their packaging frequency. Non-parametric statistical analysis showed that transducing bacteriophages φ11, φ80 and φ80α of serogroup B, in contrast to serogroup A bacteriophage φ81, efficiently package selected chromosomal genes localized in 4 various loci of the chromosome and 8 genes carried on variable elements, such as staphylococcal cassette chromosome SCCmec, staphylococcal pathogenicity island SaPI1, genomic islands vSaα and vSaβ, and plasmids with various frequency. Bacterial gene copy number per ng of DNA isolated from phage particles ranged between 1.05 × 10(2) for the tetK plasmid gene and 3.86 × 10(5) for the SaPI1 integrase gene. The new and crucial finding that serogroup B bacteriophages can package concurrently ccrA1 (1.16 × 10(4)) and mecA (1.26 × 10(4)) located at SCCmec type I into their capsids indicates that generalized transduction plays an important role in the evolution and emergence of new methicillin-resistant clones.

  3. INFLUENCE OF ROOT EXUDATES AND BACTERIAL METABOLIC ACTIVITY ON APPARENT CONJUGAL GENE TRANSFER FREQUENCIES IN THE RHIZOSPHERE OF WATER GRASS (ECHINOCLORA CRUSGALLI)

    Science.gov (United States)

    The premise that genetic exchange is primarily localized in niches characterized by dense bacterial populations and high availability of growth substrates was tested by relating conjugal gene transfer of an RP4 derivative to availability of root exudates and bacterial metabolic a...

  4. Trimeric autotransporter adhesins contribute to Actinobacillus pleuropneumoniae pathogenicity in mice and regulate bacterial gene expression during interactions between bacteria and porcine primary alveolar macrophages.

    Science.gov (United States)

    Qin, Wanhai; Wang, Lei; Zhai, Ruidong; Ma, Qiuyue; Liu, Jianfang; Bao, Chuntong; Zhang, Hu; Sun, Changjiang; Feng, Xin; Gu, Jingmin; Du, Chongtao; Han, Wenyu; Langford, P R; Lei, Liancheng

    2016-01-01

    Actinobacillus pleuropneumoniae is an important pathogen that causes respiratory disease in pigs. Trimeric autotransporter adhesin (TAA) is a recently discovered bacterial virulence factor that mediates bacterial adhesion and colonization. Two TAA coding genes have been found in the genome of A. pleuropneumoniae strain 5b L20, but whether they contribute to bacterial pathogenicity is unclear. In this study, we used homologous recombination to construct a double-gene deletion mutant, ΔTAA, in which both TAA coding genes were deleted and used it in in vivo and in vitro studies to confirm that TAAs participate in bacterial auto-aggregation, biofilm formation, cell adhesion and virulence in mice. A microarray analysis was used to determine whether TAAs can regulate other A. pleuropneumoniae genes during interactions with porcine primary alveolar macrophages. The results showed that deletion of both TAA coding genes up-regulated 36 genes, including ene1514, hofB and tbpB2, and simultaneously down-regulated 36 genes, including lgt, murF and ftsY. These data illustrate that TAAs help to maintain full bacterial virulence both directly, through their bioactivity, and indirectly by regulating the bacterial type II and IV secretion systems and regulating the synthesis or secretion of virulence factors. This study not only enhances our understanding of the role of TAAs but also has significance for those studying A. pleuropneumoniae pathogenesis.

  5. Sertoli-cell-specific knockout of connexin 43 leads to multiple alterations in testicular gene expression in prepubertal mice

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    Sarah Giese

    2012-11-01

    A significant decline in human male reproductive function has been reported for the past 20 years but the molecular mechanisms remain poorly understood. However, recent studies showed that the gap junction protein connexin-43 (CX43; also known as GJA1 might be involved. CX43 is the predominant testicular connexin (CX in most species, including in humans. Alterations of its expression are associated with different forms of spermatogenic disorders and infertility. Men with impaired spermatogenesis often exhibit a reduction or loss of CX43 expression in germ cells (GCs and Sertoli cells (SCs. Adult male transgenic mice with a conditional knockout (KO of the Gja1 gene [referred to here as connexin-43 (Cx43] in SCs (SCCx43KO show a comparable testicular phenotype to humans and are infertile. To detect possible signaling pathways and molecular mechanisms leading to the testicular phenotype in adult SCCx43KO mice and to their failure to initiate spermatogenesis, the testicular gene expression of 8-day-old SCCx43KO and wild-type (WT mice was compared. Microarray analysis revealed that 658 genes were significantly regulated in testes of SCCx43KO mice. Of these genes, 135 were upregulated, whereas 523 genes were downregulated. For selected genes the results of the microarray analysis were confirmed using quantitative real-time PCR and immunostaining. The majority of the downregulated genes are GC-specific and are essential for mitotic and meiotic progression of spermatogenesis, including Stra8, Dazl and members of the DM (dsx and map-3 gene family. Other altered genes can be associated with transcription, metabolism, cell migration and cytoskeleton organization. Our data show that deletion of Cx43 in SCs leads to multiple alterations of gene expression in prepubertal mice and primarily affects GCs. The candidate genes could represent helpful markers for investigators exploring human testicular biopsies from patients showing corresponding spermatogenic deficiencies and for

  6. BPhyOG: An interactive server for genome-wide inference of bacterial phylogenies based on overlapping genes

    Directory of Open Access Journals (Sweden)

    Lin Kui

    2007-07-01

    Full Text Available Abstract Background Overlapping genes (OGs in bacterial genomes are pairs of adjacent genes of which the coding sequences overlap partly or entirely. With the rapid accumulation of sequence data, many OGs in bacterial genomes have now been identified. Indeed, these might prove a consistent feature across all microbial genomes. Our previous work suggests that OGs can be considered as robust markers at the whole genome level for the construction of phylogenies. An online, interactive web server for inferring phylogenies is needed for biologists to analyze phylogenetic relationships among a set of bacterial genomes of interest. Description BPhyOG is an online interactive server for reconstructing the phylogenies of completely sequenced bacterial genomes on the basis of their shared overlapping genes. It provides two tree-reconstruction methods: Neighbor Joining (NJ and Unweighted Pair-Group Method using Arithmetic averages (UPGMA. Users can apply the desired method to generate phylogenetic trees, which are based on an evolutionary distance matrix for the selected genomes. The distance between two genomes is defined by the normalized number of their shared OG pairs. BPhyOG also allows users to browse the OGs that were used to infer the phylogenetic relationships. It provides detailed annotation for each OG pair and the features of the component genes through hyperlinks. Users can also retrieve each of the homologous OG pairs that have been determined among 177 genomes. It is a useful tool for analyzing the tree of life and overlapping genes from a genomic standpoint. Conclusion BPhyOG is a useful interactive web server for genome-wide inference of any potential evolutionary relationship among the genomes selected by users. It currently includes 177 completely sequenced bacterial genomes containing 79,855 OG pairs, the annotation and homologous OG pairs of which are integrated comprehensively. The reliability of phylogenies complemented by

  7. Tissue-associated bacterial alterations in rectal carcinoma patients revealed by 16S rRNA community profiling

    Directory of Open Access Journals (Sweden)

    Andrew Maltez Thomas

    2016-12-01

    Full Text Available Sporadic and inflammatory forms of colorectal cancer (CRC account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group or during surgery for tumor excision (rectal-cancer group. High throughput 16S rRNA amplicon sequencing of the V4-V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified

  8. Tissue-Associated Bacterial Alterations in Rectal Carcinoma Patients Revealed by 16S rRNA Community Profiling

    Science.gov (United States)

    Thomas, Andrew M.; Jesus, Eliane C.; Lopes, Ademar; Aguiar, Samuel; Begnami, Maria D.; Rocha, Rafael M.; Carpinetti, Paola Avelar; Camargo, Anamaria A.; Hoffmann, Christian; Freitas, Helano C.; Silva, Israel T.; Nunes, Diana N.; Setubal, João C.; Dias-Neto, Emmanuel

    2016-01-01

    Sporadic and inflammatory forms of colorectal cancer (CRC) account for more than 80% of cases. Recent publications have shown mechanistic evidence for the involvement of gut bacteria in the development of both CRC-forms. Whereas, colon and rectal cancer have been routinely studied together as CRC, increasing evidence show these to be distinct diseases. Also, the common use of fecal samples to study microbial communities may reflect disease state but possibly not the tumor microenvironment. We performed this study to evaluate differences in bacterial communities found in tissue samples of 18 rectal-cancer subjects when compared to 18 non-cancer controls. Samples were collected during exploratory colonoscopy (non-cancer group) or during surgery for tumor excision (rectal-cancer group). High throughput 16S rRNA amplicon sequencing of the V4–V5 region was conducted on the Ion PGM platform, reads were filtered using Qiime and clustered using UPARSE. We observed significant increases in species richness and diversity in rectal cancer samples, evidenced by the total number of OTUs and the Shannon and Simpson indexes. Enterotyping analysis divided our cohort into two groups, with the majority of rectal cancer samples clustering into one enterotype, characterized by a greater abundance of Bacteroides and Dorea. At the phylum level, rectal-cancer samples had increased abundance of candidate phylum OD1 (also known as Parcubacteria) whilst non-cancer samples had increased abundance of Planctomycetes. At the genera level, rectal-cancer samples had higher abundances of Bacteroides, Phascolarctobacterium, Parabacteroides, Desulfovibrio, and Odoribacter whereas non-cancer samples had higher abundances of Pseudomonas, Escherichia, Acinetobacter, Lactobacillus, and Bacillus. Two Bacteroides fragilis OTUs were more abundant among rectal-cancer patients seen through 16S rRNA amplicon sequencing, whose presence was confirmed by immunohistochemistry and enrichment verified by digital

  9. SAMP8 mice have altered hippocampal gene expression in long term potentiation, phosphatidylinositol signaling, and endocytosis pathways.

    Science.gov (United States)

    Armbrecht, Harvey J; Siddiqui, Akbar M; Green, Michael; Farr, Susan A; Kumar, Vijaya B; Banks, William A; Patrick, Ping; Shah, Gul N; Morley, John E

    2014-01-01

    The senescence-accelerated mouse (SAMP8) strain exhibits decreased learning and memory and increased amyloid beta (Aβ) peptide accumulation at 12 months. To detect differences in gene expression in SAMP8 mice, we used a control mouse that was a 50% cross between SAMP8 and CD-1 mice and which showed no memory deficits (50% SAMs). We then compared gene expression in the hippocampus of 4- and 12-month-old SAMP8 and control mice using Affymetrix gene arrays. At 12 months, but not at 4 months, pathway analysis revealed significant differences in the long term potentiation (6 genes), phosphatidylinositol signaling (6 genes), and endocytosis (10 genes) pathways. The changes in long term potentiation included mitogen-activated protein kinase (MAPK) signaling (N-ras, cAMP responsive element binding protein [CREB], protein phosphatase inhibitor 1) and Ca-dependent signaling (inositol triphosphate [ITP] receptors 1 and 2 and phospholipase C). Changes in phosphatidylinositol signaling genes suggested altered signaling through phosphatidylinositol-3-kinase, and Western blotting revealed phosphorylation changes in serine/threonine protein kinase AKT and 70S6K. Changes in the endocytosis pathway involved genes related to clathrin-mediated endocytosis (dynamin and clathrin). Endocytosis is required for receptor recycling, is involved in Aβ metabolism, and is regulated by phosphatidylinositol signaling. In summary, these studies demonstrate altered gene expression in 3 SAMP8 hippocampal pathways associated with memory formation and consolidation. These pathways might provide new therapeutic targets in addition to targeting Aβ metabolism itself.

  10. Prognostic and predictive value of VHL gene alteration in renal cell carcinoma: a meta-analysis and review.

    Science.gov (United States)

    Kim, Bum Jun; Kim, Jung Han; Kim, Hyeong Su; Zang, Dae Young

    2017-01-17

    The von Hippel-Lindau (VHL) gene is often inactivated in sporadic renal cell carcinoma (RCC) by mutation or promoter hypermethylation. The prognostic or predictive value of VHL gene alteration is not well established. We conducted this meta-analysis to evaluate the association between the VHL alteration and clinical outcomes in patients with RCC. We searched PUBMED, MEDLINE and EMBASE for articles including following terms in their titles, abstracts, or keywords: 'kidney or renal', 'carcinoma or cancer or neoplasm or malignancy', 'von Hippel-Lindau or VHL', 'alteration or mutation or methylation', and 'prognostic or predictive'. There were six studies fulfilling inclusion criteria and a total of 633 patients with clear cell RCC were included in the study: 244 patients who received anti-vascular endothelial growth factor (VEGF) therapy in the predictive value analysis and 419 in the prognostic value analysis. Out of 663 patients, 410 (61.8%) had VHL alteration. The meta-analysis showed no association between the VHL gene alteration and overall response rate (relative risk = 1.47 [95% CI, 0.81-2.67], P = 0.20) or progression free survival (hazard ratio = 1.02 [95% CI, 0.72-1.44], P = 0.91) in patients with RCC who received VEGF-targeted therapy. There was also no correlation between the VHL alteration and overall survival (HR = 0.80 [95% CI, 0.56-1.14], P = 0.21). In conclusion, this meta-analysis indicates that VHL gene alteration has no prognostic or predictive value in patients with clear cell RCC.

  11. Finding immune gene expression differences induced by mar