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Sample records for random mutagenesis electronic

  1. Generation of genetically modified rodents using random ENU mutagenesis

    NARCIS (Netherlands)

    van Boxtel, R.; Cuppen, E.

    2011-01-01

    The generation of genetically modified animals using N-ethyl-N-nitrosourea (ENU) mutagenesis is a fast and highly effective method. The technique is based on treating male animals with the supermutagen ENU, which randomly introduces mutations in the spermatogonial stem cells. By breeding these

  2. Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling

    NARCIS (Netherlands)

    Máté De Gérando, H.; Fayolle-Guichard, F.; Rudant, L.; Millah, S.K.; Monot, F.; Ferreira, Nicolas Lopes; López-Contreras, A.M.

    2016-01-01

    Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of

  3. Site-specific genomic (SSG and random domain-localized (RDL mutagenesis in yeast

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    Honigberg Saul M

    2004-04-01

    Full Text Available Abstract Background A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. Results We have modified the existing method for introducing deletions in the yeast (S. cerevisiae genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis or is synthesized by error-prone PCR (RDL mutagenesis. In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. Conclusion SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

  4. Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

    Czech Academy of Sciences Publication Activity Database

    Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

    2010-01-01

    Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

  5. Use of the Photoactic Ability of a Bacterium to Teach the Genetic Principles of Random Mutagenesis & Mutant Screening

    Science.gov (United States)

    Din, Neena; Bird, Terry H.; Berleman, James E.

    2007-01-01

    In this article, the authors present a laboratory activity that relies on the use of a very versatile bacterial system to introduce the concept of how mutagenesis can be used for molecular and genetic analysis of living organisms. They have used the techniques of random mutagenesis and selection/screening to obtain strains of the organism "R.…

  6. Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis.

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    Fedashchin, Andrij; Cernota, William H; Gonzalez, Melissa C; Leach, Benjamin I; Kwan, Noelle; Wesley, Roy K; Weber, J Mark

    2015-11-01

    A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ~3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

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    Leonard, Cory A.; Brown, Stacy D.; Hayman, J. Russell

    2013-01-01

    Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. PMID:23983696

  8. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

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    Cory A. Leonard

    2013-01-01

    Full Text Available Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS. Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

  9. Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.

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    Hu, Mei-Rong; Chao, Ya-Peng; Zhang, Guo-Qing; Yang, Xiu-Qing; Xue, Zhi-Quan; Qian, Shi-Jun

    2007-12-01

    In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mgl(-1) laccase in yeast, together with a 1.4-fold increase in K(cat). Thus, the total activity is enhanced 5-fold for 2,2'-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6mM copper, the highest activity value reached 30 Uml(-1) after a 3-day cultivation at a temperature of 30 degrees C(.) In comparison with the wild type, the best mutant enzymatic properties (K(m) for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 A. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.

  10. Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

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    Chen-Ji Huang

    2011-01-01

    Full Text Available In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123 was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3 were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.

  11. Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay

    Science.gov (United States)

    2004-01-01

    Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the ‘slow’ X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820–13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175→Gly)] resulted in the ‘slow decay’ X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between ‘slow’ and ‘fast’ luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase–flavin–oxygen intermediate. PMID:15352872

  12. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB

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    Ihssen Julian

    2012-09-01

    Full Text Available Abstract Background Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. Results In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5 were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA. We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were

  13. Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor.

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    Michael R Marit

    Full Text Available The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.

  14. Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta by Improvement of Culture Conditions and Random Mutagenesis

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    Maria Angeles Vargas

    2011-09-01

    Full Text Available Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 µmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

  15. Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta) by Improvement of Culture Conditions and Random Mutagenesis

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    Cordero, Baldo F.; Obraztsova, Irina; Couso, Inmaculada; Leon, Rosa; Vargas, Maria Angeles; Rodriguez, Herminia

    2011-01-01

    Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment. PMID:22131961

  16. Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

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    Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira; Mesecar, Andrew D.; (UIC)

    2010-11-09

    An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 C and withstanding incubation at temperatures up to 70 C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 {angstrom}. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those

  17. Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity.

    Science.gov (United States)

    Koksharov, M I; Ugarova, N N

    2008-08-01

    Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of bioluminescence spectra, and Y35N,H and F16L/A40S substitutions resulted in the enzymes with bioluminescence spectra virtually independent from pH in the range of 6.0-7.8. The structural explanation is proposed for the effect of mutations on pH-sensitivity of bioluminescence spectra.

  18. Tailoring of global transcription sigma D factor by random mutagenesis to improve Escherichia coli tolerance towards low-pHs.

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    Gao, Xi; Jiang, Ling; Zhu, Liying; Xu, Qing; Xu, Xian; Huang, He

    2016-04-20

    Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22h(-1) vs. 0.15h(-1)) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

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    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

  20. Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G.

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    Isabel Uyttendaele

    Full Text Available The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.

  1. Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries.

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    Yang, Jianhua; Ruff, Anna J; Arlt, Marcus; Schwaneberg, Ulrich

    2017-09-01

    During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance. In this report, bsla (543 bp, small lipase gene from Bacillus subtilis) was divided into three fragments (147, 192, 204 bp). Each fragment was subjected to an epPCR with a high mutation load (22, 31, and 33 mutations per kb) in order to increase the number of identified beneficial positions while maintaining a fraction of active population which can efficiently be screened in agar plate or microtiter plate format. The use of this "casting epPCR" process termed as (cepPCR), doubles the number of identified beneficial positions (from 14% to 29%), when compared to standard epPCR for the BSLA enzyme model. A further increase to 39% of beneficial positions is obtainable through combination of cepPCR with the transversion biased sequence saturation mutagenesis (SeSaM) method. Furthermore, sequencing of up to 600 mutations per fragment provided valuable insights into the correlation of total throughput and number of identified beneficial positions as well as how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns. Biotechnol. Bioeng. 2017;114: 1921-1927. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.

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    Rodrigues, Maria Beatriz Calderan; Fávaro, Léia Cecília de Lima; Pallu, Ana Paula de Souza; Ferreira, Anderson; Sebastianes, Fernanda de Souza; Rodrigues, Maria Juliana Calderan; Spósito, Marcel Bellato; de Araújo, Welington Luiz; Pizzirani-Kleiner, Aline Aparecida

    2013-01-01

    Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  3. Use of an EZ-Tn5-based random mutagenesis system to identify a novel toxin regulatory locus in Clostridium perfringens strain 13.

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    Jorge E Vidal

    2009-07-01

    Full Text Available Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO by strain 13. PFO production by the strain 13 DeltaagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 DeltaagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.

  4. Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.

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    Kang, Ji-Yeon; Ryu, Sang Hoon; Park, Sun-Ha; Cha, Gun Su; Kim, Dong-Hyun; Kim, Keon-Hee; Hong, Austin W; Ahn, Taeho; Pan, Jae-Gu; Joung, Young Hee; Kang, Hyung-Sik; Yun, Chul-Ho

    2014-07-01

    Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads. © 2014 Wiley Periodicals, Inc.

  5. Agrobacterium tumefaciens-Mediated Transformation of Valsa mali: An Efficient Tool for Random Insertion Mutagenesis

    Directory of Open Access Journals (Sweden)

    Caixia Wang

    2013-01-01

    Full Text Available Valsa mali is a causal agent of apple and pear trees canker disease, which is a destructive disease that causes serious economic losses in eastern Asia, especially in China. The lack of an efficient transformation system for Valsa mali retards its investigation, which poses difficulties to control the disease. In this research, a transformation system for this pathogen was established for the first time using A. tumefaciens-mediated transformation (ATMT, with the optimal transformation conditions as follows: 106/mL conidia suspension, cocultivation temperature 22°C, cocultivation time 72 hours, and 200 μM acetosyringone (AS in the inductive medium. The average transformation efficiency was 1015.00 ± 37.35 transformants per 106 recipient conidia. Thirty transformants were randomly selected for further confirmation and the results showed the presence of T-DNA in all hygromycin B resistant transformants and also revealed random and single gene integration with genetic stability. Compared with wild-type strain, those transformants exhibited various differences in morphology, conidia production, and conidia germination ability. In addition, pathogenicity assays revealed that 14 transformants had mitigated pathogenicity, while one had enhanced infection ability. The results suggest that ATMT of V. mali is a useful tool to gain novel insight into this economically important pathogen at molecular levels.

  6. Enhancement of thermostability of kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis.

    Science.gov (United States)

    Yamamoto, Takuo; Mukai, Kazuhisa; Yamashita, Hiroshi; Kubota, Michio; Fukuda, Shigeharu; Kurimoto, Masashi; Tsujisaka, Yoshio

    2005-08-01

    Random mutation by error-prone PCR was introduced into kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5-70 degrees C (the wild type, 65 degrees C), and thermostability up to 67.5 degrees C (the wild type, up to 60 degrees C). The half-lives of D513N were estimated to be 135 h at 60 degrees C, 110 min at 70 degrees C and 6 min at 75 degrees C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.

  7. MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids.

    Science.gov (United States)

    Miyazaki, Kentaro

    2011-01-01

    MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. After synthesis of a nicked circular plasmid, the mixture is treated with DpnI, a dam-methylated DNA-specific restriction enzyme, to digest the template plasmid. The DpnI-treated mixture is then introduced into competent Escherichia coli cells to yield plasmids carrying replaced insert fragments. Plasmids produced by the MEGAWHOP method are virtually free of contamination by species without any inserts or with multiple inserts, and also the parent. Because the fragment is usually long enough to not interfere with hybridization to the template, various types of fragments can be used with mutations at any site (either known or unknown, random, or specific). By using fragments having homologous sequences at the ends (e.g., adaptor sequence), MEGAWHOP can also be used to recombine nonhomologous sequences mediated by the adaptors, allowing rapid creation of novel constructs and chimeric genes. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

    Science.gov (United States)

    Dror, Adi; Shemesh, Einav; Dayan, Natali

    2014-01-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

  9. Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis.

    Science.gov (United States)

    Gong, Bo-Liang; Mao, Run-Qian; Xiao, Yue; Jia, Mei-Lu; Zhong, Xiao-Lin; Liu, Yan; Xu, Pei-Lin; Li, Gang

    2017-11-01

    A new protease gene (pro1437)was separated from an oil-polluted Mud flat metagenomic library. Pro1437 belongs to a peptidase M48 superfamily according to the results of sequence analysis, and it showed very low identities compared to other known proteases or peptidases. The error-prone PCR was used to introduce random mutations and improve the expression of pro1437. After two rounds of mutagenesis and screening, a mutant (Pro2T21) with a 6.6-fold higher activity and a 4.8-fold higher expression level than Pro1437 was obtained. Sequence analysis found three amino acid substitutions (A54V, L192H, F224L) in Pro2T21. 3D structure modelling analysis indicated A54V and L192H probably played a crucial role in the improvement of enzymatic activity and soluble expression level of Pro2T21. Furthermore, Pro2T21opti displayed a 5.8-fold higher expression level than the wild type under optimal pH 8.0 at 50°C after codon-optimization. Also, Pro2T21opti represented robust compatibility with several popular laundry detergents, and blood stains on white cloth pieces were completely washed away when endogenous protease-inactivated Tide and Pro2T21opti were used together. Therefore, Pro2T21opti has great potential for use as an additive in detergents after further study. Copyright © 2017. Published by Elsevier Inc.

  10. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

    Directory of Open Access Journals (Sweden)

    Gaigalat Lars

    2006-08-01

    essential L-histidinol-phosphate phosphatase (EC 3.1.3.15 in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. Conclusion The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.

  11. Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

    Directory of Open Access Journals (Sweden)

    Gray Fiona C

    2009-08-01

    Full Text Available Abstract Background DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. Results To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. Conclusion In the absence of a three-dimensional structure of the entire Pol δ complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

  12. Massively parallel single-amino-acid mutagenesis.

    Science.gov (United States)

    Kitzman, Jacob O; Starita, Lea M; Lo, Russell S; Fields, Stanley; Shendure, Jay

    2015-03-01

    Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

  13. Massively Parallel Single Amino Acid Mutagenesis

    Science.gov (United States)

    Kitzman, Jacob O.; Starita, Lea M.; Lo, Russell S.; Fields, Stanley; Shendure, Jay

    2014-01-01

    Random mutagenesis methods only partially cover the mutational space, and are constrained by DNA synthesis length limitations. Here, we demonstrate PALS, a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing. PMID:25559584

  14. 2004 Mutagenesis Gordon Conference

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  15. The effective density of randomly moving electrons and related characteristics of materials with degenerate electron gas

    Directory of Open Access Journals (Sweden)

    V. Palenskis

    2014-04-01

    Full Text Available Interpretation of the conductivity of metals, of superconductors in the normal state and of semiconductors with highly degenerate electron gas remains a significant issue if consideration is based on the classical statistics. This study is addressed to the characterization of the effective density of randomly moving electrons and to the evaluation of carrier diffusion coefficient, mobility, and other parameters by generalization of the widely published experimental results. The generalized expressions have been derived for various kinetic parameters attributed to the non-degenerate and degenerate electron gas, by analyzing a random motion of the single type carriers in homogeneous materials. The values of the most important kinetic parameters for different metals are also systematized and discussed. It has been proved that Einstein's relation between the diffusion coefficient and the drift mobility of electrons is held for any level of degeneracy if the effective density of randomly moving carriers is properly taken into account.

  16. Classical mutagenesis in higher plants

    NARCIS (Netherlands)

    Koornneef, M.

    2002-01-01

    For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power

  17. Ethyl methanesulfonate mutagenesis in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Ekwall, Karl; Thon, Genevieve

    2017-01-01

    Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells.......Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells....

  18. Electronic properties of Fibonacci and random Si-Ge chains

    Energy Technology Data Exchange (ETDEWEB)

    Vasconcelos, M S [Escola de Ciencias e Tecnologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Azevedo, David L; Hadad, A [Departamento de Fisica, Universidade Federal do Maranhao 65080-040, Sao LuIs-MA (Brazil); Galvao, D S, E-mail: mvasconcelos@ect.ufrn.br [Instituto de Fisica Gleb Wataghin, Universidade Estadual de Campinas CP 6165, 13083-970 Campinas, SP (Brazil)

    2011-10-12

    In this paper we address a theoretical calculation of the electronic spectra of an Si-Ge atomic chain that is arranged in a Fibonacci quasi-periodic sequence, by using a semi-empirical quantum method based on the Hueckel extended model. We apply the Fibonacci substitutional sequences in the atomic building blocks A(Si) and B(Ge) through the inflation rule or a recursion relation. In our ab initio calculations we use only a single point, which is sufficient for considering all the orbitals and charge distribution across the entire system. Although the calculations presented here are more complete than the models adopted in the literature which take into account the electronic interaction only up to the second and third neighbors, an interesting property remains in their electronic spectra: the fractality (which is the main signature of this kind of system). We discuss this fractality of the spectra and we compare them with the random arrangement of the Si-Ge atomic chain, and with previous results based on the tight-binding approximation of the Schroedinger equation considering up to the nearest neighbor. (paper)

  19. Electron avalanche structure determined by random walk theory

    Science.gov (United States)

    Englert, G. W.

    1973-01-01

    A self-consistent avalanche solution which accounts for collective long range Coulomb interactions as well as short range elastic and inelastic collisions between electrons and background atoms is made possible by a random walk technique. Results show that the electric field patterns in the early formation stages of avalanches in helium are close to those obtained from theory based on constant transport coefficients. Regions of maximum and minimum induced electrostatic potential phi are located on the axis of symmetry and within the volume covered by the electron swarm. As formation time continues, however, the region of minimum phi moves to slightly higher radii and the electric field between the extrema becomes somewhat erratic. In the intermediate formation periods the avalanche growth is slightly retarded by the high concentration of ions in the tail which oppose the external electric field. Eventually the formation of ions and electrons in the localized regions of high field strength more than offset this effect causing a very abrupt increase in avalanche growth.

  20. Antimicrobials, stress and mutagenesis.

    Directory of Open Access Journals (Sweden)

    Alexandro Rodríguez-Rojas

    2014-10-01

    Full Text Available Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs mostly target the cell wall, a microbial 'Achilles heel', it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient 'weapons' of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the 'Achilles heel' has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.

  1. MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites

    National Research Council Canada - National Science Library

    Tang, Lixia; Wang, Xiong; Ru, Beibei; Sun, Hengfei; Huang, Jian; Gao, Hui

    2014-01-01

    ...) for the automated design of intelligent mutagenesis libraries that can completely cover user-defined randomized sequences, especially when multiple contiguous and/or adjacent sites are targeted...

  2. Classical mutagenesis in higher plants

    OpenAIRE

    Koornneef, M.

    2002-01-01

    For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power of mutant approaches in combination with molecular techniques to investigate the molecular nature of the genes became fully appreciated

  3. Modeling and simulation of electronic structure, material interface and random doping in nano electronic devices

    Science.gov (United States)

    Chen, Duan; Wei, Guo-Wei

    2010-01-01

    The miniaturization of nano-scale electronic devices, such as metal oxide semiconductor field effect transistors (MOSFETs), has given rise to a pressing demand in the new theoretical understanding and practical tactic for dealing with quantum mechanical effects in integrated circuits. Modeling and simulation of this class of problems have emerged as an important topic in applied and computational mathematics. This work presents mathematical models and computational algorithms for the simulation of nano-scale MOSFETs. We introduce a unified two-scale energy functional to describe the electrons and the continuum electrostatic potential of the nano-electronic device. This framework enables us to put microscopic and macroscopic descriptions in an equal footing at nano scale. By optimization of the energy functional, we derive consistently-coupled Poisson-Kohn-Sham equations. Additionally, layered structures are crucial to the electrostatic and transport properties of nano transistors. A material interface model is proposed for more accurate description of the electrostatics governed by the Poisson equation. Finally, a new individual dopant model that utilizes the Dirac delta function is proposed to understand the random doping effect in nano electronic devices. Two mathematical algorithms, the matched interface and boundary (MIB) method and the Dirichlet-to-Neumann mapping (DNM) technique, are introduced to improve the computational efficiency of nano-device simulations. Electronic structures are computed via subband decomposition and the transport properties, such as the I-V curves and electron density, are evaluated via the non-equilibrium Green's functions (NEGF) formalism. Two distinct device configurations, a double-gate MOSFET and a four-gate MOSFET, are considered in our three-dimensional numerical simulations. For these devices, the current fluctuation and voltage threshold lowering effect induced by the discrete dopant model are explored. Numerical convergence

  4. Promoter analysis by saturation mutagenesis

    Directory of Open Access Journals (Sweden)

    Baliga Nitin

    2001-01-01

    Full Text Available Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

  5. Novel electronic refreshers for cardiopulmonary resuscitation: a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Magura Stephen

    2012-11-01

    Full Text Available Abstract Background Currently the American Red Cross requires that individuals renew their cardiopulmonary resuscitation (CPR certification annually; this often requires a 4- to 8-hour refresher course. Those trained in CPR often show a decrease in essential knowledge and skills within just a few months after training. New electronic means of communication have expanded the possibilities for delivering CPR refreshers to members of the general public who receive CPR training. The study’s purpose was to determine the efficacy of three novel CPR refreshers - online website, e-mail and text messaging – for improving three outcomes of CPR training - skill retention, confidence for using CPR and intention to use CPR. These three refreshers may be considered “novel” in that they are not typically used to refresh CPR knowledge and skills. Methods The study conducted two randomized clinical trials of the novel CPR refreshers. A mailed brochure was a traditional, passive refresher format and served as the control condition. In Trial 1, the refreshers were delivered in a single episode at 6 months after initial CPR training. In Trial 2, the refreshers were delivered twice, at 6 and 9 months after initial CPR training, to test the effect of a repeated delivery. Outcomes for the three novel refreshers vs. the mailed brochure were determined at 12 months after initial CPR training. Results Assignment to any of three novel refreshers did not improve outcomes of CPR training one year later in comparison with receiving a mailed brochure. Comparing outcomes for subjects who actually reviewed some of the novel refreshers vs. those who did not indicated a significant positive effect for one outcome, confidence for performing CPR. The website refresher was associated with increased behavioral intent to perform CPR. Stated satisfaction with the refreshers was relatively high. The number of episodes of refreshers (one vs. two did not have a significant effect

  6. Long-distance effects of insertional mutagenesis.

    Directory of Open Access Journals (Sweden)

    Ruchi Singhal

    2011-01-01

    Full Text Available Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance.We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected.We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation.

  7. A Mutant Mouse with a Highly Specific Contextual Fear-Conditioning Deficit Found in an N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Screen

    Science.gov (United States)

    Pletcher, Mathew T.; Wiltshire, Tim; Tarantino, Lisa M.; Mayford, Mark; Reijmers, Leon G.; Coats, Jennifer K.

    2006-01-01

    Targeted mutagenesis in mice has shown that genes from a wide variety of gene families are involved in memory formation. The efficient identification of genes involved in learning and memory could be achieved by random mutagenesis combined with high-throughput phenotyping. Here, we provide the first report of a mutagenesis screen that has…

  8. Forward and reverse mutagenesis in C. elegans.

    Science.gov (United States)

    Kutscher, Lena M; Shaham, Shai

    2014-01-17

    Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal.

  9. Cluster randomized trials utilizing primary care electronic health records : methodological issues in design, conduct, and analysis (eCRT Study)

    NARCIS (Netherlands)

    Gulliford, Martin C; van Staa, Tjeerd P; McDermott, Lisa; McCann, Gerard; Charlton, Judith; Dregan, Alex

    2014-01-01

    BACKGROUND: There is growing interest in conducting clinical and cluster randomized trials through electronic health records. This paper reports on the methodological issues identified during the implementation of two cluster randomized trials using the electronic health records of the Clinical

  10. Random walk study of electron motion in helium in crossed electromagnetic fields

    Science.gov (United States)

    Englert, G. W.

    1972-01-01

    Random walk theory, previously adapted to electron motion in the presence of an electric field, is extended to include a transverse magnetic field. In principle, the random walk approach avoids mathematical complexity and concomitant simplifying assumptions and permits determination of energy distributions and transport coefficients within the accuracy of available collisional cross section data. Application is made to a weakly ionized helium gas. Time of relaxation of electron energy distribution, determined by the random walk, is described by simple expressions based on energy exchange between the electron and an effective electric field. The restrictive effect of the magnetic field on electron motion, which increases the required number of collisions per walk to reach a terminal steady state condition, as well as the effect of the magnetic field on electron transport coefficients and mean energy can be quite adequately described by expressions involving only the Hall parameter.

  11. Electronic Properties of Random Polymers: Modelling Optical Spectra of Melanins

    Science.gov (United States)

    Bochenek, Kinga; Gudowska-Nowak, Ewa

    2003-05-01

    Melanins are a group of complex pigments of biological origin, widely spread in all species from fungi to man. Among diverse types of melanins, the human melanins, eumelanins, are brown or black nitrogen-containing pigments, mostly known for their photoprotective properties in human skin. We have undertaken theoretical studies aimed to understand absorption spectra of eumelanins and their chemical precursors. The structure of the biopigment is poorly defined, although it is believed to be composed of cross-linked heteropolymers based on indolequinones. As a basic model of the eumelanin structure, we have chosen pentamers containing hydroquinones (HQ) and/or 5,6-indolequinones (IQ) and/or semiquinones (SQ) often listed as structural melanin monomers. The eumelanin oligomers have been constructed as random compositions of basic monomers and optimized for the energy of bonding. Absorption spectra of model assemblies have been calculated within the semiempirical intermediate neglect of differential overlap (INDO) approximation. Model spectrum of eumelanin has been further obtained by sum of independent spectra of singular polymers. By comparison with experimental data it is shown that the INDO/CI method manages to reproduce well characteristic properties of experimental spectrum of synthetic eumelanins.

  12. Deep Learning the Quantum Phase Transitions in Random Two-Dimensional Electron Systems

    Science.gov (United States)

    Ohtsuki, Tomoki; Ohtsuki, Tomi

    2016-12-01

    Random electron systems show rich phases such as Anderson insulator, diffusive metal, quantum Hall and quantum anomalous Hall insulators, Weyl semimetal, as well as strong/weak topological insulators. Eigenfunctions of each matter phase have specific features, but owing to the random nature of systems, determining the matter phase from eigenfunctions is difficult. Here, we propose the deep learning algorithm to capture the features of eigenfunctions. Localization-delocalization transition, as well as disordered Chern insulator-Anderson insulator transition, is discussed.

  13. Genome-wide transposon mutagenesis in Saccharomyces cerevisiae and Candida albicans.

    Science.gov (United States)

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2011-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds.

  14. Quantum tunneling recombination in a system of randomly distributed trapped electrons and positive ions.

    Science.gov (United States)

    Pagonis, Vasilis; Kulp, Christopher; Chaney, Charity-Grace; Tachiya, M

    2017-09-13

    During the past 10 years, quantum tunneling has been established as one of the dominant mechanisms for recombination in random distributions of electrons and positive ions, and in many dosimetric materials. Specifically quantum tunneling has been shown to be closely associated with two important effects in luminescence materials, namely long term afterglow luminescence and anomalous fading. Two of the common assumptions of quantum tunneling models based on random distributions of electrons and positive ions are: (a) An electron tunnels from a donor to the nearest acceptor, and (b) the concentration of electrons is much lower than that of positive ions at all times during the tunneling process. This paper presents theoretical studies for arbitrary relative concentrations of electrons and positive ions in the solid. Two new differential equations are derived which describe the loss of charge in the solid by tunneling, and they are solved analytically. The analytical solution compares well with the results of Monte Carlo simulations carried out in a random distribution of electrons and positive ions. Possible experimental implications of the model are discussed for tunneling phenomena in long term afterglow signals, and also for anomalous fading studies in feldspars and apatite samples.

  15. Point-of-care cluster randomized trial in stroke secondary prevention using electronic health records

    NARCIS (Netherlands)

    Dregan, Alex; van Staa, Tjeerd P; McDermott, Lisa; McCann, Gerard; Ashworth, Mark; Charlton, Judith; Wolfe, Charles D A; Rudd, Anthony; Yardley, Lucy; Gulliford, Martin C

    BACKGROUND AND PURPOSE: The aim of this study was to evaluate whether the remote introduction of electronic decision support tools into family practices improves risk factor control after first stroke. This study also aimed to develop methods to implement cluster randomized trials in stroke using

  16. Magnetoresistance of a two-dimensional electron gas in a random magnetic field

    DEFF Research Database (Denmark)

    Smith, Anders; Taboryski, Rafael Jozef; Hansen, Luise Theil

    1994-01-01

    We report magnetoresistance measurements on a two-dimensional electron gas made from a high-mobility GaAs/AlxGa1-xAs heterostructure, where the externally applied magnetic field was expelled from regions of the semiconductor by means of superconducting lead grains randomly distributed...

  17. Transport properties of a two-dimensional electron gas due to a spatially random magnetic field

    Science.gov (United States)

    Rushforth, A. W.; Gallagher, B. L.; Main, P. C.; Neumann, A. C.; Marrows, C. H.; Zoller, I.; Howson, M. A.; Hickey, B. J.; Henini, M.

    2000-02-01

    We have studied the magnetoresistance of a near-surface two-dimensional electron gas (2DEG) in the presence of a random magnetic field produced by CoPd multilayers deposited onto the surface of the heterostructure. This novel method allows us to switch the random field on and off by applying an external magnetic field and also to control the amplitude and correlation length of the random field by varying the growth parameters of the multilayers. The presence of the random field is confirmed by quenching of the Shubnikov-de Haas oscillations and we see an enhanced magnetoresistance which can be interpreted semi-classically. We also observe other unusual features which may be quantum in origin.

  18. Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

    Science.gov (United States)

    Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

    2017-01-01

    Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

  19. A cluster randomized trial evaluating electronic prescribing in an ambulatory care setting

    Directory of Open Access Journals (Sweden)

    Quan Sherman

    2007-10-01

    Full Text Available Abstract Background Medication errors, adverse drug events and potential adverse drug events are common and serious in terms of the harms and costs that they impose on the health system and those who use it. Errors resulting in preventable adverse drug events have been shown to occur most often at the stages of ordering and administration. This paper describes the protocol for a pragmatic trial of electronic prescribing to reduce prescription error. The trial was designed to overcome the limitations associated with traditional study design. Design This study was designed as a 65-week, cluster randomized, parallel study. Methods The trial was conducted within ambulatory outpatient clinics in an academic tertiary care centre in Ontario, Canada. The electronic prescribing software for the study is a Canadian electronic prescribing software package which provides physician prescription entry with decision support at the point of care. Using a handheld computer (PDA the physician selects medications using an error minimising menu-based pick list from a comprehensive drug database, create specific prescription instructions and then transmit the prescription directly and electronically to a participating pharmacy via facsimile or to the physician's printer using local area wireless technology. The unit of allocation and randomization is by 'week', i.e. the system is "on" or "off" according to the randomization scheme and the unit of analysis is the prescription, with adjustment for clustering of patients within practitioners. Discussion This paper describes the protocol for a pragmatic cluster randomized trial of point-of-care electronic prescribing, which was specifically designed to overcome the limitations associated with traditional study design. Trial Registration This trial has been registered with clinicaltrials.gov (ID: NCT00252395

  20. Randomized trial of an electronic asthma monitoring system among New York City children.

    Science.gov (United States)

    Jacobson, Judith S; Lieblein, Andrea; Fierman, Arthur H; Fishkin, Edward R; Hutchinson, Vincent E; Rodriguez, Luis; Serebrisky, Denise; Chau, Michelle; Saperstein, Arnold

    2009-11-01

    To test the efficacy of an electronic asthma monitoring system (AMS) to reduce pediatric emergency department (ED) visits and hospitalizations for asthma. Randomized clinical trial. Families of pediatric patients with asthma aged 8 to 17 years were recruited at 6 medical centers. Children were randomly assigned to the American Medical Alert Corporation pediatric AMS or a paper diary. The numbers of and costs associated with ED visits and hospitalizations for the 2 groups in the year following randomization were compared using t tests of statistical significance. Of 59 children recruited to the trial, 29 were randomized to the AMS and 30 to the diary. The 2 groups were similar in demographic and clinical characteristics. During their study year, 24 AMS group members logged on a mean (SD) of 211.0 (117.3) days; 13 diary group members provided data on a mean (SD) of 136.6 (128.0) days. During the 32 months that the study was in progress, the case managers logged on a mean (SD) of 171.0 (97.2) days. Overall, 35 children had at least 1 ED visit, but only 7 children were hospitalized. The 2 groups had no statistically significant differences in the numbers of or charges associated with ED visits or hospitalizations. Electronic devices are being developed to make chronic disease management easier for patients and their families, but they should not be adopted without careful study, including randomized trials, to ascertain their use, costs, and benefits.

  1. An electronic documentation system improves the quality of admission notes: a randomized trial.

    Science.gov (United States)

    Jamieson, Trevor; Ailon, Jonathan; Chien, Vince; Mourad, Ophyr

    2017-01-01

    There are concerns that structured electronic documentation systems can limit expressivity and encourage long and unreadable notes. We assessed the impact of an electronic clinical documentation system on the quality of admission notes for patients admitted to a general medical unit. This was a prospective randomized crossover study comparing handwritten paper notes to electronic notes on different patients by the same author, generated using a semistructured electronic admission documentation system over a 2-month period in 2014. The setting was a 4-team, 80-bed general internal medicine clinical teaching unit at a large urban academic hospital. The quality of clinical documentation was assessed using the QNOTE instrument (best possible score = 100), and word counts were assessed for free-text sections of notes. Twenty-one electronic-paper note pairs (42 notes) written by 21 authors were randomly drawn from a pool of 303 eligible notes. Overall note quality was significantly higher in electronic vs paper notes (mean 90 vs 69, P quality of free-text subsections (History of Present Illness and Impression and Plan) was significantly higher in the electronic vs paper notes (mean 93 vs 78, P system improved both the quality of free-text content and the overall quality of admission notes. Authors wrote more in the free-text sections of electronic documents as compared to paper versions. © The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Electronic transport in a one-dimensional random array of scatterers

    CERN Document Server

    Filinov, V S; Varga, I; Meier, T; Bonitz, M; Fortov, V E; Koch, S W

    2003-01-01

    The quantum dynamics of an ensemble of interacting electrons in an array of random scatterers is treated using a new numerical approach for the calculation of average values of quantum operators and time correlation functions in the Wigner representation. This approach combines both molecular dynamics and Monte Carlo methods and computes numerical traces and spectra of the relevant dynamical quantities such as momentum-momentum correlation functions and spatial dispersions. Considering, as an application, a system with fixed scatterers, the results clearly demonstrate that the many-particle interaction between the electrons can lead to an enhancement of the conductivity at intermediate densities.

  3. Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers.

    Science.gov (United States)

    Tang, Lixia; Gao, Hui; Zhu, Xuechen; Wang, Xiong; Zhou, Ming; Jiang, Rongxiang

    2012-03-01

    Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel "small-intelligent" strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

  4. Metal-insulator transition of 2d electron gas in a random magnetic field

    CERN Document Server

    Wang, X R; Liu, D Z

    1999-01-01

    We study the metal-insulator transition of a two-dimensional electron gas in the presence of a random magnetic field from the localization property. The localization length is directly calculated using a transfer matrix technique and finite size scaling analysis. We argue that there is a metal-insulator transition in such a system and show strong numerical evidence that the system undergoes a disorder driven Kosterlitz-Thouless type metal-insulator transition. We will also discuss a mean field theory which maps the random field system into a two-dimensional XY-model. The vortex and antivortex excitations in the XY-model correspond to two different kinds of magnetic domains in the random field system.

  5. Effect of random vacancies on the electronic properties of graphene and T graphene: a theoretical approach

    Science.gov (United States)

    Sadhukhan, B.; Nayak, A.; Mookerjee, A.

    2017-12-01

    In this communication we present together four distinct techniques for the study of electronic structure of solids: the tight-binding linear muffin-tin orbitals, the real space and augmented space recursions and the modified exchange-correlation. Using this we investigate the effect of random vacancies on the electronic properties of the carbon hexagonal allotrope, graphene, and the non-hexagonal allotrope, planar T graphene. We have inserted random vacancies at different concentrations, to simulate disorder in pristine graphene and planar T graphene sheets. The resulting disorder, both on-site (diagonal disorder) as well as in the hopping integrals (off-diagonal disorder), introduces sharp peaks in the vicinity of the Dirac point built up from localized states for both hexagonal and non-hexagonal structures. These peaks become resonances with increasing vacancy concentration. We find that in presence of vacancies, graphene-like linear dispersion appears in planar T graphene and the cross points form a loop in the first Brillouin zone similar to buckled T graphene that originates from π and π* bands without regular hexagonal symmetry. We also calculate the single-particle relaxation time, τ (ěc {q}) of ěc {q} labeled quantum electronic states which originates from scattering due to presence of vacancies, causing quantum level broadening.

  6. Correlations in two-dimensional electron gas: Random-phase approximation with exchange and ladder results

    Science.gov (United States)

    Pederiva, F.; Lipparini, E.; Takayanagi, K.

    1997-12-01

    We have evaluated the density-density response of the two-dimensional electron gas at zero temperature by solving the Dyson equation for the particle-hole Green's function, including exchange Coulomb matrix elements and short-range contributions in the ladder approximation. We study the effect of these correlations on the total energy, compressibility per particle, local field factor G(q), static structure factor and pair-correlation function. Results are compared with the normal random-phase approximation, local field theories and quantum Monte Carlo calculations.

  7. Deducing fast electron density changes in randomly orientated uncrystallized biomolecules in a pump-probe experiment.

    Science.gov (United States)

    Pande, K; Schwander, P; Schmidt, M; Saldin, D K

    2014-07-17

    We propose a method for deducing time-resolved structural changes in uncrystallized biomolecules in solution. The method relies on measuring the angular correlations of the intensities, when averaged over a large number of diffraction patterns from randomly oriented biomolecules in solution in a liquid solvent. The experiment is somewhat like a pump-probe version of an experiment on small angle X-ray scattering, except that the data expected by the algorithm are not just the radial variation of the averaged intensities. The differences of these correlation functions as measured from a photoexcited and dark structure enable the direct calculation of the difference electron density with a knowledge of only the dark structure. We exploit a linear relation we derive between the difference in these correlation functions and the difference electron density, applicable for small structural changes.

  8. Point-of-care cluster randomized trial in stroke secondary prevention using electronic health records.

    Science.gov (United States)

    Dregan, Alex; van Staa, Tjeerd P; McDermott, Lisa; McCann, Gerard; Ashworth, Mark; Charlton, Judith; Wolfe, Charles D A; Rudd, Anthony; Yardley, Lucy; Gulliford, Martin C; Trial Steering Committee

    2014-07-01

    The aim of this study was to evaluate whether the remote introduction of electronic decision support tools into family practices improves risk factor control after first stroke. This study also aimed to develop methods to implement cluster randomized trials in stroke using electronic health records. Family practices were recruited from the UK Clinical Practice Research Datalink and allocated to intervention and control trial arms by minimization. Remotely installed, electronic decision support tools promoted intensified secondary prevention for 12 months with last measure of systolic blood pressure as the primary outcome. Outcome data from electronic health records were analyzed using marginal models. There were 106 Clinical Practice Research Datalink family practices allocated (intervention, 53; control, 53), with 11 391 (control, 5516; intervention, 5875) participants with acute stroke ever diagnosed. Participants at trial practices had similar characteristics as 47,887 patients with stroke at nontrial practices. During the intervention period, blood pressure values were recorded in the electronic health records for 90% and cholesterol values for 84% of participants. After intervention, the latest mean systolic blood pressure was 131.7 (SD, 16.8) mm Hg in the control trial arm and 131.4 (16.7) mm Hg in the intervention trial arm, and adjusted mean difference was -0.56 mm Hg (95% confidence interval, -1.38 to 0.26; P=0.183). The financial cost of the trial was approximately US $22 per participant, or US $2400 per family practice allocated. Large pragmatic intervention studies may be implemented at low cost by using electronic health records. The intervention used in this trial was not found to be effective, and further research is needed to develop more effective intervention strategies. http://www.controlled-trials.com. Current Controlled Trials identifier: ISRCTN35701810. © 2014 American Heart Association, Inc.

  9. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

    National Research Council Canada - National Science Library

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    .... Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins...

  10. Mariner-based transposon mutagenesis for Bacteroides species.

    Science.gov (United States)

    Ichimura, Minoru; Uchida, Keiko; Nakayama-Imaohji, Haruyuki; Hirakawa, Hideki; Tada, Tomoyo; Morita, Hidetoshi; Yasutomo, Koji; Okazaki, Katsuichiro; Kuwahara, Tomomi

    2014-06-01

    Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Electronic laboratory system reduces errors in National Tuberculosis Program: a cluster randomized controlled trial.

    Science.gov (United States)

    Blaya, J A; Shin, S S; Yale, G; Suarez, C; Asencios, L; Contreras, C; Rodriguez, P; Kim, J; Cegielski, P; Fraser, H S F

    2010-08-01

    To evaluate the impact of the e-Chasqui laboratory information system in reducing reporting errors compared to the current paper system. Cluster randomized controlled trial in 76 health centers (HCs) between 2004 and 2008. Baseline data were collected every 4 months for 12 months. HCs were then randomly assigned to intervention (e-Chasqui) or control (paper). Further data were collected for the same months the following year. Comparisons were made between intervention and control HCs, and before and after the intervention. Intervention HCs had respectively 82% and 87% fewer errors in reporting results for drug susceptibility tests (2.1% vs. 11.9%, P = 0.001, OR 0.17, 95%CI 0.09-0.31) and cultures (2.0% vs. 15.1%, P Chasqui users sent on average three electronic error reports per week to the laboratories. e-Chasqui reduced the number of missing laboratory results at point-of-care health centers. Clinical users confirmed viewing electronic results not available on paper. Reporting errors to the laboratory using e-Chasqui promoted continuous quality improvement. The e-Chasqui laboratory information system is an important part of laboratory infrastructure improvements to support multidrug-resistant tuberculosis care in Peru.

  12. Characterization of Electron Microscopes with Binary Pseudo-random Multilayer Test Samples

    Energy Technology Data Exchange (ETDEWEB)

    V Yashchuk; R Conley; E Anderson; S Barber; N Bouet; W McKinney; P Takacs; D Voronov

    2011-12-31

    Verification of the reliability of metrology data from high quality X-ray optics requires that adequate methods for test and calibration of the instruments be developed. For such verification for optical surface profilometers in the spatial frequency domain, a modulation transfer function (MTF) calibration method based on binary pseudo-random (BPR) gratings and arrays has been suggested [1] and [2] and proven to be an effective calibration method for a number of interferometric microscopes, a phase shifting Fizeau interferometer, and a scatterometer [5]. Here we describe the details of development of binary pseudo-random multilayer (BPRML) test samples suitable for characterization of scanning (SEM) and transmission (TEM) electron microscopes. We discuss the results of TEM measurements with the BPRML test samples fabricated from a WiSi2/Si multilayer coating with pseudo-randomly distributed layers. In particular, we demonstrate that significant information about the metrological reliability of the TEM measurements can be extracted even when the fundamental frequency of the BPRML sample is smaller than the Nyquist frequency of the measurements. The measurements demonstrate a number of problems related to the interpretation of the SEM and TEM data. Note that similar BPRML test samples can be used to characterize X-ray microscopes. Corresponding work with X-ray microscopes is in progress.

  13. Characterization of electron microscopes with binary pseudo-random multilayer test samples

    Energy Technology Data Exchange (ETDEWEB)

    Yashchuk, Valeriy V., E-mail: VVYashchuk@lbl.gov [Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Conley, Raymond [NSLS-II, Brookhaven National Laboratory, Upton, NY 11973 (United States); Anderson, Erik H. [Center for X-ray Optics, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Barber, Samuel K. [Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Bouet, Nathalie [NSLS-II, Brookhaven National Laboratory, Upton, NY 11973 (United States); McKinney, Wayne R. [Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Takacs, Peter Z. [Brookhaven National Laboratory, Upton, NY 11973 (United States); Voronov, Dmitriy L. [Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)

    2011-09-01

    Verification of the reliability of metrology data from high quality X-ray optics requires that adequate methods for test and calibration of the instruments be developed. For such verification for optical surface profilometers in the spatial frequency domain, a modulation transfer function (MTF) calibration method based on binary pseudo-random (BPR) gratings and arrays has been suggested and proven to be an effective calibration method for a number of interferometric microscopes, a phase shifting Fizeau interferometer, and a scatterometer [5]. Here we describe the details of development of binary pseudo-random multilayer (BPRML) test samples suitable for characterization of scanning (SEM) and transmission (TEM) electron microscopes. We discuss the results of TEM measurements with the BPRML test samples fabricated from a WiSi{sub 2}/Si multilayer coating with pseudo-randomly distributed layers. In particular, we demonstrate that significant information about the metrological reliability of the TEM measurements can be extracted even when the fundamental frequency of the BPRML sample is smaller than the Nyquist frequency of the measurements. The measurements demonstrate a number of problems related to the interpretation of the SEM and TEM data. Note that similar BPRML test samples can be used to characterize X-ray microscopes. Corresponding work with X-ray microscopes is in progress.

  14. Identification of a potential fibromyalgia diagnosis using random forest modeling applied to electronic medical records

    Directory of Open Access Journals (Sweden)

    Emir B

    2015-06-01

    Full Text Available Birol Emir,1 Elizabeth T Masters,1 Jack Mardekian,1 Andrew Clair,1 Max Kuhn,2 Stuart L Silverman,3 1Pfizer Inc., New York, NY, 2Pfizer Inc., Groton, CT, 3Cedars-Sinai Medical Center, Los Angeles, CA, USA Background: Diagnosis of fibromyalgia (FM, a chronic musculoskeletal condition characterized by widespread pain and a constellation of symptoms, remains challenging and is often delayed. Methods: Random forest modeling of electronic medical records was used to identify variables that may facilitate earlier FM identification and diagnosis. Subjects aged ≥18 years with two or more listings of the International Classification of Diseases, Ninth Revision, (ICD-9 code for FM (ICD-9 729.1 ≥30 days apart during the 2012 calendar year were defined as cases among subjects associated with an integrated delivery network and who had one or more health care provider encounter in the Humedica database in calendar years 2011 and 2012. Controls were without the FM ICD-9 codes. Seventy-two demographic, clinical, and health care resource utilization variables were entered into a random forest model with downsampling to account for cohort imbalances (<1% subjects had FM. Importance of the top ten variables was ranked based on normalization to 100% for the variable with the largest loss in predicting performance by its omission from the model. Since random forest is a complex prediction method, a set of simple rules was derived to help understand what factors drive individual predictions. Results: The ten variables identified by the model were: number of visits where laboratory/non-imaging diagnostic tests were ordered; number of outpatient visits excluding office visits; age; number of office visits; number of opioid prescriptions; number of medications prescribed; number of pain medications excluding opioids; number of medications administered/ordered; number of emergency room visits; and number of musculoskeletal conditions. A receiver operating

  15. New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

    Science.gov (United States)

    Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

    2013-11-13

    In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING

  16. A transdisciplinary model to inform randomized clinical trial methods for electronic cigarette evaluation

    Directory of Open Access Journals (Sweden)

    Alexa A. Lopez

    2016-03-01

    Full Text Available Abstract Background This study is a systematic evaluation of a novel tobacco product, electronic cigarettes (ECIGs using a two-site, four-arm, 6-month, parallel-group randomized controlled trial (RCT with a follow-up to 9 months. Virginia Commonwealth University is the primary site and Penn State University is the secondary site. This RCT design is important because it is informed by analytical work, clinical laboratory results, and qualitative/quantitative findings regarding the specific ECIG products used. Methods Participants (N = 520 will be randomized across sites and must be healthy smokers of >9 cigarettes for at least one year, who have not had a quit attempt in the prior month, are not planning to quit in the next 6 months, and are interested in reducing cigarette intake. Participants will be randomized into one of four 24-week conditions: a cigarette substitute that does not produce an inhalable aerosol; or one of three ECIG conditions that differ by nicotine concentration 0, 8, or 36 mg/ml. Blocked randomization will be accomplished with a 1:1:1:1 ratio of condition assignments at each site. Specific aims are to: characterize ECIG influence on toxicants, biomarkers, health indicators, and disease risk; determine tobacco abstinence symptom and adverse event profile associated with real-world ECIG use; and examine the influence of ECIG use on conventional tobacco product use. Liquid nicotine concentration-related differences on these study outcomes are predicted. Participants and research staff in contact with participants will be blinded to the nicotine concentration in the ECIG conditions. Discussion Results from this study will inform knowledge concerning ECIG use as well as demonstrate a model that may be applied to other novel tobacco products. The model of using prior empirical testing of ECIG devices should be considered in other RCT evaluations. Trial registration TRN: NCT02342795 , registered December 16, 2014.

  17. Deep Learning the Quantum Phase Transitions in Random Electron Systems: Applications to Three Dimensions

    Science.gov (United States)

    Ohtsuki, Tomi; Ohtsuki, Tomoki

    2017-04-01

    Three-dimensional random electron systems undergo quantum phase transitions and show rich phase diagrams. Examples of the phases are the band gap insulator, Anderson insulator, strong and weak topological insulators, Weyl semimetal, and diffusive metal. As in the previous paper on two-dimensional quantum phase transitions [J. Phys. Soc. Jpn. 85, 123706 (2016)], we use an image recognition algorithm based on a multilayered convolutional neural network to identify which phase the eigenfunction belongs to. The Anderson model for localization-delocalization transition, the Wilson-Dirac model for topological insulators, and the layered Chern insulator model for Weyl semimetal are studied. The situation where the standard transfer matrix approach is not applicable is also treated by this method.

  18. Handheld vs. laptop computers for electronic data collection in clinical research: a crossover randomized trial.

    Science.gov (United States)

    Haller, Guy; Haller, Dagmar M; Courvoisier, Delphine S; Lovis, Christian

    2009-01-01

    To compare users' speed, number of entry errors and satisfaction in using two current devices for electronic data collection in clinical research: handheld and laptop computers. The authors performed a randomized cross-over trial using 160 different paper-based questionnaires and representing altogether 45,440 variables. Four data coders were instructed to record, according to a random predefined and equally balanced sequence, the content of these questionnaires either on a laptop or on a handheld computer. Instructions on the kind of device to be used were provided to data-coders in individual sealed and opaque envelopes. Study conditions were controlled and the data entry process performed in a quiet environment. The authors compared the duration of the data recording process, the number of errors and users' satisfaction with the two devices. The authors divided errors into two separate categories, typing and missing data errors. The original paper-based questionnaire was used as a gold-standard. The overall duration of the recording process was significantly reduced (2.0 versus 3.3 min) when data were recorded on the laptop computer (p laptop compared to 8.4 per 1,000 with the handheld computer (p laptop was used (p laptop easier, faster and more satisfying to use than the handheld computer. Despite the increasing use of handheld computers for electronic data collection in clinical research, these devices should be used with caution. They double the duration of the data entry process and significantly increase the risk of typing errors and missing data. This may become a particularly crucial issue in studies where these devices are provided to patients or healthcare workers, unfamiliar with computer technologies, for self-reporting or research data collection processes.

  19. Improving search over Electronic Health Records using UMLS-based query expansion through random walks.

    Science.gov (United States)

    Martinez, David; Otegi, Arantxa; Soroa, Aitor; Agirre, Eneko

    2014-10-01

    Most of the information in Electronic Health Records (EHRs) is represented in free textual form. Practitioners searching EHRs need to phrase their queries carefully, as the record might use synonyms or other related words. In this paper we show that an automatic query expansion method based on the Unified Medicine Language System (UMLS) Metathesaurus improves the results of a robust baseline when searching EHRs. The method uses a graph representation of the lexical units, concepts and relations in the UMLS Metathesaurus. It is based on random walks over the graph, which start on the query terms. Random walks are a well-studied discipline in both Web and Knowledge Base datasets. Our experiments over the TREC Medical Record track show improvements in both the 2011 and 2012 datasets over a strong baseline. Our analysis shows that the success of our method is due to the automatic expansion of the query with extra terms, even when they are not directly related in the UMLS Metathesaurus. The terms added in the expansion go beyond simple synonyms, and also add other kinds of topically related terms. Expansion of queries using related terms in the UMLS Metathesaurus beyond synonymy is an effective way to overcome the gap between query and document vocabularies when searching for patient cohorts. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Pilot study of large-scale production of mutant pigs by ENU mutagenesis

    Science.gov (United States)

    Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

    2017-01-01

    N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research. DOI: http://dx.doi.org/10.7554/eLife.26248.001 PMID:28639938

  1. Perspective on mutagenesis and repair: the standard model and alternate modes of mutagenesis.

    Science.gov (United States)

    Miller, Jeffrey H

    2005-01-01

    The basic ideas of replication, mutagenesis, and repair have outlined a picture of how point mutations occur that has provided a valuable framework for theory and experiment, much as the Standard Model of particle physics has done for our concept of fundamental particles. However, alternative modes of mutagenesis are being defined that are changing our perspective of the "Standard Model" of mutagenesis, requiring an expanded model. The genome is now envisioned as being in dynamic equilibrium between a multitude of forces for mutational change and forces that counteract such change. By maintaining a delicate balance between these forces, cells avoid unwanted or excessive mutations. Yet, cells allow mutagenesis to occur under certain conditions. We can define an emerging paradigm. Namely, mechanisms exist that can direct point mutations to specific designated genes or regions of genes. In some cases, this is achieved by specific enzymes, and in other cases high mutability is programmed into the sequence of certain genes to help generate diversity. In yet additional cases, general mutability is increased under stress, and selective forces allow the recovery of favorable mutants.

  2. Dynamic response of a two-dimensional electron gas: Exact treatment of Coulomb exchange in the random-phase approximation

    Science.gov (United States)

    Takayanagi, K.; Lipparini, E.

    1995-07-01

    The Dyson equation for the particle-hole Green's function, including Coulomb exchange matrix elements, has been solved exactly for a two-dimensional electron gas. Static and dynamic dielectric functions have been calculated and compared with normal random-phase-approximation and recent quantum Monte Carlo results.

  3. Single particle electron microscopy reconstruction of the exosome complex using the random conical tilt method.

    Science.gov (United States)

    Liu, Xueqi; Wang, Hong-Wei

    2011-03-28

    of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution(1) and random conical tilt (RCT) method(2). In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method(3).

  4. Efficient multi-site-directed mutagenesis directly from genomic ...

    Indian Academy of Sciences (India)

    In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well ...

  5. Transposon Tn5 mutagenesis of pseudomonas fluorescens to isolate mutants deficient in antibacterial activity.

    Science.gov (United States)

    Rajendran, N; Jahn, D; Jayaraman, K; Marahiel, M A

    1994-01-15

    Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn5-GM into the chromosome of P. fluorescens was achieved by heat treatment of the transformed cells at 42 degrees C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens. One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis. These results introduce Tn5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens.

  6. Prediction of enzyme mutant activity using computational mutagenesis and incremental transduction.

    Science.gov (United States)

    Basit, Nada; Wechsler, Harry

    2011-01-01

    Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1). This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets.

  7. Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction

    Directory of Open Access Journals (Sweden)

    Nada Basit

    2011-01-01

    Full Text Available Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1. This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets.

  8. Scoring function to predict solubility mutagenesis

    Directory of Open Access Journals (Sweden)

    Deutsch Christopher

    2010-10-01

    Full Text Available Abstract Background Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to mutations. The scoring function captures both sequence and structure information. By exploring the literature, we have assembled a substantial database of 137 single- and multiple-point solubility mutations. Our database is the largest such collection with structural information known so far. We optimize the scoring function using linear programming (LP methods to derive its weights based on training. Starting with default values of 1, we find weights in the range [0,2] so that predictions of increase or decrease in solubility are optimized. We compare the LP method to the standard machine learning techniques of support vector machines (SVM and the Lasso. Using statistics for leave-one-out (LOO, 10-fold, and 3-fold cross validations (CV for training and prediction, we demonstrate that the LP method performs the best overall. For the LOOCV, the LP method has an overall accuracy of 81%. Availability Executables of programs, tables of weights, and datasets of mutants are available from the following web page: http://www.wsu.edu/~kbala/OptSolMut.html.

  9. A plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteria

    Directory of Open Access Journals (Sweden)

    Rita eMonson

    2015-12-01

    Full Text Available Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

  10. Integrability of the diffusion pole in the diagrammatic description of noninteracting electrons in a random potential

    OpenAIRE

    Janis, V.

    2009-01-01

    We discuss restrictions on the existence of the diffusion pole in the translationally invariant diagrammatic treatment of disordered electron systems. We use the Bethe-Salpeter equations for the two-particle vertex in the electron-hole and the electron-electron scattering channels and derive for systems with time reversal symmetry a nonlinear integral equation the two-particle irreducible vertices from both channels must obey. We use this equation to test the existence of the diffusion pole i...

  11. High-speed true random number generation based on paired memristors for security electronics

    Science.gov (United States)

    Zhang, Teng; Yin, Minghui; Xu, Changmin; Lu, Xiayan; Sun, Xinhao; Yang, Yuchao; Huang, Ru

    2017-11-01

    True random number generator (TRNG) is a critical component in hardware security that is increasingly important in the era of mobile computing and internet of things. Here we demonstrate a TRNG using intrinsic variation of memristors as a natural source of entropy that is otherwise undesirable in most applications. The random bits were produced by cyclically switching a pair of tantalum oxide based memristors and comparing their resistance values in the off state, taking advantage of the more pronounced resistance variation compared with that in the on state. Using an alternating read scheme in the designed TRNG circuit, the unbiasedness of the random numbers was significantly improved, and the bitstream passed standard randomness tests. The Pt/TaO x /Ta memristors fabricated in this work have fast programming/erasing speeds of ∼30 ns, suggesting a high random number throughput. The approach proposed here thus holds great promise for physically-implemented random number generation.

  12. Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

    Directory of Open Access Journals (Sweden)

    Laila Berg

    2013-01-01

    Full Text Available Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the promoter DNA sequence. We demonstrate that this approach can be used to select for promoter variants with abolished glucose repression in large mutant libraries. We also selected promoter variants with elevated expression level under induced conditions. The properties of the selected promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

  13. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    Science.gov (United States)

    Liu, Ning; Chen, Guo-Qing; Ning, Guo-Ao; Shi, Huan-Bin; Zhang, Chu-Long; Lu, Jian-Ping; Mao, Li-Juan; Feng, Xiao-Xiao; Liu, Xiao-Hong; Su, Zhen-Zhu; Lin, Fu-Cheng

    2016-01-01

    The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48 h at 22 °C could lead to a relatively highest frequency of transformation, and 200 μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast-Escherichia-Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae. Copyright © 2015 Elsevier GmbH. All rights reserved.

  14. Mechanisms of retroviral integration and mutagenesis.

    Science.gov (United States)

    Cavazza, Alessia; Moiani, Arianna; Mavilio, Fulvio

    2013-02-01

    Gene transfer vectors derived from oncoretroviruses or lentiviruses are the most robust and reliable tools to stably integrate therapeutic transgenes in human cells for clinical applications. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. In addressing these issues, the gene therapy community offered a spectacular example of how scientific knowledge and technology can be put to work to understand the causes of unpredicted side effects, design new vectors, and develop tools and models to predict their safety and efficacy. As an added benefit, these efforts brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. This review summarizes the current knowledge on the interactions between retroviruses and the human genome and addresses the impact of target site selection on the safety of retroviral vector-mediated gene therapy.

  15. History of attempts to quantify environmental mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hollaender, A.

    1981-01-01

    It became obvious in the early 1960's that the ready recognition of mutations produced by chemicals could have a profound influence on the refinement of methods to detect environmental mutagens. The experience derived over the previous 30 years in characterizing the effects of ionizing and ultraviolet radiation on the genetic mechanism came to serve us in good stead. Although the effects of chemicals are considerably more complicated and often require the analysis of individual substances, nonetheless, the area has developed rapidly in recent decades. The establishment and historical background of the International Association of Environmental Mutagen Societies (IAEMS) will be discussed. An attempt at the quantitation of chemical effects has been developed in comparison with radiation mutagenesis. As a first step, a definition of the Mutagen Burden or unavoidable exposure to chemicals will be discussed. A mathematical approach (Haynes/Eckhardt) will be considered and finally an outline for the comprehensive investigation of detailed interscience study will be made of less than six chemicals.

  16. A targeted mutagenesis system for Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Mulks, M H; Buysse, J M

    1995-11-07

    We describe methods for the mutagenesis of cloned Actinobacillus pleuropneumoniae (Ap) genes and for the construction of Ap mutants by allelic exchange. We used these methods to construct isogenic mutants of Ap which no longer synthesized a 48-kDa outer membrane protein (AopA). The native aopA locus was replaced with a mutated locus that had been inactivated by insertion of a gene (KmR) encoding kanamycin resistance from Tn903. The inactivated aopA locus was cloned into a conjugative, R6K-derived, lambda pir-dependent suicide vector and introduced into Ap using a filtermating technique. Southern and Western blot analyses indicated that the wild-type locus was replaced by the mutated locus through either single- or double-crossover events, and that AopA was no longer produced by either type of mutant. These methods were used successfully to construct AopA- mutants in Ap serotypes 1 and 5. These methods should be generally useful in constructing mutant loci which can be used to analyze the roles of various Ap genes in the pathogenesis of contagious pleuropneumonia in swine.

  17. Genomic approaches to DNA repair and mutagenesis.

    Science.gov (United States)

    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Symposium on molecular and cellular mechanisms of mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents. (ERB)

  19. The Roles of UmuD in Regulating Mutagenesis

    Directory of Open Access Journals (Sweden)

    Jaylene N. Ollivierre

    2010-01-01

    Full Text Available All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. Two E. coli Y-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms: UmuD2, which prevents mutagenesis, and UmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

  20. Environmental stress induces trinucleotide repeat mutagenesis in human cells

    Science.gov (United States)

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

    2015-01-01

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

  1. Synchronizing microelectrode and electronic goniometer data using a pseudo-random binary signal.

    Science.gov (United States)

    Moore, Tyler Robert; Jacobs, Rennie Underwood; Yang, Alexander Cheung; Richter, Erich Oscar

    2013-04-01

    Intra-operative investigation of the subthalamic nucleus (STN) requires concurrent measurement of microelectrode voltage, electrode depth and joint movement during deep brain stimulation (DBS) surgery. Commercial solutions to this problem exist but are more expensive. Multiple instruments from different manufacturers can collect the same data, but data from incompatible instruments are collected on disparate clocks, precluding quantitative analysis. A pseudo-random binary signal recorded simultaneously by each set of instruments allows for chronological reconciliation. A custom program collects microelectrode data while simultaneously sending a pseudo-random binary signal to instruments measuring joint movement. The record of this signal is later used to express microelectrode voltage and joint position in a single chronological frame of reference. ClockSynch was used in 15 DBS procedures. After each surgery, records of microelectrode and joint movement were successfully chronologically reconciled. In conclusion, a pseudo-random binary signal integrates disparate systems of instrumentation at a significantly decreased cost.

  2. Mott-Anderson Transition in Molecular Conductors: Influence of Randomness on Strongly Correlated Electrons in the κ-(BEDT-TTF2X System

    Directory of Open Access Journals (Sweden)

    Takahiko Sasaki

    2012-05-01

    Full Text Available The Mott-Anderson transition has been known as a metal-insulator (MI transition due to both strong electron-electron interaction and randomness of the electrons. For example, the MI transition in doped semiconductors and transition metal oxides has been investigated up to now as a typical example of the Mott-Anderson transition for changing electron correlations by carrier number control in concurrence with inevitable randomness. On the other hand, molecular conductors have been known as typical strongly correlated electron systems with bandwidth controlled Mott transition. In this paper, we demonstrate our recent studies on the randomness effect of the strongly correlated electrons of the BEDT-TTF molecule based organic conductors. X-ray irradiation on the crystals introduces molecular defects in the insulating anion layer, which cause random potential modulation of the correlated electrons in the conductive BEDT-TTF layer. In combination with hydrostatic pressure, we are able to control the parameters for randomness and correlations for electrons approaching the Mott-Anderson transition.

  3. Self-consistent electronic structure and segregation profiles of the Cu-Ni (001) random-alloy surface

    DEFF Research Database (Denmark)

    Ruban, Andrei; Abrikosov, I. A.; Kats, D. Ya.

    1994-01-01

    We have calculated the electronic structure and segregation profiles of the (001) surface of random Cu-Ni alloys with varying bulk concentrations by means of the coherent potential approximation and the linear muffin-tin-orbitals method. Exchange and correlation were included within the local......-density approximation. Temperature effects were accounted for by means of the cluster-variation method and, for comparison, by mean-field theory. The necessary interaction parameters were calculated by the Connolly-Williams method generalized to the case of a surface of a random alloy. We find the segregation profiles...... to be oscillatory with a strong preference for Cu to segregate towards the surface of the alloy....

  4. Cluster randomized trials utilizing primary care electronic health records: methodological issues in design, conduct, and analysis (eCRT Study).

    Science.gov (United States)

    Gulliford, Martin C; van Staa, Tjeerd P; McDermott, Lisa; McCann, Gerard; Charlton, Judith; Dregan, Alex

    2014-06-11

    There is growing interest in conducting clinical and cluster randomized trials through electronic health records. This paper reports on the methodological issues identified during the implementation of two cluster randomized trials using the electronic health records of the Clinical Practice Research Datalink (CPRD). Two trials were completed in primary care: one aimed to reduce inappropriate antibiotic prescribing for acute respiratory infection; the other aimed to increase physician adherence with secondary prevention interventions after first stroke. The paper draws on documentary records and trial datasets to report on the methodological experience with respect to research ethics and research governance approval, general practice recruitment and allocation, sample size calculation and power, intervention implementation, and trial analysis. We obtained research governance approvals from more than 150 primary care organizations in England, Wales, and Scotland. There were 104 CPRD general practices recruited to the antibiotic trial and 106 to the stroke trial, with the target number of practices being recruited within six months. Interventions were installed into practice information systems remotely over the internet. The mean number of participants per practice was 5,588 in the antibiotic trial and 110 in the stroke trial, with the coefficient of variation of practice sizes being 0.53 and 0.56 respectively. Outcome measures showed substantial correlations between the 12 months before, and after intervention, with coefficients ranging from 0.42 for diastolic blood pressure to 0.91 for proportion of consultations with antibiotics prescribed, defining practice and participant eligibility for analysis requires careful consideration. Cluster randomized trials may be performed efficiently in large samples from UK general practices using the electronic health records of a primary care database. The geographical dispersal of trial sites presents a difficulty for

  5. Intensive nursing care by an electronic followup system to promote secondary prevention after percutaneous coronary intervention: a randomized trial.

    Science.gov (United States)

    Hu, Xin; Zhu, Xiuqin; Gao, Lei

    2014-01-01

    To investigate the effectiveness of an intensive nursing care electronic followup system for cardiovascular risk management after percutaneous coronary intervention (PCI). In total, 840 subjects who underwent PCI in a single hospital in Beijing between January 2010 and January 2012 were enrolled. All subjects were randomized into the control and intensive nursing care groups (n = 420 each group). Both groups received standard secondary prevention according to guidelines. The control group received regular followup while the intensive nursing care group was closely monitored and followed by specific nursing staff with the electronic followup system. In total, 807 subjects were followed up for 1 year. Compared with subjects in the control group, those in the intensive group had decreased levels of total cholesterol (3.99 ± 1.08 vs 3.76 ± 0.98; P Intensive nursing care by the electronic followup system may lead to an improvement in quality of secondary prevention after PCI, including risk factor control, the use of medication, and self-management abilities.

  6. A randomized trial comparing in person and electronic interventions for improving adherence to oral medications in schizophrenia.

    Science.gov (United States)

    Velligan, Dawn; Mintz, Jim; Maples, Natalie; Xueying, Li; Gajewski, Stephanie; Carr, Heather; Sierra, Cynthia

    2013-09-01

    Poor adherence to medication leads to symptom exacerbation and interferes with the recovery process for patients with schizophrenia. Following baseline assessment, 142 patients in medication maintenance at a community mental health center were randomized to one of 3 treatments for 9 months: (1) PharmCAT, supports including pill containers, signs, alarms, checklists and the organization of belongings established in weekly home visits from a PharmCAT therapist; (2) Med-eMonitor (MM), an electronic medication monitor that prompts use of medication, cues the taking of medication, warns patients when they are taking the wrong medication or taking it at the wrong time, record complaints, and, through modem hookup, alerts treatment staff of failures to take medication as prescribed; (3) Treatment as Usual (TAU). All patients received the Med-eMonitor device to record medication adherence. The device was programmed for intervention only in the MM group. Data on symptoms, global functioning, and contact with emergency services and police were obtained every 3 months. Repeated measures analyses of variance for mixed models indicated that adherence to medication was significantly better in both active conditions than in TAU (both p<0.0001). Adherence in active treatments ranged from 90-92% compared to 73% in TAU based on electronic monitoring. In-person and electronic interventions significantly improved adherence to medication, but that did not translate to improved clinical outcomes. Implications for treatment and health care costs are discussed.

  7. Are anesthesia start and end times randomly distributed? The influence of electronic records.

    Science.gov (United States)

    Deal, Litisha G; Nyland, Michael E; Gravenstein, Nikolaus; Tighe, Patrick

    2014-06-01

    To perform a frequency analysis of start minute digits (SMD) and end minute digits (EMD) taken from the electronic, computer-assisted, and manual anesthesia billing-record systems. Retrospective cross-sectional review. University medical center. This cross-sectional review was conducted on billing records from a single healthcare institution over a 15-month period. A total of 30,738 cases were analyzed. For each record, the start time and end time were recorded. Distributions of SMD and EMD were tested against the null hypothesis of a frequency distribution equivalently spread between zero and nine. SMD and EMD aggregate distributions each differed from equivalency (P record, no differences were found between the recorded and expected equivalent distribution patterns for electronic anesthesia records for start minute (P records maintained nonequivalent distribution patterns for SMD and EMD (P record system, with automated time capture of events verified by the user, produces a more unified distribution of billing times than do more traditional methods of entering billing times. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis.

  9. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis.

  10. Advances in supramolecular electronics - from randomly self-assembled nanostructures to addressable self-organized interconnects.

    Science.gov (United States)

    Moulin, Emilie; Cid, Juan-José; Giuseppone, Nicolas

    2013-01-18

    Supramolecular organic electronics rests on the use of bottom-up chemical self-assembly processes in order to design conducting components on the 5-100 nm scale. The challenges in this field are both the construction of 1D-nanostructures displaying optimized transport properties and their precise connections to electrodes. The present Research News highlights important advances in such materials regarding their electrical performances, from semiconductors to organic metals, but also regarding their processability. In particular, by externally controlling light-responsive supramolecular polymerization processes, and by using appropriate methods of casting with an applied electric field, it becomes possible to pre-determine the accurate positioning of organic interconnects within patterned nano-circuitry. These strategies using external stimuli to obtain addressability, thus hold promising alternatives to other conducting materials such as carbon nanotubes for further technological applications in nanosciences. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Effect of Access to an Electronic Medical Resource on Performance Characteristics of a Certification Examination: A Randomized Controlled Trial.

    Science.gov (United States)

    Lipner, Rebecca S; Brossman, Bradley G; Samonte, Kelli M; Durning, Steven J

    2017-09-05

    Electronic resources are increasingly used in medical practice. Their use during high-stakes certification examinations has been advocated by many experts, but whether doing so would affect the capacity to differentiate between high and low abilities is unknown. To determine the effect of electronic resources on examination performance characteristics. Randomized controlled trial. Medical certification program. 825 physicians initially certified by the American Board of Internal Medicine (ABIM) who passed the Internal Medicine Certification examination or sat for the Internal Medicine Maintenance of Certification (IM-MOC) examination in 2012 to 2015. Participants were randomly assigned to 1 of 4 conditions: closed book using typical or additional time, or open book (that is, UpToDate [Wolters Kluwer]) using typical or additional time. All participants took the same modified version of the IM-MOC examination. Primary outcomes included item difficulty (how easy or difficult the question was), item discrimination (how well the question differentiated between high and low abilities), and average question response time. Secondary outcomes included examination dimensionality (that is, the number of factors measured) and test-taking strategy. Item response theory was used to calculate question characteristics. Analysis of variance compared differences among conditions. Closed-book conditions took significantly less time than open-book conditions (mean, 79.2 seconds [95% CI, 78.5 to 79.9 seconds] vs. 110.3 seconds [CI, 109.2 to 111.4 seconds] per question). Mean discrimination was statistically significantly higher for open-book conditions (0.34 [CI, 0.32 to 0.35] vs. 0.39 [CI, 0.37 to 0.41] per question). A strong single dimension showed that the examination measured the same factor with or without the resource. Only 1 electronic resource was evaluated. Inclusion of an electronic resource with time constraints did not adversely affect test performance and did not change

  12. Restricted second random phase approximations and Tamm-Dancoff approximations for electronic excitation energy calculations

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Degao; Yang, Yang; Zhang, Peng [Department of Chemistry, Duke University, Durham, North Carolina 27708 (United States); Yang, Weitao, E-mail: weitao.yang@duke.edu [Department of Chemistry and Department of Physics, Duke University, Durham, North Carolina 27708 (United States)

    2014-12-07

    In this article, we develop systematically second random phase approximations (RPA) and Tamm-Dancoff approximations (TDA) of particle-hole and particle-particle channels for calculating molecular excitation energies. The second particle-hole RPA/TDA can capture double excitations missed by the particle-hole RPA/TDA and time-dependent density-functional theory (TDDFT), while the second particle-particle RPA/TDA recovers non-highest-occupied-molecular-orbital excitations missed by the particle-particle RPA/TDA. With proper orbital restrictions, these restricted second RPAs and TDAs have a formal scaling of only O(N{sup 4}). The restricted versions of second RPAs and TDAs are tested with various small molecules to show some positive results. Data suggest that the restricted second particle-hole TDA (r2ph-TDA) has the best overall performance with a correlation coefficient similar to TDDFT, but with a larger negative bias. The negative bias of the r2ph-TDA may be induced by the unaccounted ground state correlation energy to be investigated further. Overall, the r2ph-TDA is recommended to study systems with both single and some low-lying double excitations with a moderate accuracy. Some expressions on excited state property evaluations, such as 〈S{sup ^2}〉 are also developed and tested.

  13. Restricted second random phase approximations and Tamm-Dancoff approximations for electronic excitation energy calculations

    Science.gov (United States)

    Peng, Degao; Yang, Yang; Zhang, Peng; Yang, Weitao

    2014-12-01

    In this article, we develop systematically second random phase approximations (RPA) and Tamm-Dancoff approximations (TDA) of particle-hole and particle-particle channels for calculating molecular excitation energies. The second particle-hole RPA/TDA can capture double excitations missed by the particle-hole RPA/TDA and time-dependent density-functional theory (TDDFT), while the second particle-particle RPA/TDA recovers non-highest-occupied-molecular-orbital excitations missed by the particle-particle RPA/TDA. With proper orbital restrictions, these restricted second RPAs and TDAs have a formal scaling of only O(N4). The restricted versions of second RPAs and TDAs are tested with various small molecules to show some positive results. Data suggest that the restricted second particle-hole TDA (r2ph-TDA) has the best overall performance with a correlation coefficient similar to TDDFT, but with a larger negative bias. The negative bias of the r2ph-TDA may be induced by the unaccounted ground state correlation energy to be investigated further. Overall, the r2ph-TDA is recommended to study systems with both single and some low-lying double excitations with a moderate accuracy. Some expressions on excited state property evaluations, such as < hat{S}2rangle are also developed and tested.

  14. Impact of Exposure to Electronic Cigarette Advertising on Susceptibility and Trial of Electronic Cigarettes and Cigarettes in US Young Adults: A Randomized Controlled Trial.

    Science.gov (United States)

    Villanti, Andrea C; Rath, Jessica M; Williams, Valerie F; Pearson, Jennifer L; Richardson, Amanda; Abrams, David B; Niaura, Raymond S; Vallone, Donna M

    2016-05-01

    This study assessed the impact of brief exposure to four electronic cigarette (e-cigarette) print advertisements (ads) on perceptions, intention, and subsequent use of e-cigarettes and cigarettes in US young adults. A randomized controlled trial was conducted in a national sample of young adults from an online panel survey in 2013. Participants were randomized to ad exposure or control. Curiosity, intentions, and perceptions regarding e-cigarettes were assessed post-exposure and e-cigarette and cigarette use at 6-month follow-up. Analyses were conducted in 2014. Approximately 6% of young adults who had never used an e-cigarette at baseline tried an e-cigarette at 6-month follow-up, half of whom were current cigarette smokers at baseline. Compared to the control group, ad exposure was associated with greater curiosity to try an e-cigarette (18.3% exposed vs. 11.3% unexposed, AOR = 1.63, 95% CI = 1.18, 2.26) among never e-cigarette users and greater likelihood of e-cigarette trial at follow-up (3.6% exposed vs. 1.2% unexposed, AOR = 2.85; 95% CI = 1.07, 7.61) among never users of cigarettes and e-cigarettes. Exploratory analyses did not find an association between ad exposure and cigarette trial or past 30-day use among never users, nor cigarette use among smokers over time. Curiosity mediated the relationship between ad exposure and e-cigarette trial among e-cigarette never users. Exposure to e-cigarette ads may enhance curiosity and limited trial of e-cigarettes in never users. Future studies are needed to examine the net effect of curiosity and trial of e-cigarettes on longer-term patterns of tobacco use. This randomized trial provides the first evidence of the effect of e-cigarette advertising on a behavioral outcome in young adults. Compared to the control group, ad exposure was associated with greater curiosity to try an e-cigarette among never e-cigarette users and greater likelihood of e-cigarette trial at follow-up in a small number of never e

  15. Methods for targetted mutagenesis in gram-positive bacteria

    Science.gov (United States)

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  16. Stationary-state mutagenesis in Escherichia coli: a model

    Indian Academy of Sciences (India)

    Stationary-phase mutagenesis in nondividing E. coli cells exposed to a nonlethal stress was, a few years ago, claimed to be a likely case of a Lamarckian mechanism capable of producing exclusively useful mutations in a directed manner. After a heated debate over the last decade it now appears to involve a Darwinian ...

  17. Model building of a thermolysin-like protease by mutagenesis

    NARCIS (Netherlands)

    Frigerio, F; Margarit, [No Value; Nogarotto, R; Grandi, G; Vriend, G; Hardy, F; Veltman, OR; Venema, G; Eijsink, VGH

    The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub), An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus

  18. Targeted mutagenesis using CRISPR/Cas in inbred potatoes

    Science.gov (United States)

    Targeted mutagenesis using sequence-specific nucleases (SSNs) has been well established in several important crop species, but is in need of improvement in potato (Solanum tuberosum L.). For over a century, potatoes have been bred as autotetraploids (2n = 4x = 48), relying on F1 selections and clona...

  19. Effect of Colchicine Induced Mutagenesis on Growth and Yield of ...

    African Journals Online (AJOL)

    Chemical mutagenesis through the use of colchicine on the seeds of two varieties of sesame (Sesamum indicum L. Var. Ex-Sudan and E-8) with the aim of inducing variability that could be exploited in the genetic improvement of its growth and yield was carried out. The sesame seeds were treated with colchicines at four ...

  20. A mariner transposon vector adapted for mutagenesis in oral streptococci

    DEFF Research Database (Denmark)

    Nilsson, Martin; Christiansen, Natalia; Høiby, Niels

    2014-01-01

    ATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via...

  1. A stochastic study of electron transfer kinetics in nano-particulate photocatalysis: a comparison of the quasi-equilibrium approximation with a random walking model.

    Science.gov (United States)

    Liu, Baoshun; Zhao, Xiujian; Yu, Jiaguo; Fujishima, Akira; Nakata, Kazuya

    2016-11-23

    In the photocatalysis of porous nano-crystalline materials, the transfer of electrons to O 2 plays an important role, which includes the electron transport to photocatalytic active centers and successive interfacial transfer to O 2 . The slowest of them will determine the overall speed of electron transfer in the photocatalysis reaction. Considering the photocatalysis of porous nano-crystalline TiO 2 as an example, although some experimental results have shown that the electron kinetics are limited by the interfacial transfer, we still lack the depth of understanding the microscopic mechanism from a theoretical viewpoint. In the present research, a stochastic quasi-equilibrium (QE) theoretical model and a stochastic random walking (RW) model were established to discuss the electron transport and electron interfacial transfer by taking the electron multi-trapping transport and electron interfacial transfer from the photocatalytic active centers to O 2 into consideration. By carefully investigating the effect of the electron Fermi level (E F ) and the photocatalytic center number on electron transport, we showed that the time taken for an electron to transport to a photocatalytic center predicated by the stochastic RW model was much lower than that predicted by the stochastic QE model, indicating that the electrons cannot reach a QE state during their transport to photocatalytic centers. The stochastic QE model predicted that the electron kinetics of a real photocatalysis for porous nano-crystalline TiO 2 should be limited by electron transport, whereas the stochastic RW model showed that the electron kinetics of a real photocatalysis can be limited by the interfacial transfer. Our simulation results show that the stochastic RW model was more in line with the real electron kinetics that have been observed in experiments, therefore it is concluded that the photoinduced electrons cannot reach a QE state before transferring to O 2 .

  2. A randomized trial evaluating Prosaptide for HIV-associated sensory neuropathies: use of an electronic diary to record neuropathic pain.

    Directory of Open Access Journals (Sweden)

    Scott R Evans

    Full Text Available OBJECTIVES: To examine the efficacy and safety of Prosaptide (PRO for the treatment of painful HIV-associated sensory neuropathies (HIV-SN. DESIGN: A randomized, double-blind, placebo-controlled, multicenter study in participants with sensory neuropathy. Pain modulating therapy was discontinued prior to baseline. Participants were stratified by sural sensory nerve action potential (SNAP amplitude. Participants were trained to use an electronic diary (ED to record pain. SETTING: Peripheral neuropathies are common complications of HIV infection. The pathogenesis is unknown and currently treatments are restricted to symptomatic measures. We examined PRO against placebo (PBO for treatment of painful HIV-SN and performed a post-hoc evaluation of an electronic diary (ED to record HIV-associated neuropathic pain. PARTICIPANTS: Eligible participants included adults with neurologist-confirmed painful HIV-SN. INTERVENTIONS: 2, 4, 8, or 16 mg/d PRO or PBO administered via subcutaneous (SC injection for six weeks. Neurotoxic antiretroviral drug usage was held constant. OUTCOME MEASURES: Changes from baseline in the weekly average of evaluable daily random prompts measuring pain using the Gracely pain scale and adverse events. RESULTS: 237 participants were randomized. The study was stopped after a planned futility analysis. There were no between-group differences in the frequency of adverse events or laboratory toxicities. The 6-week mean (sd Gracely pain scale changes were -0.12 (0.23, -0.24 (0.35, -0.15 (0.32, -0.18 (0.34, and -0.18 (0.32 for the 2, 4, 8, 16 mg, and PBO arms respectively. A similar variability of pain changes recorded using the ED were noted compared to previous trials that used paper collection methods. CONCLUSIONS: 6-week treatment with PRO was safe but not effective at reducing HIV-associated neuropathic pain. Use of an ED to record neuropathic pain is novel in HIV-SN, resulted in reasonable compliance in recording pain data, but did

  3. Addressing dental fear in children with autism spectrum disorders: a randomized controlled pilot study using electronic screen media.

    Science.gov (United States)

    Isong, Inyang A; Rao, Sowmya R; Holifield, Chloe; Iannuzzi, Dorothea; Hanson, Ellen; Ware, Janice; Nelson, Linda P

    2014-03-01

    Dental care is a significant unmet health care need for children with autism spectrum disorders (ASD). Many children with ASD do not receive dental care because of fear associated with dental procedures; oftentimes they require general anesthesia for regular dental procedures, placing them at risk of associated complications. Many children with ASD have a strong preference for visual stimuli, particularly electronic screen media. The use of visual teaching materials is a fundamental principle in designing educational programs for children with ASD. To determine if an innovative strategy using 2 types of electronic screen media was feasible and beneficial in reducing fear and uncooperative behaviors in children with ASD undergoing dental visits. We conducted a randomized controlled trial at Boston Children's Hospital dental clinic. Eighty (80) children aged 7 to 17 years with a known diagnosis of ASD and history of dental fear were enrolled in the study. Each child completed 2 preventive dental visits that were scheduled 6 months apart (visit 1 and visit 2). After visit 1, subjects were randomly assigned to 1 of 4 groups: (1) group A, control (usual care); (2) group B, treatment (video peer modeling that involved watching a DVD recording of a typically developing child undergoing a dental visit); (3) group C, treatment (video goggles that involved watching a favorite movie during the dental visit using sunglass-style video eyewear); and (4) group D, treatment (video peer modeling plus video goggles). Subjects who refused or were unable to wear the goggles watched the movie using a handheld portable DVD player. During both visits, the subject's level of anxiety and behavior were measured using the Venham Anxiety and Behavior Scales. Analyses of variance and Fisher's exact tests compared baseline characteristics across groups. Using intention to treat approach, repeated measures analyses were employed to test whether the outcomes differed significantly: (1) between

  4. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    OBJECTIVE: To assess the levels and spectrum of mitochondrial DNA (mtDNA) point mutations in synovial tissue from patients with inflammatory arthritis in relation to in vivo hypoxia and oxidative stress levels. METHODS: Random Mutation Capture assay was used to quantitatively evaluate alterations of the synovial mitochondrial genome. In vivo tissue oxygen levels (tPO(2)) were measured at arthroscopy using a Licox probe. Synovial expression of lipid peroxidation (4-hydroxynonenal [4-HNE]) and mitochondrial cytochrome c oxidase subunit II (CytcO II) deficiency were assessed by immunohistochemistry. In vitro levels of mtDNA point mutations, reactive oxygen species (ROS), mitochondrial membrane potential, and markers of oxidative DNA damage (8-oxo-7,8-dihydro-2\\'-deoxyguanine [8-oxodG]) and lipid peroxidation (4-HNE) were determined in human synoviocytes under normoxia and hypoxia (1%) in the presence or absence of superoxide dismutase (SOD) or N-acetylcysteine (NAC) or a hydroxylase inhibitor (dimethyloxalylglycine [DMOG]). Patients were categorized according to their in vivo tPO(2) level (<20 mm Hg or >20 mm Hg), and mtDNA point mutations, immunochemistry features, and stress markers were compared between groups. RESULTS: The median tPO(2) level in synovial tissue indicated significant hypoxia (25.47 mm Hg). Higher frequency of mtDNA mutations was associated with reduced in vivo oxygen tension (P = 0.05) and with higher synovial 4-HNE cytoplasmic expression (P = 0.04). Synovial expression of CytcO II correlated with in vivo tPO(2) levels (P = 0.03), and levels were lower in patients with tPO(2) <20 mm Hg (P < 0.05). In vitro levels of mtDNA mutations, ROS, mitochondrial membrane potential, 8-oxo-dG, and 4-HNE were higher in synoviocytes exposed to 1% hypoxia (P < 0.05); all of these increased levels were rescued by SOD and DMOG and, with the exception of ROS, by NAC. CONCLUSION: These findings demonstrate that hypoxia-induced mitochondrial dysfunction drives

  5. Selection of Zygosaccharomyces rouxii strains resistant to cadmium with improved removal abilities through ultraviolet-diethyl sulfate cooperative mutagenesis.

    Science.gov (United States)

    Liu, Yu; Xu, Ying; Wang, Dongfeng; Jiang, Wei

    2017-08-01

    Cd 2+ resistance and bioaccumulation capacity were selected from parental Zygosaccharomyces rouxii (CRZ-0) while maintaining NaCl tolerance using protoplast mutagenesis technology. Ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis, followed by preliminary screening and rescreening, was used to select the mutant strain CRZ-9. CRZ-9 grew better than CRZ-0 in YPD medium with 20 or 50 mg L -1 of Cd 2+ . Scanning electron microscopy observations and flow cytometry tests indicated that CRZ-9 was more effective at eliminating reactive oxygen species (ROS) generated by Cd 2+ , which led to less cellular structural damage and lower lethality. Furthermore, compared with CRZ-0, CRZ-9 exhibited increased potential for application with higher Cd 2+ removal ratio, wider working pH range, and lower biomass dosage in Cd 2+ bioaccumulation. The mutant strain CRZ-9 possessed improved Cd 2+ resistance and bioaccumulation capacity and therefore is a promising strain to remove Cd 2+ from wastewater.

  6. MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction

    NARCIS (Netherlands)

    Ahlqvist, K.J.; Leoncini, S.; Pecorelli, A.; Wortmann, S.B.; Ahola, S.; Forsstrom, S.; Guerranti, R.; Felice, C. De; Smeitink, J.; Ciccoli, L.; Hamalainen, R.H.; Suomalainen, A.

    2015-01-01

    Haematopoietic progenitor cells show special sensitivity to mitochondrial DNA (mtDNA) mutagenesis, which suggests that increased mtDNA mutagenesis could underlie anemias. Here we show that elevated mtDNA mutagenesis in mice with a proof-reading deficient mtDNA polymerase (PolG) leads to incomplete

  7. Randomized trial of automated, electronic monitoring to facilitate early detection of sepsis in the intensive care unit*.

    Science.gov (United States)

    Hooper, Michael H; Weavind, Lisa; Wheeler, Arthur P; Martin, Jason B; Gowda, Supriya Srinivasa; Semler, Matthew W; Hayes, Rachel M; Albert, Daniel W; Deane, Norment B; Nian, Hui; Mathe, Janos L; Nadas, Andras; Sztipanovits, Janos; Miller, Anne; Bernard, Gordon R; Rice, Todd W

    2012-07-01

    To determine whether automated identification with physician notification of the systemic inflammatory response syndrome in medical intensive care unit patients expedites early administration of new antibiotics or improvement of other patient outcomes in patients with sepsis. : A prospective randomized, controlled, single center study. Medical intensive care unit of an academic, tertiary care medical center. Four hundred forty-two consecutive patients admitted over a 4-month period who met modified systemic inflammatory response syndrome criteria in a medical intensive care unit. Patients were randomized to monitoring by an electronic "Listening Application" to detect modified (systemic inflammatory response syndrome) criteria vs. usual care. The listening application notified physicians in real time when modified systemic inflammatory response syndrome criteria were detected, but did not provide management recommendations. The median time to new antibiotics was similar between the intervention and usual care groups when comparing among all patients (6.0 hr vs. 6.1 hr, p = .95), patients with sepsis (5.3 hr vs. 5.1 hr; p = .90), patients on antibiotics at enrollment (5.2 hr vs. 7.0 hr, p = .27), or patients not on antibiotics at enrollment (5.2 hr vs. 5.1 hr, p = .85). The amount of fluid administered following detection of modified systemic inflammatory response syndrome criteria was similar between groups whether comparing all patients or only patients who were hypotensive at enrollment. Other clinical outcomes including intensive care unit length of stay, hospital length of stay, and mortality were not shown to be different between patients in the intervention and control groups. Realtime alerts of modified systemic inflammatory response syndrome criteria to physicians in one tertiary care medical intensive care unit were feasible and safe but did not influence measured therapeutic interventions for sepsis or significantly alter clinical outcomes.

  8. Mobile Device-Based Electronic Data Capture System Used in a Clinical Randomized Controlled Trial: Advantages and Challenges.

    Science.gov (United States)

    Zhang, Jing; Sun, Lei; Liu, Yu; Wang, Hongyi; Sun, Ningling; Zhang, Puhong

    2017-03-08

    Electronic data capture (EDC) systems have been widely used in clinical research, but mobile device-based electronic data capture (mEDC) system has not been well evaluated. The aim of our study was to evaluate the feasibility, advantages, and challenges of mEDC in data collection, project management, and telemonitoring in a randomized controlled trial (RCT). We developed an mEDC to support an RCT called "Telmisartan and Hydrochlorothiazide Antihypertensive Treatment (THAT)" study, which was a multicenter, double-blinded, RCT, with the purpose of comparing the efficacy of telmisartan and hydrochlorothiazide (HCTZ) monotherapy in high-sodium-intake patients with mild to moderate hypertension during a 60 days follow-up. Semistructured interviews were conducted during and after the trial to evaluate the feasibility, advantage, and challenge of mEDC. Nvivo version 9.0 (QSR International) was used to analyze records of interviews, and a thematic framework method was used to obtain outcomes. The mEDC was successfully used to support the data collection and project management in all the 14 study hospitals. A total of 1333 patients were recruited with support of mEDC, of whom 1037 successfully completed all 4 visits. Across all visits, the average time needed for 141 questions per patient was 53 min, which were acceptable to both doctors and patients. All the interviewees, including 24 doctors, 53 patients, 1 clinical research associate (CRA), 1 project manager (PM), and 1 data manager (DM), expressed their satisfaction to nearly all the functions of the innovative mEDC in randomization, data collection, project management, quality control, and remote monitoring in real time. The average satisfaction score was 9.2 (scale, 0-10). The biggest challenge came from the stability of the mobile or Wi-Fi signal although it was not a problem in THAT study. The innovative mEDC has many merits and is well acceptable in supporting data collection and project management in a timely

  9. Correction to: Impact of an electronic monitoring device and behavioural feedback on adherence to multiple sclerosis therapies in youth: results of a randomized trial.

    Science.gov (United States)

    Yeh, E Ann; Grover, Stephanie A; Powell, Victoria E; Alper, Gulay; Banwell, Brenda L; Edwards, Kim; Gorman, Mark; Graves, Jennifer; Lotze, Timothy E; Mah, Jean K; Mednick, Lauren; Ness, Jayne; Obadia, Maya; Slater, Ruth; Waldman, Amy; Waubant, Emmanuelle; Schwartz, Carolyn E

    2017-12-23

    The clinicaltrials.gov identifying number for the article titled "Impact of an electronic monitoring device and behavioral feedback on adherence to multiple sclerosis therapies in youth: results of a randomized trial" is NCT02234713 (https://clinicaltrials.gov/ct2/show/NCT02234713).

  10. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

    Science.gov (United States)

    Elso, Colleen M; Chu, Edward P F; Alsayb, May A; Mackin, Leanne; Ivory, Sean T; Ashton, Michelle P; Bröer, Stefan; Silveira, Pablo A; Brodnicki, Thomas C

    2015-10-04

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. Copyright © 2015 Elso et al.

  11. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes

    Science.gov (United States)

    Elso, Colleen M.; Chu, Edward P. F.; Alsayb, May A.; Mackin, Leanne; Ivory, Sean T.; Ashton, Michelle P.; Bröer, Stefan; Silveira, Pablo A.; Brodnicki, Thomas C.

    2015-01-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  12. Mutagenesis breeding research of Lactobacillus brevis of nitrite reduction

    Directory of Open Access Journals (Sweden)

    LI Zeli

    2015-10-01

    Full Text Available The pollution of nitrite in food became one of the focus of food safety issues,the use of biotechnology methods degrading nitrite became hotspot.The primitive strain was Lactobacillus brevis C2,preserved in our laboratory,had the ability to degrade nitrite,through composite mutagenesis of 15 W,254 nm,20 cm ultraviolet mutagenesis (UV for 120 s and 0.8% diethyl sulfate(DES in 37℃ mutation for 40 min,after screening,we successfully obtained high efficient strain of nitrite degradation,named UV6-DS2,relative to the starting strain,under the condition of 400 mg/L nitrite,after 12 h degradation,nitrite degradation rate increased from 92.8% to 97.8%,to explore its application in food was able to effectively reduce concentration of nitrite in food.

  13. Targeted mutagenesis in sea urchin embryos using TALENs.

    Science.gov (United States)

    Hosoi, Sayaka; Sakuma, Tetsushi; Sakamoto, Naoaki; Yamamoto, Takashi

    2014-01-01

    Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  14. UV-Induced DNA Damage and Mutagenesis in Chromatin.

    Science.gov (United States)

    Mao, Peng; Wyrick, John J; Roberts, Steven A; Smerdon, Michael J

    2017-01-01

    UV radiation induces photolesions that distort the DNA double helix and, if not repaired, can cause severe biological consequences, including mutagenesis or cell death. In eukaryotes, both the formation and repair of UV damage occur in the context of chromatin, in which genomic DNA is packaged with histones into nucleosomes and higher order chromatin structures. Here, we review how chromatin impacts the formation of UV photoproducts in eukaryotic cells. We describe the initial discovery that nucleosomes and other DNA binding proteins induce characteristic "photofootprints" during the formation of UV photoproducts. We also describe recent progress in genomewide methods for mapping UV damage, which echoes early biochemical studies, and highlights the role of nucleosomes and transcription factors in UV damage formation and repair at unprecedented resolution. Finally, we discuss our current understanding of how the distribution and repair of UV-induced DNA damage influence mutagenesis in human skin cancers. © 2016 The American Society of Photobiology.

  15. Oligonucleotide?directed mutagenesis for precision gene editing

    OpenAIRE

    Sauer, Noel J.; Mozoruk, Jerry; Miller, Ryan B.; Warburg, Zachary J.; Walker, Keith A.; Beetham, Peter R.; Sch?pke, Christian R.; Gocal, Greg F. W.

    2015-01-01

    Summary Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide?directed mutagenesis (ODM), one of the many tools of Cibus? Rapid Trait Development System ( RTDS ?) technology, offers a rapid, precise and non?transgenic breeding a...

  16. Environmental mutagenesis and radiation biology: The legacy of William Morgan.

    Science.gov (United States)

    Schwartz, Jeffrey L

    2017-12-01

    A symposium entitled Environmental Mutagenesis and Radiation Biology was held on September 27, 2016 to honor the memory of Dr. William F. Morgan who passed away unexpectedly on November 13, 2015. The speakers presented the latest reviews on homologous recombination repair, induced genetic instability, bystander effects, and risk estimate development. Their presentations are presented following the introduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Systematic identification of genetic loci required for polymyxin resistance in Campylobacter jejuni using an efficient in vivo transposon mutagenesis system.

    Science.gov (United States)

    Lin, Jun; Wang, Ying; Hoang, Ky Van

    2009-03-01

    The aim of this study was to identify genetic loci required for polymyxin (PM) resistance in Campylobacter jejuni using an efficient in vivo random mutagenesis system. PM has been widely used as a model peptide to examine mechanisms of bacterial resistance to antimicrobial peptides (AMPs), the major effectors of host innate immunity and also candidates for a new generation of antibiotics. In this study, a commercially available transposon mutagenesis approach (EZ-Tn5 Transposome; Epicentre, Madison, WI) was evaluated and used to systematically identify Campylobacter mutants with increased susceptibility to PM. This simple, yet efficient, transposon mutagenesis approach identified 12 mutants representing seven different genes of C. jejuni 81-176 involved in acquired PM resistance. Backcrossing of the transposon mutations into the parent strain confirmed that the PM-sensitive phenotype in each mutant was linked to the gene with a specific transposon insertion. The genes are identified as being involved in the synthesis of cell-surface carbohydrates, modification of intracellular targets, signal transduction, and modulation of transmembrane potential. The mutant with the highest susceptibility to PM contains a transposon insertion in a putative galU gene that is essential for production of uridine diphosphate glucose (UDP)-glucose, a precursor required for lipooligosaccharide (LOS) synthesis. LOS analysis by tricine SDSPAGE showed significant truncation of the LOS core structure in the galU mutant. Susceptibility assays also indicated that GalU contributed C. jejuni resistance to some natural AMPs. Complementation of the galU mutant in trans fully restored LOS synthesis and resistance to the levels of the parent strain. Together, these results define seven C. jejuni genetic loci that will be useful for characterizing the molecular basis of Campylobacter resistance to PM and natural AMPs, and also highlight the usefulness of the in vivo mutagenesis approach for

  18. GATMD: γ-Aminobutyric Acid Transporter Mutagenesis Database

    Science.gov (United States)

    Anderson, Cynthia M.; Kidd, Patrick D.; Eskandari, Sepehr

    2010-01-01

    Since the cloning of the first γ-aminobutyric acid (GABA) transporter (GAT1; SLC6A1) from rat brain in 1990, more than 50 published studies have provided structure–function information on investigator-designed rat and mouse GAT1 mutants. To date, more than 200 of 599 GAT1 residues have been subjected to mutagenesis experiments by substitution with different amino acids, and the resulting transporter functional properties have significantly advanced our understanding of the mechanism of Na+- and Cl–-coupled GABA transport by this important member of the neurotransmitter:sodium symporter family. Moreover, many studies have addressed the functional consequences of amino acid deletion or insertion at various positions along the primary sequence. The enormity of this growing body of structure–function information has prompted us to develop GABA Transporter Mutagenesis Database (GATMD), a web-accessible, relational database of manually annotated biochemical, functional and pharmacological data reported on GAT1—the most intensely studied GABA transporter isoform. As of the last update of GATMD, 52 GAT1 mutagenesis papers have yielded 3360 experimental records, which collectively contain a total of ∼100 000 annotated parameters. Database URL: http://physiology.sci.csupomona.edu/GATMD/ PMID:21131297

  19. Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

    Directory of Open Access Journals (Sweden)

    C.P. Nunes

    2000-12-01

    Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

  20. Effects of Home Access to Active Videogames on Child Self-Esteem, Enjoyment of Physical Activity, and Anxiety Related to Electronic Games: Results from a Randomized Controlled Trial.

    Science.gov (United States)

    Abbott, Rebecca A; Smith, Anne J; Howie, Erin K; Pollock, Clare; Straker, Leon

    2014-08-01

    Active-input videogames could provide a useful conduit for increasing physical activity by improving a child's self-confidence, physical activity enjoyment, and reducing anxiety. Therefore this study evaluated the impact of (a) the removal of home access to traditional electronic games or (b) their replacement with active-input videogames, on child self-perception, enjoyment of physical activity, and electronic game use anxiety. This was a crossover, randomized controlled trial, conducted over a 6-month period in participants' family homes in metropolitan Perth, Australia, from 2007 to 2010. Children 10-12 years old were recruited through school and community media. Of 210 children who were eligible, 74 met inclusion criteria, and 8 withdrew, leaving 66 children (33 girls) for analysis. A counterbalanced randomized order of three conditions sustained for 8 weeks each: No home access to electronic games, home access to traditional electronic games, and home access to active-input electronic games. Perception of self-esteem (Harter's Self Perception Profile for Children), enjoyment of physical activity (Physical Activity Enjoyment Scale questionnaire), and anxiety toward electronic game use (modified Loyd and Gressard Computer Anxiety Subscale) were assessed. Compared with home access to traditional electronic games, neither removal of all electronic games nor replacement with active-input games resulted in any significant change to child self-esteem, enjoyment of physical activity, or anxiety related to electronic games. Although active-input videogames have been shown to be enjoyable in the short term, their ability to impact on psychological outcomes is yet to be established.

  1. InsuOnline, an Electronic Game for Medical Education on Insulin Therapy: A Randomized Controlled Trial With Primary Care Physicians.

    Science.gov (United States)

    Diehl, Leandro Arthur; Souza, Rodrigo Martins; Gordan, Pedro Alejandro; Esteves, Roberto Zonato; Coelho, Izabel Cristina Meister

    2017-03-09

    Most patients with diabetes mellitus (DM) are followed by primary care physicians, who often lack knowledge or confidence to prescribe insulin properly. This contributes to clinical inertia and poor glycemic control. Effectiveness of traditional continuing medical education (CME) to solve that is limited, so new approaches are required. Electronic games are a good option, as they can be very effective and easily disseminated. The objective of our study was to assess applicability, user acceptance, and educational effectiveness of InsuOnline, an electronic serious game for medical education on insulin therapy for DM, compared with a traditional CME activity. Primary care physicians (PCPs) from South of Brazil were invited by phone or email to participate in an unblinded randomized controlled trial and randomly allocated to play the game InsuOnline, installed as an app in their own computers, at the time of their choice, with minimal or no external guidance, or to participate in a traditional CME session, composed by onsite lectures and cases discussion. Both interventions had the same content and duration (~4 h). Applicability was assessed by the number of subjects who completed the assigned intervention in each group. Insulin-prescribing competence (factual knowledge, problem-solving skills, and attitudes) was self-assessed through a questionnaire applied before, immediately after, and 3 months after the interventions. Acceptance of the intervention (satisfaction and perceived importance for clinical practice) was also assessed immediately after and 3 months after the interventions, respectively. Subjects' characteristics were similar between groups (mean age 38, 51.4% [69/134] male). In the game group, 69 of 88 (78%) completed the intervention, compared with 65 of 73 (89%) in the control group, with no difference in applicability. Percentage of right answers in the competence subscale, which was 52% at the baseline in both groups, significantly improved

  2. Electronic search strategies to identify reports of cluster randomized trials in MEDLINE: low precision will improve with adherence to reporting standards

    Directory of Open Access Journals (Sweden)

    Grimshaw Jeremy M

    2010-02-01

    Full Text Available Abstract Background Cluster randomized trials (CRTs present unique methodological and ethical challenges. Researchers conducting systematic reviews of CRTs (e.g., addressing methodological or ethical issues require efficient electronic search strategies (filters or hedges to identify trials in electronic databases such as MEDLINE. According to the CONSORT statement extension to CRTs, the clustered design should be clearly identified in titles or abstracts; however, variability in terminology may make electronic identification challenging. Our objectives were to (a evaluate sensitivity ("recall" and precision of a well-known electronic search strategy ("randomized controlled trial" as publication type with respect to identifying CRTs, (b evaluate the feasibility of new search strategies targeted specifically at CRTs, and (c determine whether CRTs are appropriately identified in titles or abstracts of reports and whether there has been improvement over time. Methods We manually examined a wide range of health journals to identify a gold standard set of CRTs. Search strategies were evaluated against the gold standard set, as well as an independent set of CRTs included in previous systematic reviews. Results The existing strategy (randomized controlled trial.pt is sensitive (93.8% for identifying CRTs, but has relatively low precision (9%, number needed to read 11; the number needed to read can be halved to 5 (precision 18.4% by combining with cluster design-related terms using the Boolean operator AND; combining with the Boolean operator OR maximizes sensitivity (99.4% but would require 28.6 citations read to identify one CRT. Only about 50% of CRTs are clearly identified as cluster randomized in titles or abstracts; approximately 25% can be identified based on the reported units of randomization but are not amenable to electronic searching; the remaining 25% cannot be identified except through manual inspection of the full-text article. The

  3. Electronic search strategies to identify reports of cluster randomized trials in MEDLINE: low precision will improve with adherence to reporting standards.

    Science.gov (United States)

    Taljaard, Monica; McGowan, Jessie; Grimshaw, Jeremy M; Brehaut, Jamie C; McRae, Andrew; Eccles, Martin P; Donner, Allan

    2010-02-16

    Cluster randomized trials (CRTs) present unique methodological and ethical challenges. Researchers conducting systematic reviews of CRTs (e.g., addressing methodological or ethical issues) require efficient electronic search strategies (filters or hedges) to identify trials in electronic databases such as MEDLINE. According to the CONSORT statement extension to CRTs, the clustered design should be clearly identified in titles or abstracts; however, variability in terminology may make electronic identification challenging. Our objectives were to (a) evaluate sensitivity ("recall") and precision of a well-known electronic search strategy ("randomized controlled trial" as publication type) with respect to identifying CRTs, (b) evaluate the feasibility of new search strategies targeted specifically at CRTs, and (c) determine whether CRTs are appropriately identified in titles or abstracts of reports and whether there has been improvement over time. We manually examined a wide range of health journals to identify a gold standard set of CRTs. Search strategies were evaluated against the gold standard set, as well as an independent set of CRTs included in previous systematic reviews. The existing strategy (randomized controlled trial.pt) is sensitive (93.8%) for identifying CRTs, but has relatively low precision (9%, number needed to read 11); the number needed to read can be halved to 5 (precision 18.4%) by combining with cluster design-related terms using the Boolean operator AND; combining with the Boolean operator OR maximizes sensitivity (99.4%) but would require 28.6 citations read to identify one CRT. Only about 50% of CRTs are clearly identified as cluster randomized in titles or abstracts; approximately 25% can be identified based on the reported units of randomization but are not amenable to electronic searching; the remaining 25% cannot be identified except through manual inspection of the full-text article. The proportion of trials clearly identified has

  4. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...

  5. A protocol for chemical mutagenesis in Strongyloides ratti.

    Science.gov (United States)

    Guo, Li; Chang, Zisong; Dieterich, Christoph; Streit, Adrian

    2015-11-01

    Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Environmental stress and mutagenesis in enteric and non-enteric bacteria

    OpenAIRE

    Babudri, Nora; Lancioni, Hovirag; Achilli, Alessandro

    2012-01-01

    Mutations are fundamental for evolution. For many years it has been thought that mutagenesis occurs only in dividing cells. Now it is clear that mutations arise in non-dividing or slowly dividing microorganisms. Natural populations spend most of the time in stressful environments where their growth rate is highly reduced. Thus, the existence of a mutagenesis process, independent of multiplication (stress-induced mutagenesis, SIM), might have a profound evolutionary role. In the presented pape...

  7. Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

    DEFF Research Database (Denmark)

    Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

    2015-01-01

    Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...... insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.......Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale...

  8. Cell-mediated mutagenesis and cell transformation by chemical carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.

    1977-01-01

    Results are reported from studies that showed that mutagenesis of mammalian cells can be achieved by carcinogenic polycyclic hydrocarbons, nitrosamines, and aflatoxins when tested in the presence of fibroblasts and hepatocytes which are able to metabolize these carcinogens. Further, we have found that there is a relationship between the degree of mutant induction and the degree of carcinogenicity of the different chemicals tested. By simultaneously measuring the frequency of cell transformation and the frequency of mutation at one locus (ouabain resistance) in the same cell system, it was possible to estimate the genetic target site for cell transformation. The results indicated that the target site for transformation is approximately 20 times larger than that determined for ouabain resistance. The results suggest that cell transformation may be due to a mutational event and the mutation can occur in one out of a small number of the same or different genes, and that the cell-mediated mutagenesis approach may be a valuable means of detecting tissue-specific carcinogens.

  9. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir.

    Directory of Open Access Journals (Sweden)

    Ana I de Ávila

    Full Text Available Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705 is a potent mutagenic agent for hepatitis C virus (HCV during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50. Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens.

  10. The roles of cytochrome b559 in assembly and photoprotection of Photosystem II revealed by site-directed mutagenesis studies

    Directory of Open Access Journals (Sweden)

    Hsiu-An eChu

    2016-01-01

    Full Text Available Cytochrome b559 (Cyt b559 is one of the essential components of the Photosystem II reaction center (PSII. Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b559 remains unclear. Cyt b559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b559 mutant strains and their mutant PSII complex in higher plants, green algae and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b559 and implications for revealing the physiological functions of Cyt b559 in PSII.

  11. Construction of a high-efficiency multi-site-directed mutagenesis ...

    African Journals Online (AJOL)

    Although site-directed mutagenesis has been used in many fields, it still has low rate of success and high cost because of low-yield target products. A modified method for multi-site-directed mutagenesis was developed with shifted primer design and cold-start polymerase chain reaction (PCR). The developed method was ...

  12. Quantitative studies of the mutagenesis of Toxoplasma gondii

    Energy Technology Data Exchange (ETDEWEB)

    Pfefferkorn, E.R.; Pfefferkorn, L.C.

    1979-06-01

    The induction of mutants resistant to 5-fluorodeoxyuridine (FUDR) was used to measure the efficiency of various physical and chemical mutagens on extracellular and intracellular Toxoplasma gondii. The frequency of resistant mutant was measured by plaque assay in human fibroblast cultures in the presence and absence of FUDR. When considered as a function of lethality, the most efficient mutagenesis was obtained with nitrosoguanidine treatment of extracellular parasites and with ethylmethane sulfonate treatment of actively growing intracellular parasites. Each of these treatments increased the frequency of FUDR-resistant mutants from less than one to more than 200 per million parasites. Ultraviolet irradiation, X-rays, and the alkylating mustard ICR-191 also induced FUDR-resistant mutants in a dose-dependent fashion.

  13. Role of Nicotinamide in DNA Damage, Mutagenesis, and DNA Repair

    Directory of Open Access Journals (Sweden)

    Devita Surjana

    2010-01-01

    Full Text Available Nicotinamide is a water-soluble amide form of niacin (nicotinic acid or vitamin B3. Both niacin and nicotinamide are widely available in plant and animal foods, and niacin can also be endogenously synthesized in the liver from dietary tryptophan. Nicotinamide is also commercially available in vitamin supplements and in a range of cosmetic, hair, and skin preparations. Nicotinamide is the primary precursor of nicotinamide adenine dinucleotide (NAD+, an essential coenzyme in ATP production and the sole substrate of the nuclear enzyme poly-ADP-ribose polymerase-1 (PARP-1. Numerous in vitro and in vivo studies have clearly shown that PARP-1 and NAD+ status influence cellular responses to genotoxicity which can lead to mutagenesis and cancer formation. This paper will examine the role of nicotinamide in the protection from carcinogenesis, DNA repair, and maintenance of genomic stability.

  14. The effect of permodified cyclodextrins encapsulation on the photophysical properties of a polyfluorene with randomly distributed electron-donor and rotaxane electron-acceptor units

    Directory of Open Access Journals (Sweden)

    Aurica Farcas

    2014-09-01

    Full Text Available We report on the synthesis as well as the optical, electrochemical and morphological properties of two polyrotaxanes (4a and 4b, which consist of electron-accepting 9,9-dicyanomethylenefluorene 1 as an inclusion complex in persilylated β- or γ-cyclodextrin (TMS-β-CD, TMS-γ-CD (1a, 1b and methyltriphenylamine as an electron-donating molecule. They are statistically distributed into the conjugated chains of 9,9-dioctylfluorene 3 and compared with those of the corresponding non-rotaxane 4 counterpart. Rotaxane formation results in improvements of the solubility, the thermal stability, and the photophysical properties. Polyrotaxanes 4a and 4b exhibited slightly red-shifted absorption bands with respect to the non-rotaxane 4 counterpart. The fluorescence lifetimes of polyrotaxanes follow a mono-exponential decay with a value of τ = 1.14 ns compared with the non-rotaxane, where a bi-exponential decay composed of a main component with a relative short time of τ1 = 0.88 (57.08% and a minor component with a longer lifetime of τ2 = 1.56 ns (42.92% were determined. The optical and electrochemical band gaps (ΔEg as well as the ionization potential and electronic affinity characterized by smaller values compared to the values of any of the constituents. AFM reveals that the film surface of 4a and 4b displays a granular morphology with a lower dispersity supported by a smaller roughness exponent compared with the non-rotaxane counterpart.

  15. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice.

    Science.gov (United States)

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-05-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein-gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  16. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    Directory of Open Access Journals (Sweden)

    Alexandro Rodríguez-Rojas

    2015-10-01

    Full Text Available Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection.

  17. Atomic force microscopy and scanning electron microscopy evaluation of efficacy of scaling and root planing using magnification: A randomized controlled clinical study.

    Science.gov (United States)

    Mohan, Ranjana; Agrawal, Sudhanshu; Gundappa, Mohan

    2013-07-01

    A randomized controlled clinical study was undertaken to evaluate the effectiveness of scaling and root planing (SRP) by using Magnifying Loupes (ML) and dental operating microscope (DOM). A total of 90 human teeth scheduled for extraction from 18 patients aged between 25 and 65 years suffering from generalized chronic severe periodontitis were randomly assigned to three treatment groups. Group 1 consisted SRP performed without using magnification (unaided), Group 2-SRP with ML and Group 3-SRP with DOM. Following extractions, samples were prepared for (i) evaluation of surface topography by atomic force microscopy, (ii) presence of smear layer, debris by scanning electron microscopy (iii) elemental analysis by energy dispersive X-ray analysis. Data was subjected to statistical analysis using analysis of variance, post-hoc (Tukey-HSD) and Chi-square test. Statistically significant (P Magnification tools significantly enhance the efficacy of supragingival and subgingival SRP.

  18. Electronic Monitoring-Based Counseling to Enhance Adherence Among HIV-Infected Patients: A Randomized Controlled Trial

    NARCIS (Netherlands)

    de Bruin, Marijn; Hospers, Harm J.; van Breukelen, Gerard J. P.; Kok, Gerjo; Koevoets, William M.; Prins, Jan M.

    2010-01-01

    Objective: To investigated the effectiveness of an adherence intervention (AIMS) designed to fit HIV-clinics' routine care procedures. Design: Through block randomization, patients were allocated to the intervention or control group. The study included 2 months baseline measurement, 3 months

  19. Daily electronic self-monitoring in bipolar disorder using smartphones - the MONARCA I trial: a randomized, placebo-controlled, single-blind, parallel group trial.

    Science.gov (United States)

    Faurholt-Jepsen, M; Frost, M; Ritz, C; Christensen, E M; Jacoby, A S; Mikkelsen, R L; Knorr, U; Bardram, J E; Vinberg, M; Kessing, L V

    2015-10-01

    The number of studies on electronic self-monitoring in affective disorder and other psychiatric disorders is increasing and indicates high patient acceptance and adherence. Nevertheless, the effect of electronic self-monitoring in patients with bipolar disorder has never been investigated in a randomized controlled trial (RCT). The objective of this trial was to investigate in a RCT whether the use of daily electronic self-monitoring using smartphones reduces depressive and manic symptoms in patients with bipolar disorder. A total of 78 patients with bipolar disorder according to ICD-10 criteria, aged 18-60 years, and with 17-item Hamilton Depression Rating Scale (HAMD-17) and Young Mania Rating Scale (YMRS) scores ≤17 were randomized to the use of a smartphone for daily self-monitoring including a clinical feedback loop (the intervention group) or to the use of a smartphone for normal communicative purposes (the control group) for 6 months. The primary outcomes were differences in depressive and manic symptoms measured using HAMD-17 and YMRS, respectively, between the intervention and control groups. Intention-to-treat analyses using linear mixed models showed no significant effects of daily self-monitoring using smartphones on depressive as well as manic symptoms. There was a tendency towards more sustained depressive symptoms in the intervention group (B = 2.02, 95% confidence interval -0.13 to 4.17, p = 0.066). Sub-group analysis among patients without mixed symptoms and patients with presence of depressive and manic symptoms showed significantly more depressive symptoms and fewer manic symptoms during the trial period in the intervention group. These results highlight that electronic self-monitoring, although intuitive and appealing, needs critical consideration and further clarification before it is implemented as a clinical tool.

  20. Improving Care for Patients With or at Risk for Chronic Kidney Disease Using Electronic Medical Record Interventions: A Pragmatic Cluster-Randomized Trial Protocol

    Directory of Open Access Journals (Sweden)

    Danielle M. Nash

    2017-04-01

    Full Text Available Background: Many patients with or at risk for chronic kidney disease (CKD in the primary care setting are not receiving recommended care. Objective: The objective of this study is to determine whether a multifaceted, low-cost intervention compared with usual care improves the care of patients with or at risk for CKD in the primary care setting. Design: A pragmatic cluster-randomized trial, with an embedded qualitative process evaluation, will be conducted. Setting: The study population comes from the Electronic Medical Record Administrative data Linked Database®, which includes clinical data for more than 140 000 rostered adults cared for by 194 family physicians in 34 clinics across Ontario, Canada. The 34 primary care clinics will be randomized to the intervention or control group. Intervention: The intervention group will receive resources from the “CKD toolkit” to help improve care including practice audit and feedback, printed educational materials for physicians and patients, electronic decision support and reminders, and implementation support. Measurements: Patients with or at risk for CKD within participating clinics will be identified using laboratory data in the electronic medical records. Outcomes will be assessed after dissemination of the CKD tools and after 2 rounds of feedback on performance on quality indicators have been sent to the physicians using information from the electronic medical records. The primary outcome is the proportion of patients aged 50 to 80 years with nondialysis-dependent CKD who are on a statin. Secondary outcomes include process of care measures such as screening tests, CKD recognition, monitoring tests, angiotensin-converting enzyme inhibitor or angiotensin receptor blocker prescriptions, blood pressure targets met, and nephrologist referral. Hierarchical analytic modeling will be performed to account for clustering. Semistructured interviews will be conducted with a random purposeful sample of

  1. The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

    Science.gov (United States)

    Shor, Erika; Fox, Catherine A.; Broach, James R.

    2013-01-01

    Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors. PMID:23935537

  2. The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

    Directory of Open Access Journals (Sweden)

    Erika Shor

    Full Text Available Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.

  3. Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

    Science.gov (United States)

    Stephan, B I; Alvarez Crespo, M C; Kemppainen, M J; Pardo, A G

    2017-05-01

    Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

  4. A Practical Strategy to Discover New Antitumor Compounds by Activating Silent Metabolite Production in Fungi by Diethyl Sulphate Mutagenesis

    Directory of Open Access Journals (Sweden)

    Shi-Ming Fang

    2014-03-01

    Full Text Available Many fungal biosynthetic pathways are silent in standard culture conditions, and activation of the silent pathways may enable access to new metabolites with antitumor activities. The aim of the present study was to develop a practical strategy for microbial chemists to access silent metabolites in fungi. We demonstrated this strategy using a marine-derived fungus Penicillium purpurogenum G59 and a modified diethyl sulphate mutagenesis procedure. Using this strategy, we discovered four new antitumor compounds named penicimutanolone (1, penicimutanin A (2, penicimutanin B (3, and penicimutatin (4. Structures of the new compounds were elucidated by spectroscopic methods, especially extensive 2D NMR analysis. Antitumor activities were assayed by the MTT method using human cancer cell lines. Bioassays and HPLC-photodiode array detector (PDAD-UV and HPLC-electron spray ionization (ESI-MS analyses were used to estimate the activated secondary metabolite production. Compounds 2 and 3 had novel structures, and 1 was a new compound belonging to a class of very rare natural products from which only four members are so far known. Compounds 1–3 inhibited several human cancer cell lines with IC50 values lower than 20 μM, and 4 inhibited the cell lines to some extent. These results demonstrated the effectiveness of this strategy to discover new compounds by activating silent fungal metabolic pathways. These discoveries provide rationale for the increased use of chemical mutagenesis strategies in silent fungal metabolite studies.

  5. Randomized Controlled Trial of Electronic Care Plan Alerts and Resource Utilization by High Frequency Emergency Department Users with Opioid Use Disorder

    Directory of Open Access Journals (Sweden)

    Niels Rathlev, MD

    2016-01-01

    Full Text Available Introduction: There is a paucity of literature supporting the use of electronic alerts for patients with high frequency emergency department (ED use. We sought to measure changes in opioid prescribing and administration practices, total charges and other resource utilization using electronic alerts to notify providers of an opioid-use care plan for high frequency ED patients. Methods: This was a randomized, non-blinded, two-group parallel design study of patients who had 1 opioid use disorder and 2 high frequency ED use. Three affiliated hospitals with identical electronic health records participated. Patients were randomized into “Care Plan” versus “Usual Care groups”. Between the years before and after randomization, we compared as primary outcomes the following: 1 opioids (morphine mg equivalents prescribed to patients upon discharge and administered to ED and inpatients; 2 total medical charges, and the numbers of; 3 ED visits, 4 ED visits with advanced radiologic imaging (computed tomography [CT] or magnetic resonance imaging [MRI] studies, and 5 inpatient admissions. Results: A total of 40 patients were enrolled. For ED and inpatients in the “Usual Care” group, the proportion of morphine mg equivalents received in the post-period compared with the pre-period was 15.7%, while in the “Care Plan” group the proportion received in the post-period compared with the pre-period was 4.5% (ratio=0.29, 95% CI [0.07-1.12]; p=0.07. For discharged patients in the “Usual Care” group, the proportion of morphine mg equivalents prescribed in the post-period compared with the pre-period was 25.7% while in the “Care Plan” group, the proportion prescribed in the post-period compared to the pre-period was 2.9%. The “Care Plan” group showed an 89% greater proportional change over the periods compared with the “Usual Care” group (ratio=0.11, 95% CI [0.01-0.092]; p=0.04. Care plans did not change the total charges, or, the numbers

  6. Genes Necessary for Bacterial Magnetite Biomineralization Identified by Transposon Mutagenesis

    Science.gov (United States)

    Nash, C. Z.; Komeili, A.; Newman, D. K.; Kirschvink, J. L.

    2004-12-01

    Magnetic bacteria synthesize nanoscale crystals of magnetite in intracellular, membrane-bounded organelles (magnetosomes). These crystals are preserved in the fossil record at least as far back as the late Neoproterozoic and have been tentatively identified in much older rocks (1). This fossil record may provide deep time calibration points for molecular evolution studies once the genes involved in biologically controlled magnetic mineralization (BCMM) are known. Further, a genetic and biochemical understanding of BCMM will give insight into the depositional environment and biogeochemical cycles in which magnetic bacteria play a role. The BCMM process is not well understood, though proteins have been identified from the magnetosome membrane and genetic manipulation and biochemical characterization of these proteins are underway. Most of the proteins currently thought to be involved are encoded within the mam cluster, a large cluster of genes whose products localize to the magnetosome membrane and are conserved among magnetic bacteria (2). In an effort to identify all of the genes necessary for bacterial BCMM, we undertook a transposon mutagenesis of Magnetospirillum magneticum AMB-1. Non-magnetic mutants (MNMs) were identified by growth in liquid culture followed by a magnetic assay. The insertion site of the transposon was identified two ways. First MNMs were screened with a PCR assay to determine if the transposon had inserted into the mam cluster. Second, the transposon was rescued from the mutant DNA and cloned for sequencing. The majority insertion sites are located within the mam cluster. Insertion sites also occur in operons which have not previously been suspected to be involved in magnetite biomineralization. None of the insertion sites have occurred within genes reported from previous transposon mutagenesis studies of AMB-1 (3, 4). Two of the non-mam cluster insertion sites occur in operons containing genes conserved particularly between MS-1 and MC-1. We

  7. Radiation-induced mutagenesis of antifungal metabolite producing bacillus sp. HKA-17

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Keun; Senthilkumar, M. [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-09-15

    Bacillus sp. Strain HKA-17, isolated from the surface sterilized root nodule of Glycine max, inhibited several fungal plant pathogens. It produced a diffusible extracellular antifungal metabolite that was extracted with n-butanol. The crude extract was purified through Superdex{sup TM} 75 10/300 GL FPLC column. FT-IR spectrum of the FPLC purified-antifungal metabolite confirmed the presence of peptide and glycosidic bonds in its structure. Gamma induced mutagenesis of HKA-17 was carried out at an LD{sub 99} dose (8.46 kGy) to generate a mutant library. By screening the mutant library through a duel plate assay with Alternaria alternata, we selected one mutant with enhanced biocontrol activity (HKA-17e1) and two defective mutants (HKA-17d1 and HKA-17d2). Overproducing mutant recorded the largest inhibition zone (16.25 {+-} 0.86 mm) compared to any other mutant clone as well as wild type, and could be used as a potential biocontrol agent for plant disease suppression. The effect of HKA-17 antifungal metabolite on hyphal morphology was clearly demonstrated through scanning electron microscopy. The crude extract of defective mutant HKA-17 d1 did not induce any changes in hyphal morphology of A. alternata. However, antifungal metabolites of HKA-17 induced abnormal hyphal structures such as hyphal shrivelling, the bulging and swelling of intercalary cells, fragmentation, and cell lysis.

  8. Electron and high-dose-rate brachytherapy boost in the conservative treatment of stage I-II breast cancer. First results of the randomized Budapest boost trial

    Energy Technology Data Exchange (ETDEWEB)

    Polgar, C.; Fodor, J. [National Institute of Oncology, Department of Radiotherapy, Budapest (Hungary); Orosz, Z. [National Institute of Oncology, Center of Experimental and Human Tumor Pathology Budapest (HU)] [and others

    2002-11-01

    Background and Aims: To evaluate the effect of electron and high-dose-rate brachytherapy (HDR BT) boost on local tumor control (LTC), side effects and cosmesis after breast-conserving surgery (BCS) in a prospective randomized study. Patients and Methods: 207 women with stage I-II breast cancer who underwent BCS were treated by 50 Gy irradiation to the whole breast and then randomly assigned to receive either a boost to the tumor bed (n=104) or no further radiotherapy (n=103). Boost treatments consisted of either 16 Gy electron irradiation (n=52) or 12-14.25 Gy HDR BT (n=52). Breast cancer-related events, side effects, and cosmetic results were assessed. Results: At a median follow-up of 5.3 years, the crude rate of local recurrence was 6.7% (7/104) with and 15.5% (16/103) without boost. The 5-year probability of LTC, relapse-free survival (RFS), and cancer-specific survival (CSS) was 92.7% vs 84.9% (p=0.049), 76.6% vs 66.2% (p=0.044), and 90.4% vs. 82.1% (p=0.053), respectively. There was no significant difference in LTC between patients treated with electron or HDR BT boost (94.2% vs 91.4%; p=0.74). On multivariate analysis, patient age <40 years (RR: 4.53), positive margin status (RR: 4.17), and high mitotic activity index (RR: 3.60) were found to be significant risk factors for local recurrence. The incidence of grade 2-3 side effects was higher in the boost arm (17.3% vs 7.8%; p=0.03). However, the rate of excellent/good cosmetic results was similar for the two arms (85.6% vs 91.3%; p=0.14). Cosmesis was rated as excellent/good in 88.5% of patients treated with HDR BT and 82.7% of patients with electron boost (p=0.29). Conclusions: Boost dose significantly improves LTC and RFS in patients treated with BCS and radiotherapy. In spite of the higher incidence of late side effects in the boost arm, boost dose is strongly recommended for patients at high risk for local recurrence. Positive or close margin status, high mitotic activity index, and young patient age

  9. CRISPR/Cas9-mediated targeted mutagenesis in grape.

    Science.gov (United States)

    Nakajima, Ikuko; Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi; Endo, Masaki

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

  10. Overproduction of Clavulanic Acid by UV Mutagenesis of Streptomyces clavuligerus.

    Science.gov (United States)

    Korbekandi, Hassan; Darkhal, Parisa; Hojati, Zohreh; Abedi, Daryoush; Hamedi, Javad; Pourhosein, Meraj

    2010-01-01

    Clavulanic acid is produced industrially by fermentation of Streptomyces clavuligerus and researches have increased its production by strain improvement, recombinant DNA technology, and media composition and growth condition optimization. The main objective of this study was to increase the level of clavulanic acid production from Streptomyces clavuligerus (DSM 738), using UV irradiation. After incubation, the spores and aerial mycelia were scraped off the agar plate by a sterile loop. After passing through a cotton wool, the serially diluted spore suspension was spread on GYM- agar containing caffeine. The plates were irradiated with UV light, wrapped in aluminum foil and incubated. The colonies were sub-cultured again to express the mutations. An aliquot of the spore suspension prepared from the resulted culture was poured in GYM agar plates and incubated. The plates were overlaid with nutrient-agar containing penicillin G and Klebsiela pneumoniae, and incubated. The inhibition zone diameter was measured and compared with the wild type colony. Repeating this procedure, the overproducer mutants were selected. Concentration of clavulanic acid was determined by HPLC analysis. It was concluded that secondary metabolites, mainly antibiotics containing clavulanic acid, were produced about 6-7 days after the growth, and concentration of clavulanic acid was increased up to two-folds after UV mutagenesis.

  11. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

    Directory of Open Access Journals (Sweden)

    Takashi Nakanishi

    Full Text Available The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18-47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.

  12. Overproduction of Clavulanic Acid by UV Mutagenesis of Streptomyces clavuligerus

    Science.gov (United States)

    Korbekandi, Hassan; Darkhal, Parisa; Hojati, Zohreh; Abedi, Daryoush; Hamedi, Javad; Pourhosein, Meraj

    2010-01-01

    Clavulanic acid is produced industrially by fermentation of Streptomyces clavuligerus and researches have increased its production by strain improvement, recombinant DNA technology, and media composition and growth condition optimization. The main objective of this study was to increase the level of clavulanic acid production from Streptomyces clavuligerus (DSM 738), using UV irradiation. After incubation, the spores and aerial mycelia were scraped off the agar plate by a sterile loop. After passing through a cotton wool, the serially diluted spore suspension was spread on GYM- agar containing caffeine. The plates were irradiated with UV light, wrapped in aluminum foil and incubated. The colonies were sub-cultured again to express the mutations. An aliquot of the spore suspension prepared from the resulted culture was poured in GYM agar plates and incubated. The plates were overlaid with nutrient-agar containing penicillin G and Klebsiela pneumoniae, and incubated. The inhibition zone diameter was measured and compared with the wild type colony. Repeating this procedure, the overproducer mutants were selected. Concentration of clavulanic acid was determined by HPLC analysis. It was concluded that secondary metabolites, mainly antibiotics containing clavulanic acid, were produced about 6–7 days after the growth, and concentration of clavulanic acid was increased up to two-folds after UV mutagenesis. PMID:24363725

  13. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.

    Science.gov (United States)

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Mallet, Maxime; Couchy, Gabrielle; Letouzé, Eric; Pilati, Camilla; Verret, Benjamin; Blanc, Jean-Frédéric; Balabaud, Charles; Calderaro, Julien; Laurent, Alexis; Letexier, Mélanie; Bioulac-Sage, Paulette; Calvo, Fabien; Zucman-Rossi, Jessica

    2015-10-01

    Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC.

  14. Oligonucleotide-directed mutagenesis for precision gene editing.

    Science.gov (United States)

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

    Science.gov (United States)

    Lu, Yuanyuan; Xiao, Shuqin; Wang, Fen; Sun, Jiaying; Zhao, Likun; Yan, Libin; Xue, Chunsheng

    2017-02-01

    An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. The effect of the random distribution of electronic components in the output characteristics of the Howland current source

    Science.gov (United States)

    Bertemes-Filho, P.; Felipe, A.

    2013-04-01

    When a Howland source is designed, the components are chosen so that the designed source has the desired characteristics. However, the operational amplifier limitations and resistor tolerances causes undesired behaviours. This work proposes to take in account the influence of the random distribution of the commercial resistors in the Howland circuit over the frequency range of 10 Hz to 10 MHz. The probability density function due to small changes over the resistors was calculated by using an analytical model. Results show that both output current and impedance are very sensitive to the resistor tolerances. It is shown that the output impedance is very dependent on the open-loop gain of the Opamp rather than the resistor tolerances, especially at higher frequencies. This might improve the implementations of real current source used in electrical bioimpedance.

  17. Random walk approach to spin dynamics in a two-dimensional electron gas with spin-orbit coupling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Luyi; Orenstein, J.; Lee, Dung-Hai

    2010-09-27

    We introduce and solve a semiclassical random walk (RW) model that describes the dynamics of spin polarization waves in zinc-blende semiconductor quantum wells. We derive the dispersion relations for these waves, including the Rashba, linear and cubic Dresselhaus spin-orbit interactions, as well as the effects of an electric field applied parallel to the spin polarization wave vector. In agreement with calculations based on quantum kinetic theory [P. Kleinert and V. V. Bryksin, Phys. Rev. B 76, 205326 (2007)], the RW approach predicts that spin waves acquire a phase velocity in the presence of the field that crosses zero at a nonzero wave vector, q{sub 0}. In addition, we show that the spin-wave decay rate is independent of field at q{sub 0} but increases as (q-q{sub 0}){sup 2} for q {ne} q{sub 0}. These predictions can be tested experimentally by suitable transient spin grating experiments.

  18. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

    Directory of Open Access Journals (Sweden)

    Feixiong Cheng

    2016-09-01

    Full Text Available Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase. Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline that may be potential for antiviral indication (e.g. anti-Ebola. In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

  19. Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

    OpenAIRE

    Masker, W E; Dodson, L A; Maupin, M

    1985-01-01

    We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.

  20. Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

    Science.gov (United States)

    Masker, W E; Dodson, L A; Maupin, M

    1985-01-01

    We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested. PMID:3903213

  1. Identifying novel mycobacterial stress associated genes using a random mutagenesis screen in Mycobacterium smegmatis.

    Science.gov (United States)

    Viswanathan, Gopinath; Joshi, Shrilaxmi V; Sridhar, Aditi; Dutta, Sayantanee; Raghunand, Tirumalai R

    2015-12-10

    Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. A Randomized Controlled Trial of Real-Time Electronic Adherence Monitoring With Text Message Dosing Reminders in People Starting First-Line Antiretroviral Therapy.

    Science.gov (United States)

    Orrell, Catherine; Cohen, Karen; Mauff, Katya; Bangsberg, David R; Maartens, Gary; Wood, Robin

    2015-12-15

    There are conflicting findings about whether mobile phone text message reminders impact on antiretroviral adherence. We hypothesized that text reminders sent when dosing was late would improve adherence and HIV viral suppression. Antiretroviral therapy (ART)-naive participants, from a South African outpatient ART clinic, were randomized to standard of care (SoC, 3 pretreatment education sessions), or intervention (SoC and automated text reminders if dosing >30 minutes late). Dosing time was recorded by real-time electronic adherence monitoring devices, given to participants at ART start. CD4 cell count and HIV RNA were determined at baseline, 16 and 48 weeks. Primary outcome was cumulative adherence execution by electronic adherence monitoring device. HIV-1 viral suppression (TIs) >72 hours were secondary outcomes. Analysis was by intention to treat (missing = failure). Registration was with the Pan-African Clinical Trials Registry: PACTR201311000641402. A total of 230 participants were randomly assigned to control (n = 115) or intervention (n = 115) arms. Median adherence was 82.1% (interquartile range, 56.6%-94.6%) in the intervention arm, compared with 80.4% (interquartile range, 52.8%-93.8%) for SoC [adjusted odds ratio for adherence 1.08; 95% confidence interval (CI): 0.77 to 1.52]. Suppressed HIV RNA (TIs of >72 hours was reduced (adjusted incident rate ratio, 0.84; 95% CI: 0.75 to 0.94). Text message reminders linked to late doses detected by real-time adherence monitoring reduced the number of prolonged TIs, but did not significantly improve adherence or viral suppression.

  3. Atomic force microscopy and scanning electron microscopy evaluation of efficacy of scaling and root planing using magnification: A randomized controlled clinical study

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    Ranjana Mohan

    2013-01-01

    Full Text Available Aim: A randomized controlled clinical study was undertaken to evaluate the effectiveness of scaling and root planing (SRP by using Magnifying Loupes (ML and dental operating microscope (DOM. Materials and Methods: A total of 90 human teeth scheduled for extraction from 18 patients aged between 25 and 65 years suffering from generalized chronic severe periodontitis were randomly assigned to three treatment groups. Group 1 consisted SRP performed without using magnification (unaided, Group 2-SRP with ML and Group 3-SRP with DOM. Following extractions, samples were prepared for (i evaluation of surface topography by atomic force microscopy, (ii presence of smear layer, debris by scanning electron microscopy (iii elemental analysis by energy dispersive X-ray analysis. Data was subjected to statistical analysis using analysis of variance, post-hoc (Tukey-HSD and Chi-square test. Results: Statistically significant (P < 0.001 difference was found among the different treatment groups. Group 3 was the best while Group 1 was the least effective technique for SRP. Order of efficacy in terms of the surface was found to be - Palatal < Lingual < Distal ≅ Mesial < Buccal. Efficiency in mandibular to maxillary teeth was found to be significant (P < 0.05, also anterior to posterior teeth (P < 0.05. Conclusion: Magnification tools significantly enhance the efficacy of supragingival and subgingival SRP.

  4. Evaluation of three different manual techniques of sharpening curettes through a scanning electron microscope: a randomized controlled experimental study.

    Science.gov (United States)

    Di Fiore, A; Mazzoleni, S; Fantin, F; Favero, L; De Francesco, M; Stellini, E

    2015-05-01

    The purpose of this study was to compare the effectiveness of three different techniques for manually sharpening of periodontal curettes (PCs) by examining the blades with the aid of scanning electron microscope (SEM). Three groups were considered based on three sharpening methods used: group A (moving a PC over a stationary stone); group B (moving a stone over a stationary PC) and group C (moving a PC over a stone fixed, placed on a 'sharpening horse'). After the sharpening, the blades were examined using SEM. The SEM images were assessed independently by five different independent observers. An evaluation board was used to assign a value to each image. A preliminary pilot study was conducted to establish the number of samples. Pearson's correlation test was used to assess the correlations between measurements. anova test with Bonferroni's post hoc test was used to compare the three groups. Sixty PCs (20 PCs per group) were used in this study. Statistically significant differences emerged between the three groups (P-value = 0.001). Bonferroni's test showed that the difference between groups A and B was not statistically significant (P-value = 0.80), while it was significant for the comparisons between groups A and C (P-value = 0.005) and between groups B and C (P-value = 0.001). The sharpening technique used in group C, which involved the use of the sharpening horse, proved the most effective. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

    Energy Technology Data Exchange (ETDEWEB)

    Butterfield, D. Allan; Colvin, Joshua; Liu Jiangling; Wang Jianquan; Bachas, Leonidas; Bhattacharrya, Dibakar

    2002-10-11

    The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane >> polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic

  6. Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

    Science.gov (United States)

    Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

    2017-01-01

    Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016

  7. The Seamless Transfer-of-Care Protocol: a randomized controlled trial assessing the efficacy of an electronic transfer-of-care communication tool

    Directory of Open Access Journals (Sweden)

    Okoniewska Barbara M

    2012-11-01

    Full Text Available Abstract Background The transition between acute care and community care represents a vulnerable period in health care delivery. The vulnerability of this period has been attributed to changes to patients’ medication regimens during hospitalization, failure to reconcile discrepancies between admission and discharge and the burdening of patients/families to take over care responsibilities at discharge and to relay important information to the primary care physician. Electronic communication platforms can provide an immediate link between acute care and community care physicians (and other community providers, designed to ensure consistent information transfer. This study examines whether a transfer-of-care (TOC communication tool is efficacious and cost-effective for reducing hospital readmission, adverse events and adverse drug events as well as reducing death. Methods A randomized controlled trial conducted on the Medical Teaching Unit of a Canadian tertiary care centre will evaluate the efficacy and cost-effectiveness of a TOC communication tool. Medical in-patients admitted to the unit will be considered for this study. Data will be collected upon admission, and a total of 1400 patients will be randomized. The control group’s acute care stay will be summarized using a traditional dictated summary, while the intervention group will have a summary generated using the TOC communication tool. The primary outcome will be a composite, at 3 months, of death or readmission to any Alberta acute-care hospital. Secondary outcomes will be the occurrence of post-discharge adverse events and adverse drug events at 1 month post discharge. Patients with adverse outcomes will have their cases reviewed by two Royal College certified internists or College-certified family physicians, blinded to patients’ group assignments, to determine the type, severity, preventability and ameliorability of all detected adverse outcomes. An accompanying economic

  8. The impact of self-interviews on response patterns for sensitive topics: a randomized trial of electronic delivery methods for a sexual behaviour questionnaire in rural South Africa

    Directory of Open Access Journals (Sweden)

    Guy Harling

    2017-08-01

    Full Text Available Abstract Background Self-interviews, where the respondent rather than the interviewer enters answers to questions, have been proposed as a way to reduce social desirability bias associated with interviewer-led interviews. Computer-assisted self-interviews (CASI are commonly proposed since the computer programme can guide respondents; however they require both language and computer literacy. We evaluated the feasibility and acceptability of using electronic methods to administer quantitative sexual behaviour questionnaires in the Somkhele demographic surveillance area (DSA in rural KwaZulu-Natal, South Africa. Methods We conducted a four-arm randomized trial of paper-and-pen-interview, computer-assisted personal-interview (CAPI, CASI and audio-CASI with an age-sex-urbanicity stratified sample of 504 adults resident in the DSA in 2015. We compared respondents’ answers to their responses to the same questions in previous surveillance rounds. We also conducted 48 cognitive interviews, dual-coding responses using the Framework approach. Results Three hundred forty (67% individuals were interviewed and covariates and participation rates were balanced across arms. CASI and audio-CASI were significantly slower than interviewer-led interviews. Item non-response rates were higher in self-interview arms. In single-paper meta-analysis, self-interviewed individuals reported more socially undesirable sexual behaviours. Cognitive interviews found high acceptance of both self-interviews and the use of electronic methods, with some concerns that self-interview methods required more participant effort and literacy. Conclusions Electronic data collection methods, including self-interview methods, proved feasible and acceptable for completing quantitative sexual behaviour questionnaires in a poor, rural South African setting. However, each method had both benefits and costs, and the choice of method should be based on context-specific criteria.

  9. Electronic feedback in a diet- and physical activity-based lifestyle intervention for weight loss: a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Meriwether Rebecca A

    2011-05-01

    Full Text Available Abstract Background The SenseWear™ Armband (SWA (BodyMedia, Inc. Pittsburgh, PA is a physical activity and lifestyle monitor that objectively and accurately measures free-living energy balance and sleep and includes software for self-monitoring of daily energy expenditure and energy intake. The real-time feedback of the SWA can improve individual self-monitoring and, therefore, enhance weight loss outcomes. Methods We recruited 197 sedentary overweight or obese adults (age, 46.8 ± 10.8 y; body mass index (BMI, 33.3 ± 5.2 kg/m2; 81% women, 32% African-American from the greater Columbia, South Carolina area. Participants were randomized into 1 of 4 groups, a self-directed weight loss program via an evidence-based weight loss manual (Standard Care, n = 50, a group-based behavioral weight loss program (GWL, n = 49, the armband alone (SWA-alone, n = 49, or the GWL plus the armband (GWL+SWA, n = 49, during the 9-month intervention. The primary outcome was change in body weight and waist circumference. A mixed-model repeated-measures analysis compared change in the intervention groups to the standard care group on weight and waist circumference status after adjusting for age, sex, race, education, energy expenditure, and recruitment wave. Results Body weight was available for 62% of participants at 9 months (52% standard care, 70% intervention. There was significant weight loss in all 3 intervention groups (GWL, 1.86 kg, P = 0.05; SWA-alone, 3.55 kg, P = 0.0002; GWL+SWA, 6.59 kg, P Conclusions Continuous self-monitoring from wearable technology with real-time feedback may be particularly useful to enhance lifestyle changes that promote weight loss in sedentary overweight or obese adults. This strategy, combined with a group-based behavioral intervention, may yield optimal weight loss. Trial Registration ClinicalTrials.gov: NCT00957008

  10. Electronic feedback in a diet- and physical activity-based lifestyle intervention for weight loss: a randomized controlled trial.

    Science.gov (United States)

    Shuger, Sara L; Barry, Vaughn W; Sui, Xuemei; McClain, Amanda; Hand, Gregory A; Wilcox, Sara; Meriwether, Rebecca A; Hardin, James W; Blair, Steven N

    2011-05-18

    The SenseWear™ Armband (SWA) (BodyMedia, Inc. Pittsburgh, PA) is a physical activity and lifestyle monitor that objectively and accurately measures free-living energy balance and sleep and includes software for self-monitoring of daily energy expenditure and energy intake. The real-time feedback of the SWA can improve individual self-monitoring and, therefore, enhance weight loss outcomes. We recruited 197 sedentary overweight or obese adults (age, 46.8 ± 10.8 y; body mass index (BMI), 33.3 ± 5.2 kg/m(2); 81% women, 32% African-American) from the greater Columbia, South Carolina area. Participants were randomized into 1 of 4 groups, a self-directed weight loss program via an evidence-based weight loss manual (Standard Care, n = 50), a group-based behavioral weight loss program (GWL, n = 49), the armband alone (SWA-alone, n = 49), or the GWL plus the armband (GWL+SWA, n = 49), during the 9-month intervention. The primary outcome was change in body weight and waist circumference. A mixed-model repeated-measures analysis compared change in the intervention groups to the standard care group on weight and waist circumference status after adjusting for age, sex, race, education, energy expenditure, and recruitment wave. Body weight was available for 62% of participants at 9 months (52% standard care, 70% intervention). There was significant weight loss in all 3 intervention groups (GWL, 1.86 kg, P = 0.05; SWA-alone, 3.55 kg, P = 0.0002; GWL+SWA, 6.59 kg, P < 0.0001) but not in the Standard Care group (0.89 kg, P = 0.39) at month 9. Only the GWL+SWA group achieved significant weight loss at month 9 compared to the Standard Care group (P = 0.04). Significant waist circumference reductions were achieved in all 4 groups at month 9 (Standard Care, 3.49 cm, P = 0.0004; GWL, 2.42 cm, P = 0.008; SWA-alone, 3.59 cm, P < 0.0001; GWL+SWA, 6.77 cm, P < 0.0001), but no intervention group had significantly reduced waist circumference compared to the Standard Care group. Continuous

  11. MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites.

    Science.gov (United States)

    Tang, Lixia; Wang, Xiong; Ru, Beibei; Sun, Hengfei; Huang, Jian; Gao, Hui

    2014-06-01

    Recent computational and bioinformatics advances have enabled the efficient creation of novel biocatalysts by reducing amino acid variability at hot spot regions. To further expand the utility of this strategy, we present here a tool called Multi-site Degenerate Codon Analyzer (MDC-Analyzer) for the automated design of intelligent mutagenesis libraries that can completely cover user-defined randomized sequences, especially when multiple contiguous and/or adjacent sites are targeted. By initially defining an objective function, the possible optimal degenerate PCR primer profiles could be automatically explored using the heuristic approach of Greedy Best-First-Search. Compared to the previously developed DC-Analyzer, MDC-Analyzer allows for the existence of a small amount of undesired sequences as a tradeoff between the number of degenerate primers and the encoded library size while still providing all the benefits of DC-Analyzer with the ability to randomize multiple contiguous sites. MDC-Analyzer was validated using a series of randomly generated mutation schemes and experimental case studies on the evolution of halohydrin dehalogenase, which proved that the MDC methodology is more efficient than other methods and is particularly well-suited to exploring the sequence space of proteins using data-driven protein engineering strategies.

  12. Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae.

    Science.gov (United States)

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Lin, Ling

    2014-07-01

    Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional

  13. Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

    Directory of Open Access Journals (Sweden)

    Qizhi Liu

    Full Text Available Retrovirus (RV is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.

  14. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    Science.gov (United States)

    Bulina, Maria E; Chudakov, Dmitry M; Mudrik, Nikolay N; Lukyanov, Konstantin A

    2002-01-01

    Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. PMID:11972899

  15. Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

    Science.gov (United States)

    Liu, Qizhi; Wang, Yunzhi; Lin, Fan; Zhang, Lei; Li, Yan; Ge, Ruowen; Hong, Yunhan

    2015-01-01

    Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.

  16. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    Directory of Open Access Journals (Sweden)

    Mudrik Nikolay N

    2002-04-01

    Full Text Available Abstract Background Within the family of green fluorescent protein (GFP homologs, one can mark two main groups, specifically, fluorescent proteins (FPs and non-fluorescent or chromoproteins (CPs. Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595 from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield ( Conclusions We located a novel point in asCP sequence (position 165 mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

  17. Impact of an electronic monitoring device and behavioral feedback on adherence to multiple sclerosis therapies in youth: results of a randomized trial.

    Science.gov (United States)

    Yeh, E Ann; Grover, Stephanie A; Powell, Victoria E; Alper, Gulay; Banwell, Brenda L; Edwards, Kim; Gorman, Mark; Graves, Jennifer; Lotze, Timothy E; Mah, Jean K; Mednick, Lauren; Ness, Jayne; Obadia, Maya; Slater, Ruth; Waldman, Amy; Waubant, Emmanuelle; Schwartz, Carolyn E

    2017-09-01

    To report the results of a randomized controlled trial using an electronic monitoring device (EM) plus a motivational interviewing (MI) intervention to enhance adherence to disease-modifying therapies (DMT) in pediatric MS. Fifty-two youth with MS (16.03 ± 2.2 years) were randomized to receive either MI (n = 25) (target intervention) or a MS medication video (n = 27) (attention control). Primary endpoint was change in adherence. Secondary outcomes included changes in quality of life, well-being and self-efficacy. Random effects modeling and Cohen's effect size computation evaluated intervention impact. Longitudinal random effect models revealed that the MI group decreased their EM adherence (GroupxTime interaction = -0.19), while increasing frequency of parental DMT reminder (26.01)/administration (11.69). We found decreased EM use in the MI group at 6 months (Cohen's d = -0.61), but increased pharmacy refill adherence (d = 0.23). Parental reminders about medication increased in MI subjects vs controls (d = 0.59 at 3 months; d = 0.70 at 6 months). We found increases in self-reported adherence (d = 0.21) at 3 but not 6 months, fewer barriers to adherence at three (d = -0.58) and six months (d = -0.31), better physical (d = 0.23 at 3 months; d = 0.45 at 6 months), emotional (d = 0.25 at 3 months) and self-efficacy function (d = 0.55 at 3 months; 0.48 at 6 months), but worse well-being, including self-acceptance (d = -0.53 at 6 months) and environmental mastery (d = -0.42 at 3 and 6 months) in intervention as compared to control patients. Participants receiving MI + EM experienced worsening on objective measures of adherence and increased parental involvement, but improved on some self- and parent-reported measures. MI participants reported improvements in quality of life and self-efficacy, but worsened well-being.

  18. Electronic symptom reporting between patient and provider for improved health care service quality: a systematic review of randomized controlled trials. part 1: state of the art.

    Science.gov (United States)

    Johansen, Monika Alise; Henriksen, Eva; Horsch, Alexander; Schuster, Tibor; Berntsen, Gro K Rosvold

    2012-10-03

    Over the last two decades, the number of studies on electronic symptom reporting has increased greatly. However, the field is very heterogeneous: the choices of patient groups, health service innovations, and research targets seem to involve a broad range of foci. To move the field forward, it is necessary to build on work that has been done and direct further research to the areas holding most promise. Therefore, we conducted a comprehensive review of randomized controlled trials (RCTs) focusing on electronic communication between patient and provider to improve health care service quality, presented in two parts. Part 2 investigates the methodological quality and effects of the RCTs, and demonstrates some promising benefits of electronic symptom reporting. To give a comprehensive overview of the most mature part of this emerging field regarding (1) patient groups, (2) health service innovations, and (3) research targets relevant to electronic symptom reporting. We searched Medline, EMBASE, PsycINFO, Cochrane Central Register of Controlled Trials, and IEEE Xplore for original studies presented in English-language articles published from 1990 to November 2011. Inclusion criteria were RCTs of interventions where patients or parents reported health information electronically to the health care system for health care purposes and were given feedback. Of 642 records identified, we included 32 articles representing 29 studies. The included articles were published from 2002, with 24 published during the last 5 years. The following five patient groups were represented: respiratory and lung diseases (12 studies), cancer (6), psychiatry (6), cardiovascular (3), and diabetes (1). In addition to these, 1 study had a mix of three groups. All included studies, except 1, focused on long-term conditions. We identified four categories of health service innovations: consultation support (7 studies), monitoring with clinician support (12), self-management with clinician support (9

  19. From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

    Science.gov (United States)

    Pacher, Michael; Puchta, Holger

    2017-05-01

    Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  20. Mitochondrial mutagenesis induced by tumor-specific radiation bystander effects.

    LENUS (Irish Health Repository)

    Gorman, Sheeona

    2012-02-01

    The radiation bystander effect is a cellular process whereby cells not directly exposed to radiation display cellular alterations similar to directly irradiated cells. Cellular targets including mitochondria have been postulated to play a significant role in this process. In this study, we utilized the Random Mutation Capture assay to quantify the levels of random mutations and deletions in the mitochondrial genome of bystander cells. A significant increase in the frequency of random mitochondrial mutations was found at 24 h in bystander cells exposed to conditioned media from irradiated tumor explants (p = 0.018). CG:TA mutations were the most abundant lesion induced. A transient increase in the frequency of random mitochondrial deletions was also detected in bystander cells exposed to conditioned media from tumor but not normal tissue at 24 h (p = 0.028). The increase in both point mutations and deletions was transient and not detected at 72 h. To further investigate mitochondrial dysfunction, mitochondrial membrane potential and reactive oxygen species were assessed in these bystander cells. There was a significant reduction in mitochondrial membrane potential and this was positively associated with the frequency of random point mutation and deletions in bystander cells treated with conditioned media from tumor tissue (r = 0.71, p = 0.02). This study has shown that mitochondrial genome alterations are an acute consequence of the radiation bystander effect secondary to mitochondrial dysfunction and suggests that this cannot be solely attributable to changes in ROS levels alone.

  1. 2012 Gordon Research Conference on Mutagenesis - Formal Schedule and Speaker/Poster Program

    Energy Technology Data Exchange (ETDEWEB)

    Demple, Bruce [Stony Brook Univ., NY (United States). School of Medicine

    2012-08-24

    The delicate balance among cellular pathways that control mutagenic changes in DNA will be the focus of the 2012 Mutagenesis Gordon Research Conference. Mutagenesis is essential for evolution, while genetic stability maintains cellular functions in all organisms from microbes to metazoans. Different systems handle DNA lesions at various times of the cell cycle and in different places within the nucleus, and inappropriate actions can lead to mutations. While mutation in humans is closely linked to disease, notably cancers, mutational systems can also be beneficial. The conference will highlight topics of beneficial mutagenesis, including full establishment of the immune system, cell survival mechanisms, and evolution and adaptation in microbial systems. Equal prominence will be given to detrimental mutation processes, especially those involved in driving cancer, neurological diseases, premature aging, and other threats to human health. Provisional session titles include Branching Pathways in Mutagenesis; Oxidative Stress and Endogenous DNA Damage; DNA Maintenance Pathways; Recombination, Good and Bad; Problematic DNA Structures; Localized Mutagenesis; Hypermutation in the Microbial World; and Mutation and Disease.

  2. CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (Brassica napus).

    Science.gov (United States)

    Braatz, Janina; Harloff, Hans-Joachim; Mascher, Martin; Stein, Nils; Himmelbach, Axel; Jung, Christian

    2017-06-01

    In polyploid species, altering a trait by random mutagenesis is highly inefficient due to gene redundancy. We have stably transformed tetraploid oilseed rape (Brassica napus) with a CRISPR-Cas9 construct targeting two ALCATRAZ (ALC) homoeologs. ALC is involved in valve margin development and, thus, contributes to seed shattering from mature fruits. Knocking out ALC would increase shatter resistance to avoid seed loss during mechanical harvest. We obtained a transgenic T1 plant with four alc mutant alleles by the use of a single target sequence. All mutations were stably inherited to the T2 progeny. The T2 generation was devoid of any wild-type alleles, proving that the underlying T1 was a nonchimeric double heterozygote. T-DNA and ALC loci were not linked, as indicated by random segregation in the T2 generation. Hence, we could select double mutants lacking the T-DNA already in the first offspring generation. However, whole-genome sequencing data revealed at least five independent insertions of vector backbone sequences. We did not detect any off-target effects in two genome regions homologous to the target sequence. The simultaneous alteration of multiple homoeologs by CRISPR-Cas9 mutagenesis without any background mutations will offer new opportunities for using mutant genotypes in rapeseed breeding. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Sub-lethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis

    Science.gov (United States)

    Kohanski, Michael A.; DePristo, Mark A.; Collins, James J.

    2010-01-01

    Summary Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here we show that sub-lethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS, and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sub-lethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sub-lethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics. PMID:20159551

  4. Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

    Science.gov (United States)

    Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

    2016-07-31

    Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

  5. Random nanowires of nickel doped TiO2 with high surface area and electron mobility for high efficiency dye-sensitized solar cells.

    Science.gov (United States)

    Archana, P S; Naveen Kumar, E; Vijila, C; Ramakrishna, S; Yusoff, M M; Jose, R

    2013-01-28

    Mesoporous TiO(2) with a large specific surface area (~150 m(2) g(-1)) is the most successful material in dye-sensitized solar cells so far; however, its inferior charge mobility is a major efficiency limiter. This paper demonstrates that random nanowires of Ni-doped TiO(2) (Ni:TiO(2)) have a dramatic influence on the particulate and charge transport properties. Nanowires (dia ~60 nm) of Ni:TiO(2) with a specific surface area of ~80 m(2) g(-1) were developed by an electrospinning technique. The band gap of the Ni:TiO(2) shifted to the visible region upon doping of 5 at% Ni atoms. The Mott-Schottky analysis shows that the flat band potential of Ni:TiO(2) shifts to a more negative value than the undoped samples. The electrochemical impedance spectroscopic measurements showed that the Ni:TiO(2) offer lower charge transport resistance, higher charge recombination resistance, and enhanced electron lifetime compared to the undoped samples. The dye-sensitized solar cells fabricated using the Ni:TiO(2) nanowires showed an enhanced photoconversion efficiency and short-circuit current density compared to the undoped analogue. The transient photocurrent measurements showed that the Ni:TiO(2) has improved charge mobility compared with TiO(2) and is several orders of magnitude higher compared to the P25 particles.

  6. Evaluation of sealing ability of Biodentine™ and mineral trioxide aggregate in primary molars using scanning electron microscope: A randomized controlled in vitro trial

    Directory of Open Access Journals (Sweden)

    Allwyn Samuel

    2016-01-01

    Full Text Available Objective: The aim of this study was to compare the sealing ability of mineral trioxide aggregate (MTA and Biodentine™ when used to repair the furcal perforations in primary molars using scanning electron microscope (SEM. Study Design: The study sample comprised forty recently extracted primary molars. These teeth were placed in a 5.25% sodium hypochlorite solution for 24 h and washed with tap water. Access cavities were made using a round bur in high-speed handpiece. Perforations were made in the center of the floor of the pulpal chamber using a 0.5 mm round bur. The teeth were randomly assigned into two experimental groups based on the material used to seal the perforation: Group A - MTA and Group B - Biodentine™. The packed materials were allowed to set for 24 h. The samples were sectioned longitudinally and the extent of marginal adaptation was measured by SEM. Wilcoxon-signed rank test was used for statistical analysis using SPSS software. Results: All teeth exhibited microleakage, but Biodentine™ showed significantly less leakage (0.149 compared to MTA (0.583. Conclusion: Based on the results of this study, Biodentine™ showed lesser microleakage compared to MTA and thus may be a good alternative to MTA.

  7. Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

    DEFF Research Database (Denmark)

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

    2015-01-01

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER...... cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein...

  8. Ultrafast solvation dynamics at internal site of staphylococcal nuclease investigated by site-directed mutagenesis

    CERN Document Server

    Guang-yu, Gao; Wei, Wang; Shu-feng, Wang; Zhong, Dongping; Qi-huang, Gong

    2014-01-01

    Solvation is essential for protein activities. To study internal solvation of protein, site-directed mutagenesis is applied. Intrinsic fluorescent probe, tryptophan, is inserted into desired position inside protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics researches. We introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside caves of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at internal sites of the caves indicate clear characteristics of local environment. These solvation behaviors correlated to the enzyme activity directly.

  9. Effect of a Price Transparency Intervention in the Electronic Health Record on Clinician Ordering of Inpatient Laboratory Tests: The PRICE Randomized Clinical Trial.

    Science.gov (United States)

    Sedrak, Mina S; Myers, Jennifer S; Small, Dylan S; Nachamkin, Irving; Ziemba, Justin B; Murray, Dana; Kurtzman, Gregory W; Zhu, Jingsan; Wang, Wenli; Mincarelli, Deborah; Danoski, Daniel; Wells, Brian P; Berns, Jeffrey S; Brennan, Patrick J; Hanson, C William; Dine, C Jessica; Patel, Mitesh S

    2017-07-01

    Many health systems are considering increasing price transparency at the time of order entry. However, evidence of its impact on clinician ordering behavior is inconsistent and limited to single-site evaluations of shorter duration. To test the effect of displaying Medicare allowable fees for inpatient laboratory tests on clinician ordering behavior over 1 year. The Pragmatic Randomized Introduction of Cost data through the electronic health record (PRICE) trial was a randomized clinical trial comparing a 1-year intervention to a 1-year preintervention period, and adjusting for time trends and patient characteristics. The trial took place at 3 hospitals in Philadelphia between April 2014 and April 2016 and included 98 529 patients comprising 142 921 hospital admissions. Inpatient laboratory test groups were randomly assigned to display Medicare allowable fees (30 in intervention) or not (30 in control) in the electronic health record. Primary outcome was the number of tests ordered per patient-day. Secondary outcomes were tests performed per patient-day and Medicare associated fees. The sample included 142 921 hospital admissions representing patients who were 51.9% white (74 165), 38.9% black (55 526), and 56.9% female (81 291) with a mean (SD) age of 54.7 (19.0) years. Preintervention trends of order rates among the intervention and control groups were similar. In adjusted analyses of the intervention group compared with the control group over time, there were no significant changes in overall test ordering behavior (0.05 tests ordered per patient-day; 95% CI, -0.002 to 0.09; P = .06) or associated fees ($0.24 per patient-day; 95% CI, -$0.42 to $0.91; P = .47). Exploratory subset analyses found small but significant differences in tests ordered per patient-day based on patient intensive care unit (ICU) stay (patients with ICU stay: -0.16; 95% CI, -0.31 to -0.01; P = .04; patients without ICU stay: 0.13; 95% CI, 0.08-0.17; P < .001) and the

  10. Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

    Science.gov (United States)

    Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

    2018-01-01

    We previously constructed three recombinant phyA mutant strains (PP-NPm-8, PP-NPep-6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NPep-6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NPep-6A, PP-NPm-8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NPep-6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Coherent wavepackets in the Fenna-Matthews-Olson complex are robust to excitonic-structure perturbations caused by mutagenesis

    Science.gov (United States)

    Maiuri, Margherita; Ostroumov, Evgeny E.; Saer, Rafael G.; Blankenship, Robert E.; Scholes, Gregory D.

    2018-02-01

    Femtosecond pulsed excitation of light-harvesting complexes creates oscillatory features in their response. This phenomenon has inspired a large body of work aimed at uncovering the origin of the coherent beatings and possible implications for function. Here we exploit site-directed mutagenesis to change the excitonic level structure in Fenna-Matthews-Olson (FMO) complexes and compare the coherences using broadband pump-probe spectroscopy. Our experiments detect two oscillation frequencies with dephasing on a picosecond timescale—both at 77 K and at room temperature. By studying these coherences with selective excitation pump-probe experiments, where pump excitation is in resonance only with the lowest excitonic state, we show that the key contributions to these oscillations stem from ground-state vibrational wavepackets. These experiments explicitly show that the coherences—although in the ground electronic state—can be probed at the absorption resonances of other bacteriochlorophyll molecules because of delocalization of the electronic excitation over several chromophores.

  12. Coherent wavepackets in the Fenna-Matthews-Olson complex are robust to excitonic-structure perturbations caused by mutagenesis.

    Science.gov (United States)

    Maiuri, Margherita; Ostroumov, Evgeny E; Saer, Rafael G; Blankenship, Robert E; Scholes, Gregory D

    2018-02-01

    Femtosecond pulsed excitation of light-harvesting complexes creates oscillatory features in their response. This phenomenon has inspired a large body of work aimed at uncovering the origin of the coherent beatings and possible implications for function. Here we exploit site-directed mutagenesis to change the excitonic level structure in Fenna-Matthews-Olson (FMO) complexes and compare the coherences using broadband pump-probe spectroscopy. Our experiments detect two oscillation frequencies with dephasing on a picosecond timescale-both at 77 K and at room temperature. By studying these coherences with selective excitation pump-probe experiments, where pump excitation is in resonance only with the lowest excitonic state, we show that the key contributions to these oscillations stem from ground-state vibrational wavepackets. These experiments explicitly show that the coherences-although in the ground electronic state-can be probed at the absorption resonances of other bacteriochlorophyll molecules because of delocalization of the electronic excitation over several chromophores.

  13. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

    Science.gov (United States)

    Ng, Ashley Shu Mei; Ingham, Philip W.

    2016-01-01

    CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. PMID:27832146

  14. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis.

    Directory of Open Access Journals (Sweden)

    Raymond Teck Ho Lee

    Full Text Available CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.

  15. New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis

    OpenAIRE

    Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

    2013-01-01

    Background In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagen...

  16. Effect of dose and dosing rate on the mutagenesis of nitric oxide in ...

    African Journals Online (AJOL)

    Effect of dose and dosing rate on the mutagenesis of nitric oxide in supF shuttle vector. Ji Hye Kim1 and ... Purpose: To determine how the dose and rate of NO• treatment affects mutagenic responses. Methods: Shuttle vector pSP189 was ... form a strong oxidant and nitrating agent, peroxynitrite (ONOO-), which can initiate.

  17. Highly efficient CRISPR/Cas9-mediated targeted mutagenesis of multiple genes in Populus.

    Science.gov (United States)

    Liu, Ting-ting; Fan, Di; Ran, Ling-yu; Jiang, Yuan-zhong; Liu, Rui; Luo, Ke-ming

    2015-10-01

    The typeⅡCRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats /CRISPR-associated 9) has been widely used in bacteria, yeast, animals and plants as a targeted genome editing technique. In previous work, we have successfully knocked out the endogenous phytoene dehydrogenase (PDS) gene in Populus tomentosa Carr. using this system. To study the effect of target design on the efficiency of CRISPR/Cas9-mediated gene knockout in Populus, we analyzed the efficiency of mutagenesis using different single-guide RNA (sgRNA) that target PDS DNA sequence. We found that mismatches between the sgRNA and the target DNA resulted in decreased efficiency of mutagenesis and even failed mutagenesis. Moreover, complementarity between the 3' end nucleotide of sgRNA and target DNA is especially crucial for efficient mutagenesis. Further sequencing analysis showed that two PDS homologs in Populus, PtPDS1 and PtPDS2, could be knocked out simultaneously using this system with 86.4% and 50% efficiency, respectively. These results indicated the possibility of introducing mutations in two or more endogenous genes efficiently and obtaining multi-mutant strains of Populus using this system. We have indeed generated several knockout mutants of transcription factors and structural genes in Populus, which establishes a foundation for future studies of gene function and genetic improvement of Populus.

  18. Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

    NARCIS (Netherlands)

    Varhimo, Emilia; Savijoki, Kirsi; Jalava, Jari; Kuipers, Oscar P.; Varmanen, Pekka

    Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found

  19. Workshop on ENU Mutagenesis: Planning for Saturation, July 25-28, 2002

    Energy Technology Data Exchange (ETDEWEB)

    Nadeau, Joseph H

    2002-07-25

    The goal of the conference is to enhance the development of improved technologies and new approaches to the identification of genes underlying chemically-induced mutant phenotypes. The conference brings together ENU mutagenesis experts from the United States and aborad for a small, intensive workshop to consider these issues.

  20. Mutagenesis effects of X-irradiation on the germination, growth and ...

    African Journals Online (AJOL)

    The time of exposure and mutagenesis effects of X-ray on the germination, growth and development of maize (Zea mays) seeds have been investigated employing a nuclear source, (90Sr as X-ray source; 3mA, 70kv). Five sects of zea mays seeds in different packs were irradiated in different times of exposure and later ...

  1. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    OpenAIRE

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by ...

  2. Electronic symptom reporting between patient and provider for improved health care service quality: a systematic review of randomized controlled trials. part 2: methodological quality and effects.

    Science.gov (United States)

    Johansen, Monika Alise; Berntsen, Gro K Rosvold; Schuster, Tibor; Henriksen, Eva; Horsch, Alexander

    2012-10-03

    We conducted in two parts a systematic review of randomized controlled trials (RCTs) on electronic symptom reporting between patients and providers to improve health care service quality. Part 1 reviewed the typology of patient groups, health service innovations, and research targets. Four innovation categories were identified: consultation support, monitoring with clinician support, self-management with clinician support, and therapy. To assess the methodological quality of the RCTs, and summarize effects and benefits from the methodologically best studies. We searched Medline, EMBASE, PsycINFO, Cochrane Central Register of Controlled Trials, and IEEE Xplore for original studies presented in English-language articles between 1990 and November 2011. Risk of bias and feasibility were judged according to the Cochrane recommendation, and theoretical evidence and preclinical testing were evaluated according to the Framework for Design and Evaluation of Complex Interventions to Improve Health. Three authors assessed the risk of bias and two authors extracted the effect data independently. Disagreement regarding bias assessment, extraction, and interpretation of results were resolved by consensus discussions. Of 642 records identified, we included 32 articles representing 29 studies. No articles fulfilled all quality requirements. All interventions were feasible to implement in a real-life setting, and theoretical evidence was provided for almost all studies. However, preclinical testing was reported in only a third of the articles. We judged three-quarters of the articles to have low risk for random sequence allocation and approximately half of the articles to have low risk for the following biases: allocation concealment, incomplete outcome data, and selective reporting. Slightly more than one fifth of the articles were judged as low risk for blinding of outcome assessment. Only 1 article had low risk of bias for blinding of participants and personnel. We excluded 12

  3. A randomized-controlled trial with a Canadian electronic pill dispenser used to measure and improve medication adherence in patients with schizophrenia

    Directory of Open Access Journals (Sweden)

    Emmanuel eStip

    2013-08-01

    Full Text Available Objective: Medication adherence is extremely important in preventing relapse and lowering symptoms in schizophrenic patients. However, estimates show that nearly half of these patients have poor adherence. The Brief Adherence Rating Scale (BARS seems to be the most reliable tool assessing adherence in schizophrenia and shows that the antipsychotic adherence ratio (AAR is about 49.5 % in schizophrenia. The aim of the study was to test if an electronic pill dispenser named DoPill® improved AAR of schizophrenic patients. Furthermore, we compared AAR obtained by the DoPill® and the BARS, in order to verify whether the DoPill® provides reliable assessment of medication adherence. Methods: The DoPill® is a smart pill dispenser that beeps and flashes at the appropriate time of the day. Each of its 28 compartments is covered by a plastic lamina that, when taken off, sends a signal to the pharmacist. Patients were randomized to the DoPill® or Treatment As Usual group (TAU for six weeks. The BARS was used as a reference measure. Results: Forty-six percent of patients were deemed to be non-adherent with antipsychotic medication. The mean AAR was 67 % after six weeks. DoPill® recorded better AAR than some of those found in the literature and were lower than the BARS estimate we found. Conclusion: These results suggest that DoPill® is a valid tool that provides more reliable and objective data for the clinician about their patient’s adherence, than existing assessment tools like the BARS. Furthermore, the device may help patients successfully manage their medication regimen.

  4. A randomized controlled trial with a Canadian electronic pill dispenser used to measure and improve medication adherence in patients with schizophrenia.

    Science.gov (United States)

    Stip, Emmanuel; Vincent, Philippe D; Sablier, Juliette; Guevremont, Catherine; Zhornitsky, Simon; Tranulis, Constantin

    2013-01-01

    Medication adherence is extremely important in preventing relapse and lowering symptoms in schizophrenic patients. However, estimates show that nearly half of these patients have poor adherence. The Brief Adherence Rating Scale (BARS) seems to be the most reliable tool assessing adherence in schizophrenia and shows that the antipsychotic adherence ratio (AAR) is about 49.5% in schizophrenia. The aim of the study was to test if an electronic pill dispenser named DoPill(®) improved AAR of schizophrenic patients. Furthermore, we compared AAR obtained by the DoPill(®) and the BARS, in order to verify whether the DoPill(®) provides reliable assessment of medication adherence. The DoPill(®) is a smart pill dispenser that beeps and flashes at the appropriate time of the day. Each of its 28 compartments is covered by a plastic lamina that, when taken off, sends a signal to the pharmacist. Patients were randomized to the DoPill(®) or treatment as usual groups for 6 weeks. The BARS was used as a reference measure. Forty-six percent of patients were deemed to be non-adherent with antipsychotic medication. The mean AAR was 67% after 6 weeks. DoPill(®) recorded better AAR than some of those found in the literature and were lower than the BARS estimate we found. These results suggest that DoPill(®) is a valid tool that provides more reliable and objective data for the clinician about their patient's adherence, than existing assessment tools like the BARS. Furthermore, the device may help patients successfully manage their medication regimen.

  5. Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis.

    Science.gov (United States)

    DeJesus, Michael A; Gerrick, Elias R; Xu, Weizhen; Park, Sae Woong; Long, Jarukit E; Boutte, Cara C; Rubin, Eric J; Schnappinger, Dirk; Ehrt, Sabine; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R

    2017-01-17

    For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant

  6. Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

    Directory of Open Access Journals (Sweden)

    Hughes Lori A

    2005-11-01

    Full Text Available Abstract Background Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results We produced the Cryopreserved Mutant Mouse Bank (CMMB, which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41% result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

  7. Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

    Science.gov (United States)

    Mohr, C D; Deretic, V

    1990-11-01

    A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source.

  8. Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing.

    Science.gov (United States)

    Varrelmann, Mark; Maiss, Edgar; Pilot, Ruth; Palkovics, Laszlo

    2007-03-01

    Helper component proteinase (HC-Pro) of Plum pox virus is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using Agrobacterium infiltration in transgenic Nicotiana benthamiana plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins.

  9. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

    Directory of Open Access Journals (Sweden)

    Janire Mingo

    Full Text Available Site-directed mutagenesis (SDM is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.

  10. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

    Science.gov (United States)

    Sanchis, Joaquin; Fernández, Layla; Carballeira, J Daniel; Drone, Jullien; Gumulya, Yosephine; Höbenreich, Horst; Kahakeaw, Daniel; Kille, Sabrina; Lohmer, Renate; Peyralans, Jérôme J-P; Podtetenieff, John; Prasad, Shreenath; Soni, Pankaj; Taglieber, Andreas; Wu, Sheng; Zilly, Felipe E; Reetz, Manfred T

    2008-11-01

    Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

  11. Enhancement of the lipid productivity and fatty acid methyl ester profile of Chlorella vulgaris by two rounds of mutagenesis.

    Science.gov (United States)

    Sarayloo, Ehsan; Simsek, Salim; Unlu, Yigit Sabri; Cevahir, Gul; Erkey, Can; Kavakli, Ibrahim Halil

    2017-12-02

    In this study, we applied a second round of random mutagenesis using ethyl methanesulfonate to further increase the lipid productivity of a Chlorella vulgaris mutant strain. We generated a mutant (UV715-EMS25) with a lipid content and biomass that were respectively 67% and 35% higher than those of the wild type (WT). The highest achieved lipid productivity in UV715-EMS25 was 91 mg L-1 day-1. Gas chromatography-mass spectrophotometric analysis revealed that the fatty acid methyl ester content of the mutant was 3.9-fold higher compared with that of WT cells. Amounts of saturated and monounsaturated fatty acids were also higher in the mutant, while the total amounts of polyunsaturated fatty acids were lower. Finally, the mutant displayed superior lipid productivity compared with the WT during pilot-scale cultivation in a flat panel photobioreactor. All these results demonstrate that UV715-EMS25 is highly suitable for biodiesel production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene.

    Science.gov (United States)

    Montero-Conde, Cristina; Leandro-Garcia, Luis J; Chen, Xu; Oler, Gisele; Ruiz-Llorente, Sergio; Ryder, Mabel; Landa, Iñigo; Sanchez-Vega, Francisco; La, Konnor; Ghossein, Ronald A; Bajorin, Dean F; Knauf, Jeffrey A; Riordan, Jesse D; Dupuy, Adam J; Fagin, James A

    2017-06-20

    Oncogenic RAS mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with Hras(G12V) in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-Hras(G12V) mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7, a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene.

  13. Mechanism of porcine liver xanthine oxidoreductase mediated N-oxide reduction of cyadox as revealed by docking and mutagenesis studies.

    Directory of Open Access Journals (Sweden)

    Chigang Chen

    Full Text Available Xanthine oxidoreductase (XOR is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs. To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424-434 loop, exhibited a much lower K(m and a decreased V(max respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides.

  14. Effect of Electronic Reminders, Financial Incentives, and Social Support on Outcomes After Myocardial Infarction: The HeartStrong Randomized Clinical Trial.

    Science.gov (United States)

    Volpp, Kevin G; Troxel, Andrea B; Mehta, Shivan J; Norton, Laurie; Zhu, Jingsan; Lim, Raymond; Wang, Wenli; Marcus, Noora; Terwiesch, Christian; Caldarella, Kristen; Levin, Tova; Relish, Mike; Negin, Nathan; Smith-McLallen, Aaron; Snyder, Richard; Spettell, Claire M; Drachman, Brian; Kolansky, Daniel; Asch, David A

    2017-08-01

    Adherence to medications prescribed after acute myocardial infarction (AMI) is low. Wireless technology and behavioral economic approaches have shown promise in improving health behaviors. To determine whether a system of medication reminders using financial incentives and social support delays subsequent vascular events in patients following AMI compared with usual care. Two-arm, randomized clinical trial with a 12-month intervention conducted from 2013 through 2016. Investigators were blinded to study group, but participants were not. Design was a health plan-intermediated intervention for members of several health plans. We recruited 1509 participants from 7179 contacted AMI survivors (insured with 5 large US insurers nationally or with Medicare fee-for-service at the University of Pennsylvania Health System). Patients aged 18 to 80 years were eligible if currently prescribed at least 2 of 4 study medications (statin, aspirin, β-blocker, antiplatelet agent), and were hospital inpatients for 1 to 180 days and discharged home with a principal diagnosis of AMI. Patients were randomized 2:1 to an intervention using electronic pill bottles combined with lottery incentives and social support for medication adherence (1003 patients), or to usual care (506 patients). Primary outcome was time to first vascular rehospitalization or death. Secondary outcomes were time to first all-cause rehospitalization, total number of repeated hospitalizations, medication adherence, and total medical costs. A total of 35.5% of participants were female (n = 536); mean (SD) age was 61.0 (10.3) years. There were no statistically significant differences between study arms in time to first rehospitalization for a vascular event or death (hazard ratio, 1.04; 95% CI, 0.71 to 1.52; P = .84), time to first all-cause rehospitalization (hazard ratio, 0.89; 95% CI, 0.73 to 1.09; P = .27), or total number of repeated hospitalizations (hazard ratio, 0.94; 95% CI, 0.60 to 1.48; P

  15. Evaluating a Web-Based Coaching Program Using Electronic Health Records for Patients With Chronic Obstructive Pulmonary Disease in China: Randomized Controlled Trial.

    Science.gov (United States)

    Wang, Lan; He, Lin; Tao, Yanxia; Sun, Li; Zheng, Hong; Zheng, Yashu; Shen, Yuehao; Liu, Suyan; Zhao, Yue; Wang, Yaogang

    2017-07-21

    Chronic obstructive pulmonary disease (COPD) is now the fourth leading cause of death in the world, and it continues to increase in developing countries. The World Health Organization expects COPD to be the third most common cause of death in the world by 2020. Effective and continuous postdischarge care can help patients to maintain good health. The use of electronic health records (EHRs) as an element of community health care is new technology in China. The aim of this study was to develop and evaluate a Web-based coaching program using EHRs for physical function and health-related quality of life for patients with COPD in China. A randomized controlled trial was conducted from 2008 to 2015 at two hospitals. The control group received routine care and the intervention group received routine care with the addition of the Web-based coaching program using EHRs. These were used to manage patients' demographic and clinical variables, publish relevant information, and have communication between patients and health care providers. Participants were not blinded to group assignment. The effects of the intervention were evaluated by lung function, including percent of forced expiratory volume in 1 second (FEV1%), percent of forced vital capacity (FVC%), peak expiratory flow (PEF), maximum midexpiratory flow; St George's Respiratory Questionnaire (SGRQ); Modified Medical Research Council Dyspnea Scale (MMRC); and 6-Minute Walk Test (6MWT). Data were collected before the program, and at 1, 3, 6, and 12 months after the program. Of the 130 participants, 120 (92.3%) completed the 12-month follow-up program. There were statistically significant differences in lung function (FEV1%: F1,4=5.47, P=.002; FVC%: F1,4=3.06, P=.02; PEF: F1,4=12.49, Pprogram using EHRs in China appears to be useful for patients with COPD when they are discharged from hospital into the community. It promotes the sharing of patients' medical information by hospital and community nurses, and achieves

  16. EffiCiency and Safety of an eLectronic cigAreTte (ECLAT as tobacco cigarettes substitute: a prospective 12-month randomized control design study.

    Directory of Open Access Journals (Sweden)

    Pasquale Caponnetto

    Full Text Available Electronic cigarettes (e-cigarettes are becoming increasingly popular with smokers worldwide. Users report buying them to help quit smoking, to reduce cigarette consumption, to relieve tobacco withdrawal symptoms, and to continue having a 'smoking' experience, but with reduced health risks. Research on e-cigarettes is urgently needed in order to ensure that the decisions of regulators, healthcare providers and consumers are based on science. Methods ECLAT is a prospective 12-month randomized, controlled trial that evaluates smoking reduction/abstinence in 300 smokers not intending to quit experimenting two different nicotine strengths of a popular e-cigarette model ('Categoria'; Arbi Group Srl, Italy compared to its non-nicotine choice. GroupA (n = 100 received 7.2 mg nicotine cartridges for 12 weeks; GroupB (n = 100, a 6-week 7.2 mg nicotine cartridges followed by a further 6-week 5.4 mg nicotine cartridges; GroupC (n = 100 received no-nicotine cartridges for 12 weeks. The study consisted of nine visits during which cig/day use and exhaled carbon monoxide (eCO levels were measured. Smoking reduction and abstinence rates were calculated. Adverse events and product preferences were also reviewed.Declines in cig/day use and eCO levels were observed at each study visits in all three study groups (p<0.001 vs baseline, with no consistent differences among study groups. Smoking reduction was documented in 22.3% and 10.3% at week-12 and week-52 respectively. Complete abstinence from tobacco smoking was documented in 10.7% and 8.7% at week-12 and week-52 respectively. A substantial decrease in adverse events from baseline was observed and withdrawal symptoms were infrequently reported during the study. Participants' perception and acceptance of the product under investigation was satisfactory.In smokers not intending to quit, the use of e-cigarettes, with or without nicotine, decreased cigarette consumption and elicited enduring tobacco

  17. Rationale, design, and implementation protocol of an electronic health record integrated clinical prediction rule (iCPR randomized trial in primary care

    Directory of Open Access Journals (Sweden)

    Wisnivesky Juan

    2011-09-01

    Full Text Available Abstract Background Clinical prediction rules (CPRs represent well-validated but underutilized evidence-based medicine tools at the point-of-care. To date, an inability to integrate these rules into an electronic health record (EHR has been a major limitation and we are not aware of a study demonstrating the use of CPR's in an ambulatory EHR setting. The integrated clinical prediction rule (iCPR trial integrates two CPR's in an EHR and assesses both the usability and the effect on evidence-based practice in the primary care setting. Methods A multi-disciplinary design team was assembled to develop a prototype iCPR for validated streptococcal pharyngitis and bacterial pneumonia CPRs. The iCPR tool was built as an active Clinical Decision Support (CDS tool that can be triggered by user action during typical workflow. Using the EHR CDS toolkit, the iCPR risk score calculator was linked to tailored ordered sets, documentation, and patient instructions. The team subsequently conducted two levels of 'real world' usability testing with eight providers per group. Usability data were used to refine and create a production tool. Participating primary care providers (n = 149 were randomized and intervention providers were trained in the use of the new iCPR tool. Rates of iCPR tool triggering in the intervention and control (simulated groups are monitored and subsequent use of the various components of the iCPR tool among intervention encounters is also tracked. The primary outcome is the difference in antibiotic prescribing rates (strep and pneumonia iCPR's encounters and chest x-rays (pneumonia iCPR only between intervention and control providers. Discussion Using iterative usability testing and development paired with provider training, the iCPR CDS tool leverages user-centered design principles to overcome pervasive underutilization of EBM and support evidence-based practice at the point-of-care. The ongoing trial will determine if this collaborative

  18. EffiCiency and Safety of an eLectronic cigAreTte (ECLAT) as Tobacco Cigarettes Substitute: A Prospective 12-Month Randomized Control Design Study

    Science.gov (United States)

    Caponnetto, Pasquale; Campagna, Davide; Cibella, Fabio; Morjaria, Jaymin B.; Caruso, Massimo; Russo, Cristina; Polosa, Riccardo

    2013-01-01

    Background Electronic cigarettes (e-cigarettes) are becoming increasingly popular with smokers worldwide. Users report buying them to help quit smoking, to reduce cigarette consumption, to relieve tobacco withdrawal symptoms, and to continue having a ‘smoking’ experience, but with reduced health risks. Research on e-cigarettes is urgently needed in order to ensure that the decisions of regulators, healthcare providers and consumers are based on science. Methods ECLAT is a prospective 12-month randomized, controlled trial that evaluates smoking reduction/abstinence in 300 smokers not intending to quit experimenting two different nicotine strengths of a popular e-cigarette model (‘Categoria’; Arbi Group Srl, Italy) compared to its non-nicotine choice. GroupA (n = 100) received 7.2 mg nicotine cartridges for 12 weeks; GroupB (n = 100), a 6-week 7.2 mg nicotine cartridges followed by a further 6-week 5.4 mg nicotine cartridges; GroupC (n = 100) received no-nicotine cartridges for 12 weeks. The study consisted of nine visits during which cig/day use and exhaled carbon monoxide (eCO) levels were measured. Smoking reduction and abstinence rates were calculated. Adverse events and product preferences were also reviewed. Results Declines in cig/day use and eCO levels were observed at each study visits in all three study groups (p<0.001 vs baseline), with no consistent differences among study groups. Smoking reduction was documented in 22.3% and 10.3% at week-12 and week-52 respectively. Complete abstinence from tobacco smoking was documented in 10.7% and 8.7% at week-12 and week-52 respectively. A substantial decrease in adverse events from baseline was observed and withdrawal symptoms were infrequently reported during the study. Participants’ perception and acceptance of the product under investigation was satisfactory. Conclusion In smokers not intending to quit, the use of e-cigarettes, with or without nicotine, decreased cigarette consumption and

  19. An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes.

    Science.gov (United States)

    Zhang, Zhiqiang; Xu, Kun; Xin, Ying; Zhang, Zhiying

    2015-05-01

    Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression

    Science.gov (United States)

    Rangel, Roberto; Lee, Song-Choon; Hon-Kim Ban, Kenneth; Guzman-Rojas, Liliana; Mann, Michael B.; Newberg, Justin Y.; McNoe, Leslie A.; Selvanesan, Luxmanan; Ward, Jerrold M.; Rust, Alistair G.; Chin, Kuan-Yew; Black, Michael A.; Jenkins, Nancy A.; Copeland, Neal G.

    2016-01-01

    Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC. PMID:27849608

  1. Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

    Directory of Open Access Journals (Sweden)

    Robyn N. Hall

    2012-02-01

    Full Text Available Bacterial artificial chromosome (BAC vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.

  2. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens. [Rats, hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.

    1977-01-01

    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro.

  3. Model of SOS-induced mutagenesis in bacteria Escherichia coli under ultraviolet irradiation.

    Science.gov (United States)

    Belov, Oleg V; Krasavin, Evgeny A; Parkhomenko, Alexander Yu

    2009-12-07

    A mathematical model of the mutation process in bacteria Escherichia coli induced by ultraviolet radiation is developed. Our model is based on the experimental data characterizing the main processes of the bacterial SOS response. Here we have modeled a whole sequence of the events leading to the fixation of the primary DNA lesion as a point mutation. A quantitative analysis of the key ways of the SOS mutagenesis was performed in terms of modern system biology. The dynamic changes of the basic SOS protein concentrations and the process of the translesion synthesis by the modified replication complex are described quantitatively. We have also demonstrated the applicability of the developed model to the description of the mutagenesis in individual genes. As an example, an estimation of the mutation frequency in E. coli's lacI gene is performed.

  4. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis

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    T.A. Beacham

    2015-09-01

    Full Text Available Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed.

  5. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    Science.gov (United States)

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation. PMID:11172002

  6. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis.

    Science.gov (United States)

    Beacham, T A; Macia, V Mora; Rooks, P; White, D A; Ali, S T

    2015-09-01

    Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed.

  7. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome

    OpenAIRE

    Messerle, Martin; Crnkovic, Irena; Hammerschmidt, Wolfgang; Ziegler, Heike; Koszinowski, Ulrich H

    1997-01-01

    A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete constructi...

  8. Prevention of mutagenesis: new potential mechanisms of metformin action in neoplastic cells.

    Science.gov (United States)

    Bost, Frédéric; Ben-Sahra, Issam; Tanti, Jean-François

    2012-04-01

    Several experimental and epidemiologic studies have shown that the antidiabetes drug metformin has antitumor properties. The report by Algire and colleagues in this issue of the journal (beginning on page 536) shows for the first time that metformin reduces mutagenesis induced by reactive oxygen species. This report offers new perspectives on metformin in cancer prevention and provides a new mechanism for the reduction of cancer risk in diabetic patients treated with this drug. 2012 AACR

  9. General Mutagenesis of F Plasmid TraI Reveals Its Role in Conjugative Regulation

    OpenAIRE

    Haft, Rembrandt J. F.; Palacios, Gilberto; Nguyen, Tran; Mally, Manuela; Gachelet, Eliora G.; Zechner, Ellen L.; Traxler, Beth

    2006-01-01

    Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI...

  10. Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion

    OpenAIRE

    Batista, Rita; Saibo, Nelson; Lourenço, Tiago; Oliveira, Maria Margarida

    2008-01-01

    Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring ...

  11. Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

    Directory of Open Access Journals (Sweden)

    Grégory Caignard

    2014-09-01

    Full Text Available Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses.

  12. mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

    Directory of Open Access Journals (Sweden)

    Riikka H. Hämäläinen

    2015-06-01

    Full Text Available mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function.

  13. Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat.

    Science.gov (United States)

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W; Xiao, Ningna; Overbeek, Paul A; Behringer, Richard R

    2012-12-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

  14. Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

    Science.gov (United States)

    Huang, Renhua; Fang, Pete; Kay, Brian K

    2012-09-01

    Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects

    Science.gov (United States)

    Zhang, Ying; Zhou, Junqing; Baldwin, Joseph; Held, Kathryn D; Prise, Kevin M; Redmond, Robert W.; Liber, Howard L.

    2009-01-01

    This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naïve recipient cells. Kinetic studies revealed that it required up to one hour to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least one hour of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12–24 hours to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent γ-ray exposures. PMID:19695271

  16. Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Ying; Zhou Junqing; Baldwin, Joseph [Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States); Held, Kathryn D. [Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA (United States); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast (United Kingdom); Redmond, Robert W. [Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); Liber, Howard L., E-mail: howard.liber@colostate.edu [Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States); University of Colorado Cancer Center, Denver, CO (United States)

    2009-12-01

    This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naive recipient cells. Kinetic studies revealed that it required up to 1 h to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least 1 h of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12-24 h to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent {gamma}-ray exposures.

  17. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    Science.gov (United States)

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-06

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    Directory of Open Access Journals (Sweden)

    Martine Aubert

    2014-01-01

    Full Text Available Following acute infection, herpes simplex virus (HSV establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs for cure of chronic viral infections.

  19. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.

    Science.gov (United States)

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-02-04

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3'-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.Molecular Therapy-Nucleic Acids (2014) 3, e1; doi:10.1038/mtna.2013.75; published online 4 February 2014.

  20. CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.

    Science.gov (United States)

    Bi, Hong-Lun; Xu, Jun; Tan, An-Jiang; Huang, Yong-Ping

    2016-06-01

    Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  1. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    Science.gov (United States)

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  2. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Directory of Open Access Journals (Sweden)

    Moskowitz Ivan

    2009-03-01

    Full Text Available Abstract Background ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. Results We isolated 11 lethal lines that map to the region of chromosome 4 between D4Mit117 and D4Mit281. These lines form 10 complementation groups. The majority of lines die during embryonic development between E5.5 and E12.5 and display defects in gastrulation, cardiac development, and craniofacial development. One line displayed postnatal lethality and neurological defects, including ataxia and seizures. Conclusion These eleven mutants allow us to query gene function within the distal region of mouse chromosome 4 and demonstrate that new mouse models of mammalian developmental defects can easily and quickly be generated and mapped with the use of ENU-mutagenesis in combination with balancer chromosomes. The low number of mutations isolated in this screen compared with other balancer chromosome screens indicates that the functions of genes in different regions of the genome vary widely.

  3. Yeasts acquire resistance secondary to antifungal drug treatment by adaptive mutagenesis.

    Directory of Open Access Journals (Sweden)

    David Quinto-Alemany

    Full Text Available Acquisition of resistance secondary to treatment both by microorganisms and by tumor cells is a major public health concern. Several species of bacteria acquire resistance to various antibiotics through stress-induced responses that have an adaptive mutagenesis effect. So far, adaptive mutagenesis in yeast has only been described when the stress is nutrient deprivation. Here, we hypothesized that adaptive mutagenesis in yeast (Saccharomyces cerevisiae and Candida albicans as model organisms would also take place in response to antifungal agents (5-fluorocytosine or flucytosine, 5-FC, and caspofungin, CSP, giving rise to resistance secondary to treatment with these agents. We have developed a clinically relevant model where both yeasts acquire resistance when exposed to these agents. Stressful lifestyle associated mutation (SLAM experiments show that the adaptive mutation frequencies are 20 (S. cerevisiae -5-FC, 600 (C. albicans -5-FC or 1000 (S. cerevisiae--CSP fold higher than the spontaneous mutation frequency, the experimental data for C. albicans -5-FC being in agreement with the clinical data of acquisition of resistance secondary to treatment. The spectrum of mutations in the S. cerevisiae -5-FC model differs between spontaneous and acquired, indicating that the molecular mechanisms that generate them are different. Remarkably, in the acquired mutations, an ectopic intrachromosomal recombination with an 87% homologous gene takes place with a high frequency. In conclusion, we present here a clinically relevant adaptive mutation model that fulfils the conditions reported previously.

  4. Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

    Science.gov (United States)

    Zhang, Chaolei; Shen, Hongwei; Zhang, Xibin; Yu, Xue; Wang, Han; Xiao, Shan; Wang, Jihui; Zhao, Zongbao K

    2016-10-01

    To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

  5. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

    National Research Council Canada - National Science Library

    Svitashev, Sergei; Young, Joshua K; Schwartz, Christine; Gao, Huirong; Falco, S Carl; Cigan, A Mark

    2015-01-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas...

  6. Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h

    OpenAIRE

    Chiu, Joyce; March, Paul E.; Lee, Ryan; Tillett, Daniel

    2004-01-01

    Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. U...

  7. Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis.

    Science.gov (United States)

    He, Zhiming; Kosinska, Wieslawa; Zhao, Zhong-Lin; Wu, Xue-Ru; Guttenplan, Joseph B

    2012-02-18

    Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue™) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue™) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. [IXR1 and HMO1 genes jointly control the level of spontaneous mutagenesis in yeast Saccharomyces cerevisiae].

    Science.gov (United States)

    Fedorov, D V; Koval'tsova, S V; Peshekhonov, V T; Korolev, V G

    2010-06-01

    The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, gamma-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo 1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo 1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo 1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo 1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.

  9. Effect of pay-for-performance incentives on quality of care in small practices with electronic health records: a randomized trial.

    Science.gov (United States)

    Bardach, Naomi S; Wang, Jason J; De Leon, Samantha F; Shih, Sarah C; Boscardin, W John; Goldman, L Elizabeth; Dudley, R Adams

    2013-09-11

    Most evaluations of pay-for-performance (P4P) incentives have focused on large-group practices. Thus, the effect of P4P in small practices, where many US residents receive care, is largely unknown. Furthermore, whether electronic health records (EHRs) with chronic disease management capabilities support small-practice response to P4P has not been studied. To assess the effect of P4P incentives on quality in EHR-enabled small practices in the context of an established quality improvement initiative. A cluster-randomized trial of small (<10 clinicians) primary care clinics in New York City from April 2009 through March 2010. A city program provided all participating clinics with the same EHR software with decision support and patient registry functionalities and quality improvement specialists offering technical assistance. Incentivized clinics were paid for each patient whose care met the performance criteria, but they received higher payments for patients with comorbidities, who had Medicaid insurance, or who were uninsured (maximum payments: $200/patient; $100,000/clinic). Quality reports were given quarterly to both the intervention and control groups. Comparison of differences in performance improvement, from the beginning to the end of the study, between control and intervention clinics for aspirin or antithrombotic prescription, blood pressure control, cholesterol control, and smoking cessation interventions. Mixed-effects logistic regression was used to account for clustering of patients within clinics, with a treatment by time interaction term assessing the statistical significance of the effect of the intervention. Participating clinics (n = 42 for each group) had similar baseline characteristics, with a mean of 4592 (median, 2500) patients at the intervention group clinics and 3042 (median, 2000) at the control group clinics. Intervention clinics had greater adjusted absolute improvement in rates of appropriate antithrombotic prescription (12.0% vs 6

  10. Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

    Science.gov (United States)

    Bierle, Craig J; Anderholm, Kaitlyn M; Wang, Jian Ben; McVoy, Michael A; Schleiss, Mark R

    2016-08-01

    The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial

  11. Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction.

    Directory of Open Access Journals (Sweden)

    Dušan Turek

    Full Text Available Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

  12. A mariner transposon-based signature-tagged mutagenesis system for the analysis of oral infection by Listeria monocytogenes.

    Science.gov (United States)

    Cummins, Joanne; Casey, Pat G; Joyce, Susan A; Gahan, Cormac G M

    2013-01-01

    Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM) system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610), lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842), lmOh7858_2579 (encoding the HupDGC hemin transport system) and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system). We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes.

  13. A mariner transposon-based signature-tagged mutagenesis system for the analysis of oral infection by Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Joanne Cummins

    Full Text Available Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610, lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842, lmOh7858_2579 (encoding the HupDGC hemin transport system and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system. We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes.

  14. Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Alex S Nord

    2007-07-01

    Full Text Available High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.The findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

  15. Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

    Science.gov (United States)

    Liu, Yang; Merrick, Paul; Zhang, Zhengzhi; Ji, Chonghui; Yang, Bing; Fei, Shui-Zhang

    2018-02-01

    The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  16. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

    Science.gov (United States)

    LeBlanc, Chantal; Zhang, Fei; Mendez, Josefina; Lozano, Yamile; Chatpar, Krishna; Irish, Vivian F; Jacob, Yannick

    2018-01-01

    The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  17. Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes.

    Science.gov (United States)

    Patel, Jinesh D; Wright, Robert J; Auld, Dick; Chandnani, Rahul; Goff, Valorie H; Ingles, Jennifer; Pierce, Gary J; Torres, Manuel J; Paterson, Andrew H

    2014-04-01

    Genetic improvements for many fiber traits are obtained by mutagenesis of elite cottons, mitigating genetic uniformity in this inbred polyploid by contributing novel alleles important to ongoing crop improvement. The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate (EMS) mutagenesis of two G. hirsutum breeding lines, TAM 94L-25 and Acala 1517-99, were characterized for basic components of fiber quality and selected yield components. Across all measured traits, the ranges of phenotypic values among the mutant lines were consistently larger than could be explained by chance (5.27-10.1 for TAM 94 L-25 and 5.29-7.94 standard deviations for Acala 1517-99-derived lines). Multi-year replicated studies confirmed a genetic basis for these differences, showing significant correlations between lines across years and environments. A subset of 157 lines selected for superior fiber qualities, including fiber elongation (22 lines), length (22), lint percent (17), fineness (23), Rd value (21), strength (19), uniformity (21) and multiple attributes in a selection index (26) were compared to 55 control lines in replicated trials in both Texas and Georgia. For all traits, mutant lines showing substantial and statistically significant improvements over control lines were found, in most cases from each of the two genetic backgrounds. This indicates that genetic improvements for a wide range of fiber traits may be obtained from mutagenesis of elite cottons. Indeed, lines selected for one fiber trait sometimes conferred additional attributes, suggesting pleiotropic effects of some mutations and offering multiple benefits for the incorporation of some alleles into mainstream breeding programs.

  18. CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

    Science.gov (United States)

    Sugano, Shigeo S; Shirakawa, Makoto; Takagi, Junpei; Matsuda, Yoriko; Shimada, Tomoo; Hara-Nishimura, Ikuko; Kohchi, Takayuki

    2014-03-01

    Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

  19. Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis.

    Science.gov (United States)

    Xu, Liang; Wang, Wei; Wu, Jiabin; Shin, Ji Hyun; Wang, Pengcheng; Unarta, Ilona Christy; Chong, Jenny; Wang, Yinsheng; Wang, Dong

    2017-08-22

    Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O 2 -, N 3-, and O 4 -ethylthymidine ( O 2 -, N 3-, and O 4 -EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O 2 -EtdT and N 3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O 2 -EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O 4 -EtdT lesion. For the N 3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

  20. Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

    Science.gov (United States)

    Denzin, L K; Voss, E W

    1992-05-05

    In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active

  1. Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis.

    Science.gov (United States)

    Milano, Shawn K; Pace, Helen C; Kim, You-Me; Brenner, Charles; Benovic, Jeffrey L

    2002-03-12

    Arrestin binding to activated, phosphorylated G protein-coupled receptors (GPCRs) represents a critical step in regulation of light- and hormone-dependent signaling. Nonvisual arrestins, such as arrestin-2, interact with multiple proteins for the purpose of propagating and terminating signaling events. Using a combination of X-ray crystallography, molecular modeling, mutagenesis, and binding analysis, we reveal structural features of arrestin-2 that may enable simultaneous binding to phosphorylated receptor, SH3 domains, phosphoinositides, and beta-adaptin. The structure of full-length arrestin-2 thus provides a uniquely oriented scaffold for assembly of multiple, diverse molecules involved in GPCR signal transduction.

  2. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    OpenAIRE

    Hoskins, J.T.; Zhou, Z.; Harding, P A

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

  3. Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

    Directory of Open Access Journals (Sweden)

    Kathryn E. Tiller

    2017-09-01

    Full Text Available The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease with the greatest gains in affinity (>5-fold have several (four to six CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability while other mutations enhance some of these properties (e.g., increased specificity and display trade-offs in others (e.g., reduced affinity and/or stability. Computational modeling reveals that improvements in affinity

  4. Induction of apomixis and fixation of heterosis in Egyptian rice Hybrid1 line using colchicine mutagenesis

    OpenAIRE

    Reda M. Gaafar; Adel R. El Shanshoury; Ahmad A. El Hisseiwy; Mahmoud A. AbdAlhak; Aimn F. Omar; Mohammad M. Abd El Wahab; Randa S. Nofal

    2017-01-01

    It is known that hybrid rice yields 15–20% over inbred varieties in first generation because of heterosis. However, heterosis is normally broken due to segregation. Applying apomixis produces plants as a clone of mother plant and overcomes the problem of breaking heterosis. In order to fix heterosis in the Egyptian rice Hybrid1, their seeds were mutagenized in 0.2% colchicine for two time periods 24 and 50 h. After colchicine mutagenesis, rice seedlings were grown in the field till maturation...

  5. Impact of Laboratory Charge Display Within the Electronic Health Record Across an Entire Academic Medical Center: Results of a Randomized Controlled Trial.

    Science.gov (United States)

    Schmidt, Robert L; Colbert-Getz, Jorie M; Milne, Caroline K; Vargo, Daniel J; Hussong, Jerry W; Hoidal, John R; Markewitz, Boaz A; Walker, Brandon S; Kawamoto, Kensaku

    2017-11-20

    To determine the impact of systemwide charge display on laboratory utilization. This was a randomized controlled trial with a baseline period and an intervention period. Tests were randomized to a control arm or an active arm. The maximum allowable Medicare reimbursement rate was displayed for tests in the active arm during the intervention period. Total volume of tests in the active arm was compared with those in the control arm. Residents were surveyed before and after the intervention to assess charge awareness. Charge display had no effect on order behavior. This result held for patient type (inpatient vs outpatient) and for insurance category (commercial, government, self-pay). Residents overestimated the charges of tests both before and after the intervention. Many residents failed to notice the charge display in the computerized order entry system. The impact of charge display depends on context. Charge display is not always effective.

  6. Electronics and electronic systems

    CERN Document Server

    Olsen, George H

    1987-01-01

    Electronics and Electronic Systems explores the significant developments in the field of electronics and electronic devices. This book is organized into three parts encompassing 11 chapters that discuss the fundamental circuit theory and the principles of analog and digital electronics. This book deals first with the passive components of electronic systems, such as resistors, capacitors, and inductors. These topics are followed by a discussion on the analysis of electronic circuits, which involves three ways, namely, the actual circuit, graphical techniques, and rule of thumb. The remaining p

  7. A randomized-controlled trial with a Canadian electronic pill dispenser used to measure and improve medication adherence in patients with schizophrenia

    OpenAIRE

    Emmanuel eStip; Emmanuel eStip; Emmanuel eStip; Philippe D. Vincent; Philippe D. Vincent; Philippe D. Vincent; Catherine eGuevremont; Simon eZhornitsky; Constantin eTranulis; Constantin eTranulis; Constantin eTranulis; Juliette eSablier; Juliette eSablier

    2013-01-01

    Objective: Medication adherence is extremely important in preventing relapse and lowering symptoms in schizophrenic patients. However, estimates show that nearly half of these patients have poor adherence. The Brief Adherence Rating Scale (BARS) seems to be the most reliable tool assessing adherence in schizophrenia and shows that the antipsychotic adherence ratio (AAR) is about 49.5 % in schizophrenia. The aim of the study was to test if an electronic pill dispenser named DoPill® improv...

  8. A randomized controlled trial with a Canadian electronic pill dispenser used to measure and improve medication adherence in patients with schizophrenia

    OpenAIRE

    Stip, Emmanuel; Vincent, Philippe D.; Sablier, Juliette; Guevremont, Catherine; Zhornitsky, Simon; Tranulis, Constantin

    2013-01-01

    Objective: Medication adherence is extremely important in preventing relapse and lowering symptoms in schizophrenic patients. However, estimates show that nearly half of these patients have poor adherence. The Brief Adherence Rating Scale (BARS) seems to be the most reliable tool assessing adherence in schizophrenia and shows that the antipsychotic adherence ratio (AAR) is about 49.5% in schizophrenia. The aim of the study was to test if an electronic pill dispenser named DoPill® improved AAR...

  9. Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

    Directory of Open Access Journals (Sweden)

    Régine Janel-Bintz

    Full Text Available Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G and 5'-GGCGCC-3' (NarI site, induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.

  10. Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

    Science.gov (United States)

    Nomura, Toshihisa; Sakurai, Tetsuya; Osakabe, Yuriko; Osakabe, Keishi; Sakakibara, Hitoshi

    2016-12-01

    Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution. © The Authors 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Comparative Analysis of Context-Dependent Mutagenesis Using Human and Mouse Models

    Directory of Open Access Journals (Sweden)

    Sofya A. Medvedeva

    2013-01-01

    Full Text Available Substitution rates strongly depend on their nucleotide context. One of the most studied examples is the excess of C > T mutations in the CG context in various groups of organisms, including vertebrates. Studies on the molecular mechanisms underlying this mutation regularity have provided insights into evolution, mutagenesis, and cancer development. Recently several other hypermutable motifs were identified in the human genome. There is an increased frequency of T > C mutations in the second position of the words ATTG and ATAG and an increased frequency of A > C mutations in the first position of the word ACAA. For a better understanding of evolution, it is of interest whether these mutation regularities are human specific or present in other vertebrates, as their presence might affect the validity of currently used substitution models and molecular clocks. A comprehensive analysis of mutagenesis in 4 bp mutation contexts requires a vast amount of mutation data. Such data may be derived from the comparisons of individual genomes or from single nucleotide polymorphism (SNP databases. Using this approach, we performed a systematical comparison of mutation regularities within 2–4 bp contexts in Mus musculus and Homo sapiens and uncovered that even closely related organisms may have notable differences in context-dependent mutation regularities.

  12. A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

    Directory of Open Access Journals (Sweden)

    Ashlee J. Conway

    2017-08-01

    Full Text Available A genome-wide ethyl-N-nitrosourea (ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX. A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

  13. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Puck, T.P.; Johnson, R.; Waldren, C.A. (Eleanor Roosevelt Institute for Cancer Research, Denver, CO (United States)); Morse, H. (Univ. of Colorado Cancer Center, Denver, CO (United States))

    1993-09-01

    The well-known action of caffeine in synergizing mutagenesis (including chromosome aberrations) of agents like ionizing radiation by inhibition of cellular repair processes has been incorporated into a rapid procedure for detection of mutagenicity with high sensitivity. Effects of 5-10 rads of [gamma]-irradiation, which approximate the human lifetime dose accumulation from background radiation, can be detected in a two-day procedure using an immortalized human WBC culture. Chromosomally visible lesions are scored on cells incubated for 2 h after irradiation in the presence and absence of 1.0 mg/ml of caffeine. An eightfold amplification of scorable lesions is achieved over the action of radiation alone. This approach provides a closer approximation to absolute mutagenicity unmitigated by repair processes, which can vary in different situations. It is proposed that mutagenesis testing of this kind, using caffiene or other repair-inhibitory agents, be employed to identify mutagens in their effective concentrations to which human populations may be exposed; to detect agents such as caffeine that may synergize mutagenic actions and pose epidemiologic threats; and to discover effective anti-mutagens. Information derived from the use of such procedures may help prevent cancer and newly acquired genetic disease.

  14. Identification of 17 hearing impaired mouse strains in the TMGC ENU-mutagenesis screen

    Energy Technology Data Exchange (ETDEWEB)

    Kermany, Mohammad [St. Jude Children' s Research Hospital; Parker, Lisan [St. Jude Children' s Research Hospital; Guo, Yun-Kai [St. Jude Children' s Research Hospital; Miller, Darla R [ORNL; Swanson, Douglas J [ORNL; Yoo, Tai-June [Neuroscience Institute, Memphis, TN; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis; Zuo, Jian [St. Jude Children' s Research Hospital

    2006-01-01

    The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding of presbycusis and noise-induced hearing loss in humans.

  15. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    Science.gov (United States)

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

    Science.gov (United States)

    Ranzani, Marco; Cesana, Daniela; Bartholomae, Cynthia C.; Sanvito, Francesca; Pala, Mauro; Benedicenti, Fabrizio; Gallina, Pierangela; Sergi, Lucia Sergi; Merella, Stefania; Bulfone, Alessandro; Doglioni, Claudio; von Kalle, Christof; Kim, Yoon Jun; Schmidt, Manfred; Tonon, Giovanni; Naldini, Luigi; Montini, Eugenio

    2013-01-01

    Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells, hampering the identification of early cancer-driving events amongst bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVV) by which we could efficiently induce hepatocellular carcinoma (HCC) in 3 different mouse models. By virtue of LVV’s replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of 4 new liver cancer genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. Our newly identified cancer genes are likely to play a role in human disease, since they are upregulated and/or amplified/deleted in human HCCs and can predict clinical outcome of patients. PMID:23314173

  17. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

    Science.gov (United States)

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

    2016-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Laboratory Activity to Promote Student Understanding of UV Mutagenesis and DNA Repair

    Directory of Open Access Journals (Sweden)

    Joshua Ernest Kouassi

    2017-05-01

    Full Text Available Changes in DNA molecules are common, due to the effects of UV light and other external and internal mutagens. Cells have a variety of repair mechanisms which serve to maintain the accuracy of the genetic code. This activity includes a low-cost, safe and technically feasible experiment, which allows students to observe the effects of UV mutagenesis and DNA photorepair in the halophilc archaeon, Haloferax volcanii. An optional extension links this activity to topics of immediate concern to students – how exposure to UVC light contributes to skin cancer risk and the protective effects of sunscreen. Students design and carry out an experiment to test whether SPF 15 sunscreen increases the lethal exposure time for H. volcanii by a factor of 15. Throughout the activity, discussion questions engage students in actively thinking about the biological phenomena and experimental procedures and analysis. This activity is designed for students in college or university genetics, microbiology, or introductory biology courses as well as in high school honors biology courses. Teachers report that this activity was valuable in helping students understand mutagenesis and photorepair and in developing student skills in designing and analyzing experiments.

  19. Increased activity of β-glucuronidase variants produced by site-directed mutagenesis.

    Science.gov (United States)

    Zhang, Xiaolei; Sitasuwan, Pongkwan; Horvath, Gary; Yang, Jia; Nie, Yuzhe; Marinova, Margarita; Lee, L Andrew; Wang, Qian

    2018-02-01

    β-glucuronidase (BGus) is an essential glycosyl hydrolase which has been widely used in biological and biomedical applications. In this paper, we report the construction and screening of nineteen Escherichia coli BGus (EBGus) mutants using site-directed mutagenesis. The mutants G559N, G559S and G559T showed a 3-5 fold increase in enzyme activity in comparison to wild type EBGus. In particular, G559S, with the highest activity, showed 2-6 fold enhanced activity compared to abalone and snail BGus extracts. Moreover, the glycine to serine mutagenesis for the same site in Staphylococcus sp. RLH1 BGus (StBGus) exhibited significantly enhanced activity, which indicated the importance of the G559→S mutation on BGus function. Based on this structural analysis, we postulate that the mutation at G559 plays an important role in the stabilization of the enzyme conformation, and thereby facilitates the effective binding of substrate. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    Science.gov (United States)

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. © The Author(s).

  1. Randomization tests

    CERN Document Server

    Edgington, Eugene

    2007-01-01

    Statistical Tests That Do Not Require Random Sampling Randomization Tests Numerical Examples Randomization Tests and Nonrandom Samples The Prevalence of Nonrandom Samples in Experiments The Irrelevance of Random Samples for the Typical Experiment Generalizing from Nonrandom Samples Intelligibility Respect for the Validity of Randomization Tests Versatility Practicality Precursors of Randomization Tests Other Applications of Permutation Tests Questions and Exercises Notes References Randomized Experiments Unique Benefits of Experiments Experimentation without Mani

  2. Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations.

    Science.gov (United States)

    Ambriz-Aviña, Verónica; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2016-11-01

    Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B. subtilis cultures. Using this approach, we demonstrate that (i) high levels of transcription correlates with augmented mutation frequency, and (ii) mutation frequency is enhanced in nongrowing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.

  3. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART for genetic screens in mice.

    Directory of Open Access Journals (Sweden)

    Sean F Landrette

    Full Text Available Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

  4. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    Science.gov (United States)

    Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed similar growth rate, colony morphology, and sclerotial and oxalate ...

  5. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

    Directory of Open Access Journals (Sweden)

    Huifen Xu

    2015-01-01

    Full Text Available Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene was confirmed to have a high tolerance for mutagenesis by this new assay.

  6. Electronic monitoring of symptoms and syndromes associated with cancer: methods of a randomized controlled trial SAKK 95/06 E-MOSAIC

    Directory of Open Access Journals (Sweden)

    Blum David

    2012-09-01

    Full Text Available Abstract Background In patients with advanced, incurable cancer, anticancer treatment may be used to alleviate cancer-related symptoms, but monitoring of them in daily practice is rarely done. We aim to test the effectiveness of a real-time symptom and syndrome assessment using the E-MOSAIC software installed in handheld computer generating a longitudinal monitoring sheet (LoMoS provided to the oncologists in a phase III setting. Methods In this prospective multicentre cluster randomized phase-III trial patients with any incurable solid tumor and having defined cancer related symptoms, who receive new outpatient chemotherapy in palliative intention (expected tumor-size response rate ≤20% are eligible. Immediately before the weekly visit to oncologists, all patients complete with nurse assistance the E-MOSAIC Assessment: Edmonton Symptom Assessment Scale, ≤3 additional symptoms, estimated nutritional intake, body weight, Karnofsky and medications for pain and cachexia. Experienced oncologists will be randomized to receive the LoMoS or not. To minimize contamination, LoMoS are removed from the medical charts after visits. Primary endpoint is the difference in global quality of life (items 29 & 30 of EORTC-QlQ-C30 between baseline and last study visit at week 6, with a 10 point between-arm difference considered to be clinically relevant. 20 clusters (=oncologists per treatment arm with 4–8 patients each are aimed for to achieve a significance level of 5% and a power of 80% in a mixed model approach. Selected co- variables are included in the model for adjustment. Secondary endpoints include patient-perceived patient-physician communication symptom burden over time, and oncologists’ symptom management performance (predefined thresholds of symptoms compared to oncologists’ pharmacological, diagnostic or counselling actions [structured chart review]. Discussion This trial will contribute to the research question, whether structured

  7. Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

    Science.gov (United States)

    Breyer, Didier; Herman, Philippe; Brandenburger, Annick; Gheysen, Godelieve; Remaut, Erik; Soumillion, Patrice; Van Doorsselaere, Jan; Custers, René; Pauwels, Katia; Sneyers, Myriam; Reheul, Dirk

    2009-01-01

    In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

  8. Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

    Directory of Open Access Journals (Sweden)

    Andrew R. Bassett

    2013-07-01

    Full Text Available Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated. We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

  9. Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides.

    Science.gov (United States)

    Liu, Guangfei; Zhou, Jiti; Wang, Jing; Yan, Bin; Li, Jingmei; Lu, Hong; Qu, Yuanyuan; Jin, Ruofei

    2008-05-01

    Comparison of three-dimensional structures of flavin-dependent azoreductases revealed two conserved loops around the flavin mononucleotide (FMN) cofactor. Tyr74, His75 and Lys109 in the two loops of azoreductase AZR from Rhodobacter sphaeroides were replaced with Trp, Asn and Ala/His by site-directed mutagenesis, respectively. The optimal pH values of K109H and H75N were pH 6, and those of K109A and Y74W were pH 9. The optimal temperature (30 degrees C) was not affected by mutation. Positively charged residues at position 109 is critical for the binding of methyl red. K109 might only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH. Y74W and H75N mutations decreased the binding of methyl red/nitrofurazone and had no affect on the binding of NADPH.

  10. Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

    Directory of Open Access Journals (Sweden)

    Nicole M Anderson

    Full Text Available Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X, resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-. Timed pregnancy experiments revealed that Jak2(K915X/K915X and Jak2(-/- displayed embryonic lethality; however, Jak2(K915X/K915X embryos were viable an additional two days compared to Jak2(-/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

  11. Highly Efficient Site-Specific Mutagenesis in Malaria Mosquitoes Using CRISPR

    Directory of Open Access Journals (Sweden)

    Ming Li

    2018-02-01

    Full Text Available Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, A. coluzzii, and A. funestus. When guide RNAs (gRNAs and Cas9 protein are injected at high concentration, germline mutations are common and usually biallelic, allowing for the rapid creation of stable mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control.

  12. Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes.

    Science.gov (United States)

    Li, Yanhua; Gao, Zengqiang; Hou, Haifeng; Li, Lei; Zhang, Jihui; Yang, Haihua; Dong, Yuhui; Tan, Huarong

    2011-02-18

    Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli

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    Clayton Tincher

    2017-10-01

    Full Text Available Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria.

  14. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

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    Audrey Olshefsky

    Full Text Available Two-component signaling (2CS systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.

  15. Directed mutagenesis under the action of mobile elements in unstable lines of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Gerasimova, T.I.; Mizrokhi, L.Yu.; Obolenkova, L.A.; Georgiev, P.G.

    1985-07-01

    The unstable mutation ct/sup MR2/, obtained under hybrid dysgenesis and caused by the incorporation of MDG 4 into locus cut, was described earlier. A peculiarity of this line and its derivatives is that multiple transpositions of different mobile elements, the transposition spurts, take place in them with a frequency of 10/sup -2/-10/sup -4/. A consequence of this process is active insertion mutagenesis taking place under the action of different transposons with high locus specifity. The mutations are unstable and frequently revert to wild type. However, there are some unstable revertants in which the mobile element is eliminated almost completely, and yet unstable mutations appear at the same locus at a high rate. In the case of cut locus, this happens due to repeated integration of MDG 4. The authors report cases of such repeated directed mutagenesis for several loci. Reversions were observed in the homozygous line as well as during the process of obtaining homozygous lines of new mutations using the chromosome with multiple inversions FM4, Y/sup 31d/sc/sup 8/ dmB. In situ hybridization with the (/sup 3/H)-labeled DNA was carried out as described earlier. The plasmid DNA containing cloned MDG4 and their long terminal repeats were used as probes for hybridization. Hybridization was done on the salivary gland polytene chromosomes of larvae. The new spontaneous mutations appearing in the process of hybridization were tested for allelism with the standard mutations y, w, cm, ct, D, and g.

  16. The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

    Science.gov (United States)

    Maisnier-Patin, Sophie; Roth, John R.

    2015-01-01

    Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

  17. Mutagenesis in sequence encoding of human factor VII for gene therapy of hemophilia

    Directory of Open Access Journals (Sweden)

    B Kazemi

    2009-12-01

    Full Text Available "nBackground: Current treatment of hemophilia which is one of the most common bleeding disorders, involves replacement therapy using concentrates of FVIII and FIX .However, these concentrates have been associated with viral infections and thromboembolic complications and development of antibodies. "nThe use of recombinant human factor VII (rhFVII is effective  for the treatment of patients with  hemophilia A or B, who develop antibodies ( referred as inhibitors against  replacement therapy , because it induces coagulation independent of FVIII and FIX. However, its short half-life and high cost have limited its use. One potential solution to this problem may be the use of FVIIa gene transfer, which would attain continuing therapeutic levels of expression from a single injection. The aim of this study was to engineer a novel hFVII (human FVII gene containing a cleavage site for the intracellular protease and furin, by PCR mutagenesis "nMethods: The sequence encoding light and heavy chains of hFVII, were amplified by using hFVII/pTZ57R and specific primers, separately. The PCR products were cloned in pTZ57R vector. "nResults and discussion: Cloning was confirmed by restriction analysis or PCR amplification using specific primers and plasmid universal primers. Mutagenesis of sequence encoding light and heavy chain was confirmed by restriction enzyme. "nConclusion: In the present study, it was provided recombinant plasmids based on mutant form of DNA encoding light and heavy chains.  Joining mutant form of DNA encoding light chain with mutant heavy chain led to a new variant of hFVII. This variant can be activated by furin and an increase in the proportion of activated form of FVII. This mutant form of hFVII may be used for gene therapy of hemophilia.

  18. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    Science.gov (United States)

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  19. Characterization of a new allele of Ames waltzer generated by ENU mutagenesis.

    Science.gov (United States)

    Washington, Jesse L; Pitts, Darrell; Wright, Charles G; Erway, L C; Davis, Rickie R; Alagramam, Kumar

    2005-04-01

    Mutation in the protocadherin 15 (Pcdh15) gene causes hair cell dysfunction and is associated with abnormal stereocilia development. We have characterized the first allele (Pcdh15(av-nmf19)) of Ames waltzer (av) obtained by N-ethyl-N-nitrosourea (ENU) mutagenesis. Pcdh15(av-nmf19) was generated in the Neuroscience Mutagenesis Facility (NMF) at The Jackson Lab (Bar Habor, USA). Pcdh15(av-nmf19) mutants display circling and abnormal swimming behavior along with lack of auditory-evoked brainstem response at the highest intensities tested. Mutation analysis shows base substitution (A--> G) in the consensus splice donor sequence linked to exon 14 resulting in the skipping of exon 14 and the splicing of exon 13-15. This results in the introduction of a stop codon in the coding sequence of exon 15 due to shift in the reading frame. The effect of nmf19 mutation is expected to be severe since the expressed Pcdh15 protein is predicted to truncate in the 5th cadherin domain. Abnormalities of cochlear hair cell stereocilia are apparent in Pcdh15(av-nmf19) mutants near the time of birth and by about P15 (15 days after birth) there is evidence of sensory cell degeneration. Disorganization of outer hair cell stereocilia is observed as early as P2. Inner hair cell stereocilia are also affected, but less severely than those of the outer hair cells. These results are consistent with characteristics of the mutation in the Pcdh15(av-nmf19) allele and they support our previous finding that Protocadherin 15 plays an important role in hair-bundle morphogenesis.

  20. Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Maura McGrail

    2011-04-01

    Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

  1. Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

    Science.gov (United States)

    Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2017-02-15

    The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H2O2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

  2. Effect of radiation-sensitive mutations and mutagens/carcinogens on bacterial recombination and mutagenesis. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Matney, T.S.

    1978-01-01

    Progress is reported on effects of temperature sensitive DNA-initiation mutation in E. coli K-12 mutants; the use of Bacillus subtilis transforming system as an in vitro mutagenesis system; characteristics of the E. coli lysogen used to test the permeability to polycyclic aromatic hydrocarbons; and the genetic toxicology of gentian violet. (PCS)

  3. USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells

    Directory of Open Access Journals (Sweden)

    Shu-ichiro Kashiwaba

    2015-12-01

    Full Text Available Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair.

  4. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    Science.gov (United States)

    Giron, Maria D.; Salto, Rafael

    2011-01-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

  5. Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

    NARCIS (Netherlands)

    Michielse, C.B.; van Wijk, R.; Reijnen, L.; Cornelissen, B.J.C.; Rep, M.

    2009-01-01

    Background: Fusarium oxysporum f. sp. lycopersici is the causal agent of vascular wilt disease in tomato. In order to gain more insight into the molecular processes in F. oxysporum necessary for pathogenesis and to uncover the genes involved, we used Agrobacterium-mediated insertional mutagenesis to

  6. Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

    NARCIS (Netherlands)

    Huser, C.A.; Gilroy, K.L.; De Ridder, J.; Kilbey, A.; Borland, G.; Mackay, N.; Jenkins, A.; Bell, M.; Herzyk, P.; Van der Weyden, L.

    2014-01-01

    Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC,

  7. Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Gilquin, Bernard; Le Du, Marie Hélène

    2006-01-01

    a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were...

  8. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  9. Feasibility, design and conduct of a pragmatic randomized controlled trial to reduce overweight and obesity in children: The electronic games to aid motivation to exercise (eGAME study

    Directory of Open Access Journals (Sweden)

    Rodgers Anthony

    2009-05-01

    Full Text Available Abstract Background Childhood obesity has reached epidemic proportions in developed countries. Sedentary screen-based activities such as video gaming are thought to displace active behaviors and are independently associated with obesity. Active video games, where players physically interact with images onscreen, may have utility as a novel intervention to increase physical activity and improve body composition in children. The aim of the Electronic Games to Aid Motivation to Exercise (eGAME study is to determine the effects of an active video game intervention over 6 months on: body mass index (BMI, percent body fat, waist circumference, cardio-respiratory fitness, and physical activity levels in overweight children. Methods/Design Three hundred and thirty participants aged 10–14 years will be randomized to receive either an active video game upgrade package or to a control group (no intervention. Discussion An overview of the eGAME study is presented, providing an example of a large, pragmatic randomized controlled trial in a community setting. Reflection is offered on key issues encountered during the course of the study. In particular, investigation into the feasibility of the proposed intervention, as well as robust testing of proposed study procedures is a critical step prior to implementation of a large-scale trial. Trial registration Australian New Zealand Clinical Trials Registry ACTRN12607000632493

  10. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes Internalin A for enhanced infectivity in the murine oral infection model

    LENUS (Irish Health Repository)

    Monk, Ian R

    2010-12-13

    Abstract Background Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human

  11. Improving the care of children with advanced cancer by using an electronic patient-reported feedback intervention: results from the PediQUEST randomized controlled trial.

    Science.gov (United States)

    Wolfe, Joanne; Orellana, Liliana; Cook, E Francis; Ullrich, Christina; Kang, Tammy; Geyer, Jeffrey Russell; Feudtner, Chris; Weeks, Jane C; Dussel, Veronica

    2014-04-10

    This study aimed to determine whether feeding back patient-reported outcomes (PROs) to providers and families of children with advanced cancer improves symptom distress and health-related quality of life (HRQoL). This study was a parallel, multicentered pilot randomized controlled trial. At most once per week, children age ≥ 2 years old with advanced cancer or their parent completed the computer-based Pediatric Quality of Life and Evaluation of Symptoms Technology (PediQUEST) survey consisting of age- and respondent-adapted versions of the Memorial Symptom Assessment Scale (MSAS), Pediatric Quality of Life Inventory 4.0 Generic Core Scales (PedsQL4.0), and an overall Sickness question. In the intervention group (n = 51), oncologists and families received printed reports summarizing PROs; e-mails were sent to oncologists and subspecialists when predetermined scores were exceeded. No feedback was provided in the control group (n = 53). Primary outcomes included linear trends of MSAS, PedsQL4.0 total and subscale scores, and Sickness scores during 20 weeks of follow-up, along with child, parent, and provider satisfaction with PediQUEST feedback. Feedback did not significantly affect average MSAS, PedsQL4.0, or Sickness score trends. Post hoc subgroup analyses among children age ≥ 8 years who survived 20 weeks showed that feedback improved PedsQL4.0 emotional (+8.1; 95% CI, 1.8 to 14.4) and Sickness (-8.2; 95% CI, -14.2 to -2.2) scores. PediQUEST reports were valued by children, parents, and providers and contributed at least sometimes to physician initiation of a psychosocial consult (56%). Although routine feedback of PROs did not significantly affect the child's symptoms or HRQoL, changes were in expected directions and improvements observed in emotional HRQoL through exploratory analyses were encouraging. Importantly, children, parents, and providers value PRO feedback.

  12. Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

    Science.gov (United States)

    Eyre, Nicholas S; Johnson, Stephen M; Eltahla, Auda A; Aloi, Maria; Aloia, Amanda L; McDevitt, Christopher A; Bull, Rowena A; Beard, Michael R

    2017-12-01

    Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the

  13. Randomized Trial Comparing the Electronic Composite Psychosocial Screener YouthCHAT With a Clinician-Interview Assessment for Young People: A Study Protocol.

    Science.gov (United States)

    Thabrew, Hiran; Corter, Arden; Goodyear-Smith, Felicity; Goldfinch, Mary

    2017-07-31

    Psychosocial problems such as depression, anxiety, and substance abuse are common and burdensome in young people, particularly those with long-term physical conditions such as asthma and diabetes. In New Zealand, "screening" for such problems is undertaken routinely only with Year 9 students in low-decile schools and opportunistically in pediatric settings using a nonvalidated and time-consuming clinician-administered Home, Education/employment, Eating, Activity, Drugs, Sexuality, Suicide/depression, Safety (HEEADSSS) interview. The Youth version, Case-finding and Help Assessment Tool (YouthCHAT) is a relatively new, locally developed, eTablet-based composite screener for identifying similar psychosocial issues to HEEADSSS. Based on individually validated screening instruments, it is self-administered within minutes. Preliminary testing has revealed its acceptability to young people, but further research is required to expand its modules to cover all HEEADSSS domains, to evaluate its acceptability for young people with and without long-term physical conditions, and to compare its effectiveness against HEEADSSS. Our aim is to (1) ascertain acceptability and utility of YouthCHAT for children with long-term physical illness and high school students, (2) validate three additional YouthCHAT domains against comparable HEEADSSS domains, and (3) compare the performance of YouthCHAT and HEEADSSS in the high school setting. During the first phase of the study, three additional YouthCHAT domains were codesigned with high school students. During the second phase of the study, the updated version of YouthCHAT will be administered to 30 young people with long-term physical conditions, and to 150 high school students either before or after HEEADSSS in the form of a randomized trial with counter-balanced design. Primary outcomes include comparability between HEEADSSS and YouthCHAT in detecting psychosocial issues, and time to administer; acceptability of YouthCHAT as an acceptable

  14. Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

    Science.gov (United States)

    Jin, Liang; Wu, Xuri; Xue, Yanjiu; Jin, Yue; Wang, Shuzhen

    2016-01-01

    ABSTRACT Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the

  15. Structure-based mutagenesis of Penicillium griseofulvum xylanase using computational design.

    Science.gov (United States)

    André-Leroux, Gwénaëlle; Berrin, Jean-Guy; Georis, Jacques; Arnaut, Filip; Juge, Nathalie

    2008-09-01

    Penicillium griseofulvum xylanase (PgXynA) belongs to family 11 glycoside hydrolase. It exhibits unique amino acid features but its three-dimensional structure is not known. Based upon the X-ray structure of Penicillium funiculosum xylanase (PfXynC), we generated a three-dimensional model of PgXynA by homology modeling. The native structure of PgXynA displayed the overall beta-jelly roll folding common to family 11 xylanases with two large beta-pleated sheets and a single alpha-helix that form a structure resembling a partially closed right hand. Although many features of PgXynA were very similar to previously described enzymes from this family, crucial differences were observed in the loop forming the "thumb" and at the edge of the binding cleft. The robustness of the xylanase was challenged by extensive in silico-based mutagenesis analysis targeting mutations retaining stereochemical and energetical control of the protein folding. On the basis of structural alignments, modeled three-dimensional structure, in silico mutations and docking analysis, we targeted several positions for the replacement of amino acids by site-directed mutagenesis to change substrate and inhibitor specificity, alter pH profile and improve overall catalytic activity. We demonstrated the crucial role played by Ser44(PgXynA) and Ser129(PgXynA), two residues unique to PgXynA, in conferring distinct specificity to P. griseofulvum xylanase. We showed that the pH optimum of PgXynA could be shifted by -1 to +0.5 units by mutating Ser44(PgXynA) to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in K(m) and V(max) whereas a S44A mutant lost both pH-dependence profile and activity. We were able to produce PgXynA S129G mutants with acquired sensitivity to the Xylanase Inhibitor Protein, XIP-I. The replacement of Gln121(PgXynA), located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity. 2008 Wiley

  16. Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis.

    Science.gov (United States)

    Jin, Liang; Wu, Xuri; Xue, Yanjiu; Jin, Yue; Wang, Shuzhen; Chen, Yijun

    2017-02-15

    Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the

  17. Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

    Directory of Open Access Journals (Sweden)

    Chen Tsung-Chieh

    2012-05-01

    Full Text Available Abstract Background N-ethyl-N-nitrosourea mutagenesis was used to induce a point mutation in C57BL/6 J mice. Pain-related phenotype screening was performed in 915 G3 mice. We report the detection of a heritable recessive mutant in meiotic recombinant N1F1 mice that caused an abnormal pain sensitivity phenotype with spontaneous skin inflammation in the paws and ears. Methods We investigated abnormal sensory processing, neuronal peptides, and behavioral responses after the induction of autoinflammatory disease. Single-nucleotide polymorphism (SNP markers and polymerase chain reaction product sequencing were used to identify the mutation site. Results All affected mice developed paw inflammation at 4–8 weeks. Histological examinations revealed hyperplasia of the epidermis in the inflamed paws and increased macrophage expression in the spleen and paw tissues. Mechanical and thermal nociceptive response thresholds were reduced in the affected mice. Locomotor activity was decreased in affected mice with inflamed hindpaws, and this reduction was attributable to the avoidance of contact of the affected paw with the floor. Motor strength and daily activity in the home cage in the affected mice did not show any significant changes. Although Fos immunoreactivity was normal in the dorsal horn of affected mice, calcitonin gene-related peptide immunoreactivity significantly increased in the deep layer of the dorsal horn. The number of microglia increased in the spinal cord, hippocampus, and cerebral cortex in affected mice, and the proliferation of microglia was maintained for a couple of months. Two hundred eighty-five SNP markers were used to reveal the affected gene locus, which was found on the distal part of chromosome 18. A point mutation was detected at A to G in exon 8 of the pstpip2 gene, resulting in a conserved tyrosine residue at amino acid 180 replaced by cysteine (Y180 C. Conclusions The data provide definitive evidence that a mutation

  18. Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels.

    Science.gov (United States)

    Goldstein, Matthias; Rinné, Susanne; Kiper, Aytug K; Ramírez, David; Netter, Michael F; Bustos, Daniel; Ortiz-Bonnin, Beatriz; González, Wendy; Decher, Niels

    2016-01-22

    Two-pore-domain potassium (K2P) channels have a large extracellular cap structure formed by two M1-P1 linkers, containing a cysteine for dimerization. However, this cysteine is not present in the TASK-1/3/5 subfamily. The functional role of the cap is poorly understood and it remained unclear whether K2P channels assemble in the domain-swapped orientation or not. Functional alanine-mutagenesis screens of TASK-1 and TRAAK were used to build an in silico model of the TASK-1 cap. According to our data the cap structure of disulfide-bridge free TASK channels is similar to that of other K2P channels and is most likely assembled in the domain-swapped orientation. As the conserved cysteine is not essential for functional expression of all K2P channels tested, we propose that hydrophobic residues at the inner leaflets of the cap domains can interact with each other and that this way of stabilizing the cap is most likely conserved among K2P channels.

  19. Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis▿

    Science.gov (United States)

    Vidales, Luz E.; Cárdenas, Lluvia C.; Robleto, Eduardo; Yasbin, Ronald E.; Pedraza-Reyes, Mario

    2009-01-01

    Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism. PMID:19011023

  20. Dopamine D1 receptor-agonist interactions: A mutagenesis and homology modeling study.

    Science.gov (United States)

    Mente, Scot; Guilmette, Edward; Salafia, Michelle; Gray, David

    2015-01-01

    The dopamine D1 receptor is a G protein-coupled receptor that regulates intracellular signaling via agonist activation. Although the number of solved GPCR X-ray structures has been steadily increasing, still no structure of the D1 receptor exists. We have used site-directed mutagenesis of 12 orthosteric vicinity residues of possible importance to G protein-coupled activation to examine the function of prototypical orthosteric D1 agonists and partial agonists. We find that residues from four different regions of the D1 receptor make significant contributions to agonist function. All compounds studied, which are catechol-amines, are found to interact with the previously identified residues: the conserved D103(3.32), as well as the trans-membrane V serine residues. Additional key interactions are found for trans-membrane VI residues F288(6.51), F289(6.52) and N292(6.55), as well as the extra-cellular loop residue L190(ECL2). Molecular dynamics simulations of a D1 homology model have been used to help put the ligand-residue interactions into context. Finally, we considered the rescaling of fold-shift data as a method to account for the change in the size of the mutated side-chain and found that this rescaling helps to relate the calculated ligand-residue energies with observed experimental fold-shifts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma.

    Science.gov (United States)

    Mann, Karen M; Ward, Jerrold M; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W; Black, Michael A; Brett, Benjamin T; Sheetz, Todd E; Dupuy, Adam J; Chang, David K; Biankin, Andrew V; Waddell, Nicola; Kassahn, Karin S; Grimmond, Sean M; Rust, Alistair G; Adams, David J; Jenkins, Nancy A; Copeland, Neal G

    2012-04-17

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.

  2. Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

    Science.gov (United States)

    Kowalsky, Caitlin A; Faber, Matthew S; Nath, Aritro; Dann, Hailey E; Kelly, Vince W; Liu, Li; Shanker, Purva; Wagner, Ellen K; Maynard, Jennifer A; Chan, Christina; Whitehead, Timothy A

    2015-10-30

    Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Apparent resistance to mutagenesis by ionizing radiation, and some other unusual responses

    Energy Technology Data Exchange (ETDEWEB)

    Faberge, A.C.

    1983-11-01

    It is pointed out that in some species it is very difficult or even apparently impossible to get genetic visible marker mutants by means of ionizing radiation. Such results are not as a rule published. Species in which no mutant at all could be obtained, after what seem to be adequate efforts by several geneticists are Limnaea peregra, Rhynchosciara angelae, Coelopa frigida, Oncopeltus fasciatus, Dermestes maculatus. Published data on Sciara, Tribolium, Blattella, in which a relative difficulty in getting mutants is reported, are discussed and compared to many other forms in which obtaining marker mutants is on the contrary easy. What appears to be very abnormal mutagenesis is described in Drosophila busckii, which yielded an unusual frequency of autosomal dominants, and Drosophila nebulosa, in which only very poor mutants could be obtained. Possible reasons for all these departures from the usual are discussed. No firm conclusions are reached, but one is led to consider the possible effects of overall chromosome structure, presence or absence of heterochromatin, and multiplicity at the single locus or single polytene band level. There seems to be no relation between the rates of induction of dominant lethality and of point mutation for visible markers.

  4. CRISPR/Cas9-Mediated In Vitro Mutagenesis in GC-Like B Cells.

    Science.gov (United States)

    Chu, Van Trung; Graf, Robin; Rajewsky, Klaus

    2017-01-01

    The CRISPR/Cas9 technology has developed into a powerful tool for genome editing, both in terms of gene silencing and the insertion of precise mutations. However, the application of CRISPR/Cas9-mediated mutagenesis in primary immune cells, in particular in B cells, is still in its infancy because of the difficulty to deliver the CRISPR/Cas9 system into these cells. Here, we describe a new method to use CRISPR/Cas9 for manipulating genes in germinal center (GC)-like B cells in vitro. We isolated Cas9-expressing B cells from R26-Cas9iGFP/+ mice (expressing Cas9 constitutively from the Rosa26 locus) and mixed them with control B cells. Primary B cells were cultured on CD40L- and BAFF-expressing feeder cells and transduced with retroviral particles expressing the sgRNAs of interest. Using this system, we have achieved complete gene knockouts in up to 92% of activated B cells.

  5. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    Science.gov (United States)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  6. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis

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    Daisuke Nojima

    2017-05-01

    Full Text Available Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively were exposed to ethyl methane sulfonate (EMS or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG, and pale green mutants (PMs were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

  7. ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus by treating mature sperm.

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    Xia-Yun Jiang

    Full Text Available N-ethyl-N-nitrosourea (ENU mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1 and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.

  8. In vitro mutagenesis in embryogenic cell suspensions of banana cv. Grande naine (Musa AAA

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    Idalmis Bermúdez-Caraballoso

    2016-04-01

    Full Text Available Somatic embryogenesis is a useful process for clonal propagation and genetic improvement by induction of mutations. This work was carried out with the objective of determining the effect of 60Co source Gamma radiations on embryogenic cell suspensions of banana cv. 'Grande naine' (Musa AAA until conversion to plants. Different doses of radiation (0, 30, 40, 50, 60, 70 and 80 Gy were applied to embryogenic cell suspensions in the multiplication phase and the embryos were later formed, matured and germinated. To determine the ex vitro response of the population of plants obtained these were transferred to greenhouse. The results showed that with somatic embryos formed fresh mass no differences were observed between the effect of the different doses of radiation applied and the control. However, the radiation dose affected the percentage of somatic embryo formation and germination. Plants with phenotypic variations were regenerated with 40 Gy. The results at the greenhouse showed that as radiation doses increased up to 50 Gy, the frequency of variations increased. With higher doses of radiation the survival of the plants was affected.   Keywords: Gamma radiation, in vitro mutagenesis, radiation dose, radiosensibility, somatic embryo

  9. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.

    Science.gov (United States)

    Liang, Zhen; Zhang, Kang; Chen, Kunling; Gao, Caixia

    2014-02-20

    Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize. Copyright © 2013. Published by Elsevier Ltd.

  10. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pletneva, Nadya V.; Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Souslova, Ekaterina; Chudakov, Dmitry M. [Russian Academy of Sciences, Moscow (Russian Federation); Lukyanov, Sergey [Russian Academy of Sciences, Moscow (Russian Federation); Nizhny Novgorod State Medical Academy, Nizhny Novgorod (Russian Federation); Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor [Russian Academy of Sciences, Moscow (Russian Federation); Wlodawer, Alexander [National Cancer Institute, Frederick, MD 21702 (United States); Dauter, Zbigniew [National Cancer Institute, Argonne, IL 60439 (United States); Pletnev, Sergei [National Cancer Institute, Argonne, IL 60439 (United States); SAIC-Frederick, 9700 South Cass Avenue, Argonne, IL 60439 (United States); Russian Academy of Sciences, Moscow (Russian Federation)

    2013-06-01

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  11. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    Science.gov (United States)

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-03

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

  12. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

    Directory of Open Access Journals (Sweden)

    Yvonne Jing Mei Liew

    2015-02-01

    Full Text Available Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1 has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

  13. General mutagenesis of F plasmid TraI reveals its role in conjugative regulation.

    Science.gov (United States)

    Haft, Rembrandt J F; Palacios, Gilberto; Nguyen, Tran; Mally, Manuela; Gachelet, Eliora G; Zechner, Ellen L; Traxler, Beth

    2006-09-01

    Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.

  14. Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen.

    Science.gov (United States)

    Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

    2011-12-01

    Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. © 2011 John Wiley & Sons A/S.

  15. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

    Science.gov (United States)

    Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

    2016-01-01

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

  16. Sleeping Beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumor.

    Science.gov (United States)

    Tschida, Barbara R; Temiz, Nuri A; Kuka, Timothy P; Lee, Lindsey A; Riordan, Jesse D; Tierrablanca, Carlos A; Hullsiek, Robert; Wagner, Sandra; Hudson, Wendy A; Linden, Michael A; Amin, Khalid; Beckmann, Pauline J; Heuer, Rachel A; Sarver, Aaron L; Yang, Ju Dong; Roberts, Lewis R; Nadeau, Joseph H; Dupuy, Adam J; Keng, Vincent W; Largaespada, David

    2017-10-09

    Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis, and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Copyright ©2017, American Association for Cancer Research.

  17. Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.

    Science.gov (United States)

    Starita, Lea M; Pruneda, Jonathan N; Lo, Russell S; Fowler, Douglas M; Kim, Helen J; Hiatt, Joseph B; Shendure, Jay; Brzovic, Peter S; Fields, Stanley; Klevit, Rachel E

    2013-04-02

    Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.

  18. Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis.

    Science.gov (United States)

    Fuller, T E; Kennedy, M J; Lowery, D E

    2000-07-01

    P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets. Copyright 2000 Academic Press.

  19. An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions.

    Directory of Open Access Journals (Sweden)

    Carolien Wansleeben

    Full Text Available BACKGROUND: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila] and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. CONCLUSIONS/SIGNIFICANCE: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

  20. Molecular Mechanisms for High Hydrostatic Pressure-Induced Wing Mutagenesis in Drosophila melanogaster.

    Science.gov (United States)

    Wang, Hua; Wang, Kai; Xiao, Guanjun; Ma, Junfeng; Wang, Bingying; Shen, Sile; Fu, Xueqi; Zou, Guangtian; Zou, Bo

    2015-10-08

    Although High hydrostatic pressure (HHP) as an important physical and chemical tool has been increasingly applied to research of organism, the response mechanisms of organism to HHP have not been elucidated clearly thus far. To identify mutagenic mechanisms of HHP on organisms, here, we treated Drosophila melanogaster (D. melanogaster) eggs with HHP. Approximately 75% of the surviving flies showed significant morphological abnormalities from the egg to the adult stages compared with control flies (p melanogaster induced by HHP were used to investigate the mutagenic mechanisms of HHP on organism. Thus 285 differentially expressed genes associated with wing mutations were identified using Affymetrix Drosophila Genome Array 2.0 and verified with RT-PCR. We also compared wing development-related central genes in the mutant flies with control flies using DNA sequencing to show two point mutations in the vestigial (vg) gene. This study revealed the mutagenic mechanisms of HHP-induced mutagenesis in D. melanogaster and provided a new model for the study of evolution on organisms.

  1. Promoter mutagenesis for fine-tuning expression of essential genes in Mycobacterium tuberculosis.

    Science.gov (United States)

    Boldrin, Francesca; Degiacomi, Giulia; Serafini, Agnese; Kolly, Gaëlle S; Ventura, Marcello; Sala, Claudia; Provvedi, Roberta; Palù, Giorgio; Cole, Stewart T; Manganelli, Riccardo

    2018-01-01

    A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  2. Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation

    Directory of Open Access Journals (Sweden)

    Jianqiang Wu

    2016-03-01

    Full Text Available To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1 neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs and Schwann cells (SCs prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3fl/fl;Nf1fl/fl;DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

  3. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.

    Science.gov (United States)

    Horton, John R; Nugent, Rebecca L; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-03-07

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

  4. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

    Science.gov (United States)

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C; Johnson, Jennifer L; Entzminger, Kevin; Jain, Avni; Heaner, David P; Morales, Ivan A; Truskett, Thomas M; Maynard, Jennifer A; Lieberman, Raquel L

    2014-09-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. © 2014 Wiley Periodicals, Inc.

  5. Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria.

    Science.gov (United States)

    Ilmjärv, Tanel; Naanuri, Eve; Kivisaar, Maia

    2017-01-01

    Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source.

  6. Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria.

    Directory of Open Access Journals (Sweden)

    Tanel Ilmjärv

    Full Text Available Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2 and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source.

  7. ADA1 and NET1 Genes of Yeast Mediate Both Chromosome Maintenance and Mitochondrial $\\rho^{-}$ Mutagenesis

    CERN Document Server

    Koltovaya, N A; Tchekhouta, I A; Devin, A B

    2002-01-01

    An increase in the mitochondrial (mt) rho^- mutagenesis is a well-known respose of yeast cells to mutations in the numerous nuclear genes as well as to various kinds of stress. Notwithstanding the extensive studies during several decades the biological significance of this response is not yet fully understood. The genetic approach to solution of this subject includes the study of genes that are required for the high incidence of spontaneous rho^- mutants. Previously we found that mutations in certain nuclear genes including CDC28, the central cell-cycle regulation gene, may decrease the spontaneous rho^- mutability and simultaneously affect maintenance of the yeast chromosomes and plasmids. The present work provides data on identification of two more genes, resembling CDC28 in this respect. These genes NET1 and ADA1 mediate important regulatory protein-protein interactions in the yeast cell. The effects of net1 and ada1 mutations on the maintenance of yeast mt genome, chromosomes and plasmids as well as on ce...

  8. FHIT loss-induced DNA damage creates optimal APOBEC substrates: Insights into APOBEC-mediated mutagenesis.

    Science.gov (United States)

    Waters, Catherine E; Saldivar, Joshua C; Amin, Zaynab A; Schrock, Morgan S; Huebner, Kay

    2015-02-20

    APOBEC cytidine deaminase activity is a major source of hypermutation in cancer. But previous studies have shown that the TC context signature of these enzymes is not observed in sizable fractions of cancers with overexpression of APOBEC, suggesting that cooperating factors that contribute to this mutagenesis should be identified. The fragile histidine triad protein (Fhit) is a tumor suppressor and DNA caretaker that is deleted or silenced in >50% of cancers. Loss of Fhit protein activity causes replication stress through reduced Thymidine Kinase 1 expression, increased DNA breaks, and global genome instability in normal and cancer cells. Using data from The Cancer Genome Atlas (TCGA), we show that FHIT-low/APOBEC3B-high expressing lung adenocarcinomas display significantly increased numbers of APOBEC signature mutations. Tumor samples in this cohort with normal FHIT expression do not exhibit APOBEC hypermutation, despite having high APOBEC3B expression. In vitro, silencing Fhit expression elevates APOBEC3B-directed C > T mutations in the TP53 gene. Furthermore, inhibition of Fhit loss-induced DNA damage via thymidine supplementation decreases the TP53 mutation burden in FHIT-low/APOBEC3B-high cells. We conclude that APOBEC3B overexpression and Fhit-loss induced DNA damage are independent events that, when occurring together, result in a significantly increased frequency of APOBEC-induced mutations that drive cancer progression.

  9. Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

    Directory of Open Access Journals (Sweden)

    Régine Janel-Bintz

    2017-07-01

    Full Text Available It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM, exhibits several characteristics distinct from mutations that occur within the course of replication: i following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min than replication dependent mutations (t ½ ≈ 80-100 min ii NERiM specifically requires DNA Pol IV in addition to Pol V iii NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

  10. Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h.

    Science.gov (United States)

    Chiu, Joyce; March, Paul E; Lee, Ryan; Tillett, Daniel

    2004-12-07

    Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.

  11. Daily electronic monitoring of subjective and objective measures of illness activity in bipolar disorder using smartphones--the MONARCA II trial protocol: a randomized controlled single-blind parallel-group trial.

    Science.gov (United States)

    Faurholt-Jepsen, Maria; Vinberg, Maj; Frost, Mads; Christensen, Ellen Margrethe; Bardram, Jakob; Kessing, Lars Vedel

    2014-11-25

    Patients with bipolar disorder often show decreased adherence with mood stabilizers and frequently interventions on prodromal depressive and manic symptoms are delayed. Recently, the MONARCA I randomized controlled trial investigated the effect of electronic self-monitoring using smartphones on depressive and manic symptoms. The findings suggested that patients using the MONARCA system had more sustained depressive symptoms than patients using a smartphone for normal communicative purposes, but had fewer manic symptoms during the trial. It is likely that the ability of these self-monitored measures to detect prodromal symptoms of depression and mania may be insufficient compared to automatically generated objective data on measures of illness activity such as phone usage, social activity, physical activity, and mobility. The Monsenso system, for smartphones integrating subjective and objective measures of illness activity was developed and will be tested in the present trial. The MONARCA II trial uses a randomized controlled single-blind parallel-group design. Patients with bipolar disorder according to ICD-10 who previously have been treated at the Copenhagen Clinic for Affective Disorder, Denmark are included and randomized to either daily use of the Monsenso system including an feedback loop between patients and clinicians (the intervention group) or to the use of a smartphone for normal communicative purposes (the control group) for a 9-month trial period. The trial was started in September 2014 and recruitment is ongoing. The outcomes are: differences in depressive and manic symptoms; rate of depressive and manic episodes (primary); automatically generated objective data on measures of illness activity; number of days hospitalized; psychosocial functioning (secondary); perceived stress; quality of life; self-rated depressive symptoms; self-rated manic symptoms; recovery; empowerment and adherence to medication (tertiary) between the intervention group and the

  12. Signature-Tagged Mutagenesis of Pasteurella multocida Identifies Mutants Displaying Differential Virulence Characteristics in Mice and Chickens

    OpenAIRE

    Harper, Marina; Boyce, John D.; Wilkie, Ian W.; Adler, Ben

    2003-01-01

    Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants ident...

  13. The role of Dbf4-dependent protein kinase in DNA polymerase ζ-dependent mutagenesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brandão, Luis N; Ferguson, Rebecca; Santoro, Irma; Jinks-Robertson, Sue; Sclafani, Robert A

    2014-08-01

    The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK's essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate "complex" spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication "gap filling" rather than during "polymerase switching" by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32. Copyright © 2014 by the Genetics Society of America.

  14. Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

    Science.gov (United States)

    Peterson, A R; Peterson, H

    1982-01-01

    Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms. PMID:6951203

  15. Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity.

    Science.gov (United States)

    Mao, Peng; Brown, Alexander J; Malc, Ewa P; Mieczkowski, Piotr A; Smerdon, Michael J; Roberts, Steven A; Wyrick, John J

    2017-10-01

    DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers. © 2017 Mao et al.; Published by Cold Spring Harbor Laboratory Press.

  16. A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2‐deoxyglucose

    National Research Council Canada - National Science Library

    Dillon, A.J.P; Bettio, M; Pozzan, F.G; Andrighetti, T; Camassola, M

    2011-01-01

    Aims:  The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods...

  17. The Electron

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, George

    1972-01-01

    Electrons are elementary particles of atoms that revolve around and outside the nucleus and have a negative charge. This booklet discusses how electrons relate to electricity, some applications of electrons, electrons as waves, electrons in atoms and solids, the electron microscope, among other things.

  18. Electron radiography

    Science.gov (United States)

    Merrill, Frank E.; Morris, Christopher

    2005-05-17

    A system capable of performing radiography using a beam of electrons. Diffuser means receive a beam of electrons and diffuse the electrons before they enter first matching quadrupoles where the diffused electrons are focused prior to the diffused electrons entering an object. First imaging quadrupoles receive the focused diffused electrons after the focused diffused electrons have been scattered by the object for focusing the scattered electrons. Collimator means receive the scattered electrons and remove scattered electrons that have scattered to large angles. Second imaging quadrupoles receive the collimated scattered electrons and refocus the collimated scattered electrons and map the focused collimated scattered electrons to transverse locations on an image plane representative of the electrons' positions in the object.

  19. Principle and application of plant mutagenesis in crop improvement: a review

    Directory of Open Access Journals (Sweden)

    Yusuff Oladosu

    2016-01-01

    Full Text Available The first step in plant breeding is to identify suitable genotypes containing the desired genes among existing varieties, or to create one if it is not found in nature. In nature, variation occurs mainly as a result of mutations and without it, plant breeding would be impossible. In this context, the major aim in mutation-based breeding is to develop and improve well-adapted plant varieties by modifying one or two major traits to increase their productivity or quality. Both physical and chemical mutagenesis is used in inducing mutations in seeds and other planting materials. Then, selection for agronomic traits is done in the first generation, whereby most mutant lines may be discarded. The agronomic traits are confirmed in the second and third generations through evident phenotypic stability, while other evaluations are carried out in the subsequent generations. Finally, only the mutant lines with desirable traits are selected as a new variety or as a parent line for cross breeding. New varieties derived by induced mutatgenesis are used worldwide: rice in Vietnam, Thailand, China and the United States; durum wheat in Italy and Bulgaria; barley in Peru and European nations; soybean in Vietnam and China; wheat in China; as well as leguminous food crops in Pakistan and India. This paper integrates available data about the impact of mutation breeding-derived crop varieties around the world and highlights the potential of mutation breeding as a flexible and practicable approach applicable to any crop provided that appropriate objectives and selection methods are used.

  20. A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries.

    Science.gov (United States)

    Tang, Lixia; Li, Yang; Wang, Xiong

    2010-06-01

    Here we have reported a high throughput pH indicator-based assay to measure the activity of halohydrin dehalogenases (HheC). The assay relies upon the absorbance change at 560nm and the visual color change of phenol red in a weakly buffered system, due to the release of protons from the enzyme-catalyzed ring-closure reactions. The assay can be performed in a microplate format using whole cells, making the assay simple and robust. Thus, it is suitable for library screening. The assay has been further validated using two previously studied HheC variants, D80N and W249F, which exhibit 200-fold lower and 2-fold higher k(cat) values, respectively, toward 1,3-dichloro-2-propanol than the wild-type HheC. In addition, a saturation mutagenesis library of HheC was screened using the developed assay for its ability to efficiently catalyze the conversion of 1,3-dichloro-2-propanol. After screening of 500 colonies, one mutant W139C was identified and was further purified and characterized. Kinetic analysis indicates that the resulting mutant shows 2- and 5-fold improvement in k(cat) value toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, respectively, although it exhibits higher K(m) values than the wild-type enzyme. The method described herein represents a useful tool given the need for the high throughput screening of halohydrin dehalogenase mutants. 2010 Elsevier B.V. All rights reserved.

  1. Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.

    Science.gov (United States)

    Yoon, Taejin; Macmillan, Henriette; Mortimer, Sarah E; Jiang, Wei; Rinderknecht, Cornelia H; Stern, Lawrence J; Mellins, Elizabeth D

    2012-07-10

    HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ∼6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.

  2. Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ε-toxin.

    Directory of Open Access Journals (Sweden)

    Susan E Ivie

    Full Text Available The Clostridium perfringens ε-toxin is an extremely potent toxin associated with lethal toxemias in domesticated ruminants and may be toxic to humans. Intoxication results in fluid accumulation in various tissues, most notably in the brain and kidneys. Previous studies suggest that the toxin is a pore-forming toxin, leading to dysregulated ion homeostasis and ultimately cell death. However, mammalian host factors that likely contribute to ε-toxin-induced cytotoxicity are poorly understood. A library of insertional mutant Madin Darby canine kidney (MDCK cells, which are highly susceptible to the lethal affects of ε-toxin, was used to select clones of cells resistant to ε-toxin-induced cytotoxicity. The genes mutated in 9 surviving resistant cell clones were identified. We focused additional experiments on one of the identified genes as a means of validating the experimental approach. Gene expression microarray analysis revealed that one of the identified genes, hepatitis A virus cellular receptor 1 (HAVCR1, KIM-1, TIM1, is more abundantly expressed in human kidney cell lines than it is expressed in human cells known to be resistant to ε-toxin. One human kidney cell line, ACHN, was found to be sensitive to the toxin and expresses a larger isoform of the HAVCR1 protein than the HAVCR1 protein expressed by other, toxin-resistant human kidney cell lines. RNA interference studies in MDCK and in ACHN cells confirmed that HAVCR1 contributes to ε-toxin-induced cytotoxicity. Additionally, ε-toxin was shown to bind to HAVCR1 in vitro. The results of this study indicate that HAVCR1 and the other genes identified through the use of gene-trap mutagenesis and RNA interference strategies represent important targets for investigation of the process by which ε-toxin induces cell death and new targets for potential therapeutic intervention.

  3. ENU mutagenesis reveals a novel phenotype of reduced limb strength in mice lacking fibrillin 2.

    Directory of Open Access Journals (Sweden)

    Gaynor Miller

    2010-02-01

    Full Text Available Fibrillins 1 (FBN1 and 2 (FBN2 are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.As part of a large-scale N-ethyl-N-nitrosourea (ENU mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2(fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.

  4. Genome-wide mutagenesis reveals that ORF7 is a novel VZV skin-tropic factor.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2010-07-01

    Full Text Available The Varicella Zoster Virus (VZV is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes. We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7 has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles.

  5. Pre-breeding of lentil (Lens culinaris Medik.) for herbicide resistance through seed mutagenesis.

    Science.gov (United States)

    Rizwan, Muhammad; Aslam, Muhammad; Asghar, Muhammad Jawad; Abbas, Ghulam; Shah, Tariq Mahmud; Shimelis, Hussein

    2017-01-01

    Lentil is a poor competitor of weeds and its sensitivity to herbicides is a major hurdle for large scale production. The present study was conducted to select herbicide resistant lentil genotypes through seed mutagenesis. Seeds of three advanced lentil genotypes (LPP 11001, LPP 11100 and LPP 11116) were treated with two different concentrations of ethyl methanesulfonate (EMS; 0.1 and 0.2%), hydrazine hydrate (HH; 0.02 and 0.03%) and sodium azide (SA; 0.01 and 0.02%) to develop M1 seed. The M2 was screened against two herbicides including Ally Max 28.6% SG (X = 34.58 g/ha and 1.5X = 51.87 g/ha) and Atlantis 3.6% WG (X = 395.2 g/ha and 1.5X = 592.8 g/ha) using the following three screening methods: post plant emergence (PPE), pre-plant incorporation (PPI) and seed priming (SP). Data were recorded on survival index and survival percentage from each experimental unit of every population. Plants in all populations were categorized following their reaction to herbicides. The newly developed populations showed greater variation for herbicide resistance when compared to their progenitors. Phenotypic traits were significantly reduced in all the screening environments. Overall, 671 herbicide resistant mutants were selected from all testing environments. The seeds from selected plants were re-mutagenized at 150 Gy of gamma radiation and evaluated against higher dose of herbicides. This allowed selection of 134 herbicide resistant mutants. The selected mutants are useful germplasm for herbicide resistance breeding of lentil.

  6. WR-2721 protects against cytoxan-induced hprt mutagenesis without affecting therapeutic effectiveness

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Yasushi [Univ. of Chicago, Chicago, IL (United States). Dept. of Radiation and Cellular Oncology; Perrin, J. [Argonne National Lab., IL (United States); Hunter, N.; Milas, L. [Univ. of Texas, Houston, TX (United States); Grdina, D. [Univ. of Chicago, Chicadgo, IL (United States). Dept. of Radiation and Cellular Oncology]|[Argonne National Lab., IL (United States)]|[Argonne National Lab., IL (United States)

    1995-12-31

    The radioprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) was evaluated for its ability to protect against cytoxan-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in mouse splenocytes under conditions that would not interfere with the therapeutic effectiveness of cytoxan in the treatment of fibrosarcoma lung tumors. Mutations at the hprt locus increase in frequency as a function of the dose of cytoxan used. With a spontaneous mutation frequency in C3H mice of 1.5 {times} 10{sup {minus}6}, mutation frequencies increased from 6.2 {times} 10{sup {minus}6} to 2.0 {times} 10{sup {minus}5} as the dose of cytoxan increased from 50 to 200 mg/kg. C3H male mice were injected in their tail veins with 3.5 {times} 10{sup 5} viable fibrosarcoma (FSa) cells. This protocol gave rise to an average of 68 tumor colonies per mouse. Four days following injection animals were treated with cytoxan at a dose of 100 mg/kg, which gave rise to significant tumor cell killing and a reduction in tumor colony number to less than an average of one per animal. WR-2721 at a concentration of 100 mg/kg did not affect on cytoxan`s therapeutic effectiveness. However, a 100 mg/kg dose of WR-2721 was effective in reducing the cytoxan induced hprt mutation frequency in mice from 160 to 35 per 10{sup 5} viable cells regardless of whether it was administered 30 min before or 2 h following cytoxan treatment.

  7. Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

    Science.gov (United States)

    Kodama, Takahiro; Newberg, Justin Y.; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C.; Parsons, Pamela H.; Wu, Hao; Finegold, Milton J.; Copeland, Neal G.; Jenkins, Nancy A.

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

  8. Intensive mutagenesis of the nisin hinge leads to the rational design of enhanced derivatives.

    Science.gov (United States)

    Healy, Brian; Field, Des; O'Connor, Paula M; Hill, Colin; Cotter, Paul D; Ross, R Paul

    2013-01-01

    Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid 'hinge' region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.

  9. Intensive mutagenesis of the nisin hinge leads to the rational design of enhanced derivatives.

    Directory of Open Access Journals (Sweden)

    Brian Healy

    Full Text Available Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid 'hinge' region (N 20, M 21 and K 22 which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.

  10. Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis.

    Science.gov (United States)

    Rasmussen, Tim; Rasmussen, Akiko; Singh, Shivani; Galbiati, Heloisa; Edwards, Michelle D; Miller, Samantha; Booth, Ian R

    2015-07-28

    Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau ), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating.

  11. Effect of mutagenesis on the stereochemistry of enoyl-CoA hydratase.

    Science.gov (United States)

    Feng, Yuguo; Hofstein, Hilary A; Zwahlen, Jacque; Tonge, Peter J

    2002-10-22

    Enoyl-CoA hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)-HB-CoA, 3(S)-hydroxybutyryl-CoA with a stereospecificity (k(S)/k(R)) of 400000 to 1 [Wu, W. J., Feng, Y., He, X., Hofstein, H. S., Raleigh, D. P., and Tonge, P. J. (2000) J. Am. Chem. Soc. 122, 3987-3994]. Replacement of E164, one of the catalytic glutamates in the active site, with either aspartate or glutamine reduces the rate of formation of the 3(S) product enantiomer (k(S)) without affecting the rate of formation of the 3(R) product (k(R)). Consequently, k(S)/k(R) is 1000 and 0.33 for E164D and E164Q, respectively. In contrast, mutagenesis of E144, the second catalytic glutamate, reduces the rate of formation of both product enantiomers. Thus, only E144 is required for the formation of 3(R)-HB-CoA, 3(R)-hydroxybutyryl-CoA. Modeling studies together with analysis of alpha-proton exchange rates and experiments with crotonyl-oxyCoA, a substrate analogue in which the alpha-proton acidity has been reduced 10000-fold, support a mechanism of 3(R)-hydroxybutyryl-CoA formation that involves the E144-catalyzed stepwise addition of water to crotonyl-CoA which is bound in an s-trans conformation in the active site. Finally, we also demonstrate that hydrogen bonds in the oxyanion hole, provided by the backbone amide groups of G141 and A98, are important for the formation of both product enantiomers.

  12. Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis.

    Science.gov (United States)

    Mudahi-Orenstein, Sigalit; Levisohn, Sharon; Geary, Steven J; Yogev, David

    2003-07-01

    Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

  13. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola.

    Directory of Open Access Journals (Sweden)

    Gabriel Scalliet

    Full Text Available A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1 is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen

  14. Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus.

    Science.gov (United States)

    Hansen, Thomas; Schlichting, Bettina; Grötzinger, Joachim; Swan, Michael K; Davies, Christopher; Schönheit, Peter

    2005-12-01

    Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by site-directed mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and/or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni2+ saturating conditions. Further, the Ni2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.

  15. Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.

    Science.gov (United States)

    Zhu, Jinjie; Song, Ning; Sun, Silong; Yang, Weilong; Zhao, Haiming; Song, Weibin; Lai, Jinsheng

    2016-01-20

    CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  16. ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

    Science.gov (United States)

    Miller, Gaynor; Neilan, Monica; Chia, Ruth; Gheryani, Nabeia; Holt, Natalie; Charbit, Annabelle; Wells, Sara; Tucci, Valter; Lalanne, Zuzanne; Denny, Paul; Fisher, Elizabeth M. C.; Cheeseman, Michael; Askew, Graham N.; Dear, T. Neil

    2010-01-01

    Background Fibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes. Methodology/Principal Findings As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice. Conclusions These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further. PMID

  17. Phenotypic and biochemical profile changes in calendula (Calendula officinalis L.) plants treated with two chemical mutagenesis.

    Science.gov (United States)

    El-Nashar, Y I; Asrar, A A

    2016-05-06

    Chemical mutagenesis is an efficient tool used in mutation-breeding programs to improve the vital characters of the floricultural crops. This study aimed to estimate the effects of different concentrations of two chemical mutagens; sodium azide (SA) and diethyl sulfate (DES). The vegetative growth and flowering characteristics in two generations (M1 and M2) of calendula plants were investigated. Seeds were treated with five different concentrations of SA and DES (at the same rates) of 1000, 2000, 3000, 4000, and 5000 ppm, in addition to a control treatment of 0 ppm. Results showed that lower concentrations of SA mutagen had significant effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements in plants of both generations. Calendula plants tended to flower earlier under low mutagen concentrations (1000 ppm), whereas higher concentrations delayed flowering significantly. Positive results on seed germination, plant height, number of branches, plant fresh weight, and leaf area were observed in the M2-generation at lower concentrations of SA (1000 ppm), as well as at 4000 ppm DES on number of leaves and inflorescences. The highest total soluble protein was detected at the concentrations of 1000 ppm SA and 2000 ppm DES. DES showed higher average of acid phosphatase activity than SA. Results indicated that lower concentrations of SA and DES mutagens had positive effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements. Thus, lower mutagen concentrations could be recommended for better floral and physio-chemical performance.

  18. Charged particle mutagenesis at low dose and fluence in mouse splenic T cells

    Energy Technology Data Exchange (ETDEWEB)

    Grygoryev, Dmytro [Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239 (United States); Gauny, Stacey [Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Lasarev, Michael; Ohlrich, Anna [Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239 (United States); Kronenberg, Amy [Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Turker, Mitchell S., E-mail: turkerm@ohsu.edu [Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239 (United States); Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR 97239 (United States)

    2016-06-15

    Highlights: • Densely ionizing forms of space radiation induce mutations in splenic T cells at low fluence. • Large interstitial deletions and discontinuous LOH patterns are radiation signature mutations. • Space radiation mutagenesis suggests a cancer risk from deep space travel. - Abstract: High-energy heavy charged particles (HZE ions) found in the deep space environment can significantly affect human health by inducing mutations and related cancers. To better understand the relation between HZE ion exposure and somatic mutation, we examined cell survival fraction, Aprt mutant frequencies, and the types of mutations detected for mouse splenic T cells exposed in vivo to graded doses of densely ionizing {sup 48}Ti ions (1 GeV/amu, LET = 107 keV/μm), {sup 56}Fe ions (1 GeV/amu, LET = 151 keV/μm) ions, or sparsely ionizing protons (1 GeV, LET = 0.24 keV/μm). The lowest doses for {sup 48}Ti and {sup 56}Fe ions were equivalent to a fluence of approximately 1 or 2 particle traversals per nucleus. In most cases, Aprt mutant frequencies in the irradiated mice were not significantly increased relative to the controls for any of the particles or doses tested at the pre-determined harvest time (3–5 months after irradiation). Despite the lack of increased Aprt mutant frequencies in the irradiated splenocytes, a molecular analysis centered on chromosome 8 revealed the induction of radiation signature mutations (large interstitial deletions and complex mutational patterns), with the highest levels of induction at 2 particles nucleus for the {sup 48}Ti and {sup 56}Fe ions. In total, the results show that densely ionizing HZE ions can induce characteristic mutations in splenic T cells at low fluence, and that at least a subset of radiation-induced mutant cells are stably retained despite the apparent lack of increased mutant frequencies at the time of harvest.

  19. Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions*

    Science.gov (United States)

    Wojciechowski, Daniel; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    CLC-K chloride channels are expressed in the kidney and in the inner ear and require the accessory subunit barttin for proper function and membrane insertion. Barttin exerts multiple functions on CLC-proteins: it modifies protein stability and intracellular trafficking as well as channel activity, ion conduction, and gating. So far, the molecular determinants of these distinct barttin functions have remained elusive. Here we performed serial perturbation mutagenesis to identify the sequence determinants of barttin function. Barttin consists of two transmembrane helices followed by a long intracellular carboxyl terminus, and earlier work demonstrated that the transmembrane core of barttin suffices for most effects on the α-subunit. We individually substituted every amino acid of the predicted transmembrane core (amino acids 9–26 and 35–55) with tryptophan, co-expressed mutant barttin with hClC-Ka or V166E rClC-K1, and characterized CLC-K/barttin channels by patch clamp techniques, biochemistry, and confocal microscopy. The majority of mutations left the chaperone function of barttin, i.e. the effects on endoplasmic reticulum exit and surface membrane insertion, unaffected. In contrast, tryptophan insertion at multiple positions resulted in impaired activity of hClC-Ka/barttin and changes in gating of V166E rClC-K1/barttin. These results demonstrate that mutations in a cluster of hydrophobic residues within transmembrane domain 1 affect barttin-CLC-K interaction and impair gating modification by the accessory subunit. Whereas tight interaction is necessary for functional modification, even impaired association of barttin and CLC-K suffices for normal intracellular trafficking. Our findings allow definition of a likely interaction surface and clarify the mechanisms underlying CLC-K channel modification by barttin. PMID:26063802

  20. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

    Directory of Open Access Journals (Sweden)

    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  1. Induction of apomixis and fixation of heterosis in Egyptian rice Hybrid1 line using colchicine mutagenesis

    Directory of Open Access Journals (Sweden)

    Reda M. Gaafar

    2017-06-01

    Full Text Available It is known that hybrid rice yields 15–20% over inbred varieties in first generation because of heterosis. However, heterosis is normally broken due to segregation. Applying apomixis produces plants as a clone of mother plant and overcomes the problem of breaking heterosis. In order to fix heterosis in the Egyptian rice Hybrid1, their seeds were mutagenized in 0.2% colchicine for two time periods 24 and 50 h. After colchicine mutagenesis, rice seedlings were grown in the field till maturation and the resulted M1 seeds were sown in season 2 and plants were selected based on yield and homogeneity. Then, seeds were sown to be evaluated in season 3. Pollen fertility test, esterase isozyme analysis, and flow cytometry seed screening were performed to confirm the results of field selection of populations identical to control. Pollen fertility examination was performed on the populations of the third season. Pollens of populations 304, 298, 292, 284, 281, 154 and 149 were found to be completely sterile. However, these plants had high seed set percentage. The flow cytometry screening of the six yield-based identical populations and the control seeds showed that populations 220, 339, 351 and 298 have higher nuclear DNA content (C2 than untreated hybrid (C2 & C3. Results of flow cytometry clearly showed that population 298 has one peak (C2 and its endosperm was formed autonomously without fertilization. Although its pollen grains were sterile, it showed high seed set percentage. This indicates that heterosis was completely fixed by apomixis in this population.

  2. The HIV mutation browser: a resource for human immunodeficiency virus mutagenesis and polymorphism data.

    Directory of Open Access Journals (Sweden)

    Norman E Davey

    2014-12-01

    Full Text Available Huge research effort has been invested over many years to determine the phenotypes of natural or artificial mutations in HIV proteins--interpretation of mutation phenotypes is an invaluable source of new knowledge. The results of this research effort are recorded in the scientific literature, but it is difficult for virologists to rapidly find it. Manually locating data on phenotypic variation within the approximately 270,000 available HIV-related research articles, or the further 1,500 articles that are published each month is a daunting task. Accordingly, the HIV research community would benefit from a resource cataloguing the available HIV mutation literature. We have applied computational text-mining techniques to parse and map mutagenesis and polymorphism information from the HIV literature, have enriched the data with ancillary information and have developed a public, web-based interface through which it can be intuitively explored: the HIV mutation browser. The current release of the HIV mutation browser describes the phenotypes of 7,608 unique mutations at 2,520 sites in the HIV proteome, resulting from the analysis of 120,899 papers. The mutation information for each protein is organised in a residue-centric manner and each residue is linked to the relevant experimental literature. The importance of HIV as a global health burden advocates extensive effort to maximise the efficiency of HIV research. The HIV mutation browser provides a valuable new resource for the research community. The HIV mutation browser is available at: http://hivmut.org.

  3. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

    Science.gov (United States)

    Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

    2016-11-01

    Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T1/2, respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Rationale and design of a randomized trial of home electronic symptom and lung function monitoring to detect cystic fibrosis pulmonary exacerbations: the early intervention in cystic fibrosis exacerbation (eICE) trial.

    Science.gov (United States)

    Lechtzin, N; West, N; Allgood, S; Wilhelm, E; Khan, U; Mayer-Hamblett, N; Aitken, M L; Ramsey, B W; Boyle, M P; Mogayzel, P J; Goss, C H

    2013-11-01

    Acute pulmonary exacerbations are central events in the lives of individuals with cystic fibrosis (CF). Pulmonary exacerbations lead to impaired lung function, worse quality of life, and shorter survival. We hypothesized that aggressive early treatment of acute pulmonary exacerbation may improve clinical outcomes. Describe the rationale of an ongoing trial designed to determine the efficacy of home monitoring of both lung function measurements and symptoms for early detection and subsequent early treatment of acute CF pulmonary exacerbations. A randomized, non-blinded, multi-center trial in 320 individuals with CF aged 14 years and older. The study compares usual care to a twice a week assessment of home spirometry and CF respiratory symptoms using an electronic device with data transmission to the research personnel to identify and trigger early treatment of CF pulmonary exacerbation. Participants will be enrolled in the study for 12 months. The primary endpoint is change in FEV1 (L) from baseline to 12 months determined by a linear mixed effects model incorporating all quarterly FEV1 measurements. Secondary endpoints include time to first acute protocol-defined pulmonary exacerbation, number of acute pulmonary exacerbations, number of hospitalization days for acute pulmonary exacerbation, time from the end of acute pulmonary exacerbation to onset of subsequent pulmonary exacerbation, change in health related quality of life, change in treatment burden, change in CF respiratory symptoms, and adherence to the study protocol. This study is a first step in establishing alternative approaches to the care of CF pulmonary exacerbations. We hypothesize that early treatment of pulmonary exacerbations has the potential to slow lung function decline, reduce respiratory symptoms and improve the quality of life for individuals with CF. © 2013.

  5. Implementing an evidence-based computerized decision support system linked to electronic health records to improve care for cancer patients: the ONCO-CODES study protocol for a randomized controlled trial.

    Science.gov (United States)

    Moja, Lorenzo; Passardi, Alessandro; Capobussi, Matteo; Banzi, Rita; Ruggiero, Francesca; Kwag, Koren; Liberati, Elisa Giulia; Mangia, Massimo; Kunnamo, Ilkka; Cinquini, Michela; Vespignani, Roberto; Colamartini, Americo; Di Iorio, Valentina; Massa, Ilaria; González-Lorenzo, Marien; Bertizzolo, Lorenzo; Nyberg, Peter; Grimshaw, Jeremy; Bonovas, Stefanos; Nanni, Oriana

    2016-11-25

    Computerized decision support systems (CDSSs) are computer programs that provide doctors with person-specific, actionable recommendations, or management options that are intelligently filtered or presented at appropriate times to enhance health care. CDSSs might be integrated with patient electronic health records (EHRs) and evidence-based knowledge. The Computerized DEcision Support in ONCOlogy (ONCO-CODES) trial is a pragmatic, parallel group, randomized controlled study with 1:1 allocation ratio. The trial is designed to evaluate the effectiveness on clinical practice and quality of care of a multi-specialty collection of patient-specific reminders generated by a CDSS in the IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) hospital. We hypothesize that the intervention can increase clinician adherence to guidelines and, eventually, improve the quality of care offered to cancer patients. The primary outcome is the rate at which the issues reported by the reminders are resolved, aggregating specialty and primary care reminders. We will include all the patients admitted to hospital services. All analyses will follow the intention-to-treat principle. The results of our study will contribute to the current understanding of the effectiveness of CDSSs in cancer hospitals, thereby informing healthcare policy about the potential role of CDSS use. Furthermore, the study will inform whether CDSS may facilitate the integration of primary care in cancer settings, known to be usually limited. The increasing use of and familiarity with advanced technology among new generations of physicians may support integrated approaches to be tested in pragmatic studies determining the optimal interface between primary and oncology care. ClinicalTrials.gov, NCT02645357.

  6. Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

    Science.gov (United States)

    Rusconi, B; Maranhao, A C; Fuhrer, J P; Krotee, P; Choi, S H; Grun, F; Thireou, T; Dimitratos, S D; Woods, D F; Marinotti, O; Walter, M F; Eliopoulos, E

    2012-08-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

    Science.gov (United States)

    Rusconi, B; Maranhao, AC; Fuhrer, JP; Krotee, P; Choi, SH; Grun, F; Thireou, T; Dimitratos, SD; Woods, DF; Marinotti, O; Walter, MF; Eliopoulos, E

    2012-01-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to 22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. PMID:22564768

  8. Insertional mutagenesis enables cleistothecial formation in a non-mating strain of Histoplasma capsulatum

    Directory of Open Access Journals (Sweden)

    Laskowski Meggan C

    2010-02-01

    Full Text Available Abstract Background Histoplasma capsulatum is a pathogenic ascomycete fungus that rapidly loses mating ability in culture. Loss of mating ability, as well as the organism's low rate of targeted gene replacement, limits techniques available for genetic studies in H. capsulatum. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in H. capsulatum strains, introducing a variety of classical genetics techniques to the field. We generated a strain, UC1, by insertional mutagenesis of the laboratory strain G217B, and found that UC1 acquired the ability to form mating structures called cleistothecia. The aim of this study was to determine the mechanism by which UC1 gained the ability to form cleistothecia. We also present initial studies demonstrating that UC1 can be used as a tool to determine molecular correlates of mating in H. capsulatum. Results The strain UC1 was found to have increased RNA levels of the mating locus transcription factor (MAT1-1-1, and the putative alpha pheromone (PPG1 compared to G217B. Agrobacterium-mediated transformation and integration of T-DNA from the vector pCB301-GFP-HYG were found to be partially responsible for the increased RNA levels of these genes; however, the site of integration appeared to play the largest role in the strain's ability to form cleistothecia. Silencing HMK1, a putative FUS3/KSS1 homolog, had no effect on cleistothecial production by UC1. Protein kinase C (PKC1 RNA and protein levels were increased in UC1 compared to G217B, and pheromone production was found to be linked with Pkc1 activity in H. capsulatum. Conclusions The site of the T-DNA integration event appears to play the largest role in UC1's ability to form cleistothecia. We show that the UC1 strain can be used as a tool to study cleistothecia production in H. capsulatum by manipulating the strain, or by identifying differences between UC1 and G217B. Using these approaches

  9. Implementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Norberto Villegas-Negrete

    Full Text Available A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS and confer a trimethoprim resistant (Tmpr phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM. In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10-9. Genetic disruption of the canonical mismatch (MMR and guanine oxidized (GO repair pathways increased the Tmpr frequency of mutation by ~2-3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10-7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.

  10. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

    Science.gov (United States)

    Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

    2017-02-01

    CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Quest of novel GH20 N-acetyl hexosaminidasetransglycosylating catalysts: functional screening, data mining and semi-rational mutagenesis

    DEFF Research Database (Denmark)

    Teze, David; Visnapuu, Triinu; Kjeldsen, Christian

    and the fact that the products are also substrates, thus needing a kinetic control of the reaction. Several approaches have been developed to overcome these, including mechanism modifications (e.g. glycosynthases, chemical rescue), functional screening and data mining to find natural transglycosidases...... been reported. Thus, we turned to discovery and characterization of new GH20s and performing a systematic mutagenesis study. Several new GH20s of bacterial origin were isolated and described by functional screening and data mining, including transglycosidases able to synthesize lacto...

  12. Chemical mutagenesis of Gluconobacter frateurii to construct methanol-resistant mutants showing glyceric acid production from methanol-containing glycerol.

    Science.gov (United States)

    Sato, Shun; Kitamoto, Dai; Habe, Hiroshi

    2014-02-01

    To produce glyceric acid (GA) from methanol-containing glycerol, resistance to methanol of Gluconobacter frateurii NBRC103465 was improved by chemical mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The obtained mutant Gf398 produced 6.3 g/L GA in 5% (v/v) methanol-containing 17% (w/v) glycerol medium, in which the wild-type strain neither grew nor produced GA. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

    OpenAIRE

    Shiau, Rong-Jen; Hung, Hui-Chen; Jeang, Chii-Ling

    2003-01-01

    A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca2+-independent mechanism.

  14. Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

    Science.gov (United States)

    Shiau, Rong-Jen; Hung, Hui-Chen; Jeang, Chii-Ling

    2003-01-01

    A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca2+-independent mechanism. PMID:12676725

  15. Random thoughts

    Science.gov (United States)

    ajansen; kwhitefoot; panteltje1; edprochak; sudhakar, the

    2014-07-01

    In reply to the physicsworld.com news story “How to make a quantum random-number generator from a mobile phone” (16 May, http://ow.ly/xFiYc, see also p5), which describes a way of delivering random numbers by counting the number of photons that impinge on each of the individual pixels in the camera of a Nokia N9 smartphone.

  16. Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

    Directory of Open Access Journals (Sweden)

    Silverman Mel

    2005-09-01

    Full Text Available Abstract Background Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. Results A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. Conclusion We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

  17. A novel electronic algorithm using host biomarker point-of-care tests for the management of febrile illnesses in Tanzanian children (e-POCT): A randomized, controlled non-inferiority trial.

    Science.gov (United States)

    Keitel, Kristina; Kagoro, Frank; Samaka, Josephine; Masimba, John; Said, Zamzam; Temba, Hosiana; Mlaganile, Tarsis; Sangu, Willy; Rambaud-Althaus, Clotilde; Gervaix, Alain; Genton, Blaise; D'Acremont, Valérie

    2017-10-01

    The management of childhood infections remains inadequate in resource-limited countries, resulting in high mortality and irrational use of antimicrobials. Current disease management tools, such as the Integrated Management of Childhood Illness (IMCI) algorithm, rely solely on clinical signs and have not made use of available point-of-care tests (POCTs) that can help to identify children with severe infections and children in need of antibiotic treatment. e-POCT is a novel electronic algorithm based on current evidence; it guides clinicians through the entire consultation and recommends treatment based on a few clinical signs and POCT results, some performed in all patients (malaria rapid diagnostic test, hemoglobin, oximeter) and others in selected subgroups only (C-reactive protein, procalcitonin, glucometer). The objective of this trial was to determine whether the clinical outcome of febrile children managed by the e-POCT tool was non-inferior to that of febrile children managed by a validated electronic algorithm derived from IMCI (ALMANACH), while reducing the proportion with antibiotic prescription. We performed a randomized (at patient level, blocks of 4), controlled non-inferiority study among children aged 2-59 months presenting with acute febrile illness to 9 outpatient clinics in Dar es Salaam, Tanzania. In parallel, routine care was documented in 2 health centers. The primary outcome was the proportion of clinical failures (development of severe symptoms, clinical pneumonia on/after day 3, or persistent symptoms at day 7) by day 7 of follow-up. Non-inferiority would be declared if the proportion of clinical failures with e-POCT was no worse than the proportion of clinical failures with ALMANACH, within statistical variability, by a margin of 3%. The secondary outcomes included the proportion with antibiotics prescribed on day 0, primary referrals, and severe adverse events by day 30 (secondary hospitalizations and deaths). We enrolled 3,192 patients

  18. A novel electronic algorithm using host biomarker point-of-care tests for the management of febrile illnesses in Tanzanian children (e-POCT: A randomized, controlled non-inferiority trial.

    Directory of Open Access Journals (Sweden)

    Kristina Keitel

    2017-10-01

    Full Text Available The management of childhood infections remains inadequate in resource-limited countries, resulting in high mortality and irrational use of antimicrobials. Current disease management tools, such as the Integrated Management of Childhood Illness (IMCI algorithm, rely solely on clinical signs and have not made use of available point-of-care tests (POCTs that can help to identify children with severe infections and children in need of antibiotic treatment. e-POCT is a novel electronic algorithm based on current evidence; it guides clinicians through the entire consultation and recommends treatment based on a few clinical signs and POCT results, some performed in all patients (malaria rapid diagnostic test, hemoglobin, oximeter and others in selected subgroups only (C-reactive protein, procalcitonin, glucometer. The objective of this trial was to determine whether the clinical outcome of febrile children managed by the e-POCT tool was non-inferior to that of febrile children managed by a validated electronic algorithm derived from IMCI (ALMANACH, while reducing the proportion with antibiotic prescription.We performed a randomized (at patient level, blocks of 4, controlled non-inferiority study among children aged 2-59 months presenting with acute febrile illness to 9 outpatient clinics in Dar es Salaam, Tanzania. In parallel, routine care was documented in 2 health centers. The primary outcome was the proportion of clinical failures (development of severe symptoms, clinical pneumonia on/after day 3, or persistent symptoms at day 7 by day 7 of follow-up. Non-inferiority would be declared if the proportion of clinical failures with e-POCT was no worse than the proportion of clinical failures with ALMANACH, within statistical variability, by a margin of 3%. The secondary outcomes included the proportion with antibiotics prescribed on day 0, primary referrals, and severe adverse events by day 30 (secondary hospitalizations and deaths. We enrolled 3

  19. Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation

    Directory of Open Access Journals (Sweden)

    Zhiqian Yi

    2015-09-01

    Full Text Available Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.

  20. System-dependent regulations of colour-pattern development: a mutagenesis study of the pale grass blue butterfly.

    Science.gov (United States)

    Iwata, Masaki; Hiyama, Atsuki; Otaki, Joji M

    2013-01-01

    Developmental studies on wing colour patterns have been performed in nymphalid butterflies, but efficient genetic manipulations, including mutagenesis, have not been well established. Here, we have performed mutagenesis experiments in a lycaenid butterfly, the pale grass blue Zizeeria maha, to produce colour-pattern mutants. We fed the P-generation larvae an artificial diet containing the mutagen ethyl methane sulfonate (EMS), and the F1- and F2-generation adults showed various aberrant colour patterns: dorsoventral transformation, anterioposterior background colouration gap, weak contrast, disarrangement of spots, reduction of the size of spots, loss of spots, fusion of spots, and ectopic spots. Among them, the disarrangement, reduction, and loss of spots were likely produced by the coordinated changes of many spots of a single wing around the discal spot in a system-dependent manner, demonstrating the existence of the central symmetry system. The present study revealed multiple genetic regulations for system-dependent and wing-wide colour-pattern determination in lycaenid butterflies.

  1. Effects of vitamins A and E on methylazoxymethanol-induced mutagenesis in Salmonella typhimurium strain TA100.

    Science.gov (United States)

    Tavan, E; Maziere, S; Narbonne, J F; Cassand, P

    1997-07-03

    The aim of this study is to report the antimutagenic effect of vitamin A and vitamin E towards methylazoxymethanol (MAM)-induced mutagenesis in Salmonella typhimurium strain TA100 sensitive to alkylating agents. In order to characterize different levels of action of these two fat-soluble vitamins towards the mutagenicity of MAM, several assays have been considered to show the antimutagenic effect and the possible interactions of vitamins with MAM or with the bacteria. Thus, for each vitamin, three different assays with three different incubations have been conducted: (i) MAM, bacteria and vitamins together, (ii) MAM and vitamins, (iii) bacteria and vitamins. The results showed that both vitamins A and E present an antimutagenic effect towards MAM induced mutagenesis. alpha-Tocopherol seems to have an action directly on to the mutagenic agent, whereas the action of retinol is likely due to a protection of the bacterial genoma against MAM. These in vitro results could help to interpret results of colon carcinogenesis studies using animals induced by 1,2-dimethylhydrazine and fed vitamins supplemented diet.

  2. Mutation breeding of extracellular polysaccharide-producing microalga Crypthecodinium cohnii by a novel mutagenesis with atmospheric and room temperature plasma.

    Science.gov (United States)

    Liu, Bin; Sun, Zheng; Ma, Xiaonian; Yang, Bo; Jiang, Yue; Wei, Dong; Chen, Feng

    2015-04-13

    Extracellular polysaccharides (EPS) produced by marine microalgae have the potential to be used as antioxidants, antiviral agents, immunomodulators, and anti-inflammatory agents. Although the marine microalga Crypthecodinium cohnii releases EPS during the process of docosahexaenoic acid (DHA) production, the yield of EPS remains relatively low. To improve the EPS production, a novel mutagenesis of C. cohnii was conducted by atmospheric and room temperature plasma (ARTP). Of the 12 mutants obtained, 10 mutants exhibited significantly enhanced EPS yield on biomass as compared with the wild type strain. Among them, mutant M7 was the best as it could produce an EPS volumetric yield of 1.02 g/L, EPS yield on biomass of 0.39 g/g and EPS yield on glucose of 94 mg/g, which were 33.85%, 85.35% and 57.17% higher than that of the wild type strain, respectively. Results of the present study indicated that mutagenesis of the marine microalga C. cohnii by ARTP was highly effective leading to the high-yield production of EPS.

  3. Mutation Breeding of Extracellular Polysaccharide-Producing Microalga Crypthecodinium cohnii by a Novel Mutagenesis with Atmospheric and Room Temperature Plasma

    Directory of Open Access Journals (Sweden)

    Bin Liu

    2015-04-01

    Full Text Available Extracellular polysaccharides (EPS produced by marine microalgae have the potential to be used as antioxidants, antiviral agents, immunomodulators, and anti-inflammatory agents. Although the marine microalga Crypthecodinium cohnii releases EPS during the process of docosahexaenoic acid (DHA production, the yield of EPS remains relatively low. To improve the EPS production, a novel mutagenesis of C. cohnii was conducted by atmospheric and room temperature plasma (ARTP. Of the 12 mutants obtained, 10 mutants exhibited significantly enhanced EPS yield on biomass as compared with the wild type strain. Among them, mutant M7 was the best as it could produce an EPS volumetric yield of 1.02 g/L, EPS yield on biomass of 0.39 g/g and EPS yield on glucose of 94 mg/g, which were 33.85%, 85.35% and 57.17% higher than that of the wild type strain, respectively. Results of the present study indicated that mutagenesis of the marine microalga C. cohnii by ARTP was highly effective leading to the high-yield production of EPS.

  4. High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease

    KAUST Repository

    Eid, Ayman

    2016-05-28

    Key message: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. Abstract: The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination. © 2016, Springer-Verlag Berlin Heidelberg.

  5. Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    Ashok A. Nikam

    2015-02-01

    Full Text Available Gamma ray-induced in vitro mutagenesis and selection for salt (NaCl tolerance were investigated in sugarcane (Saccharum officinarum L.. Embryogenic callus cultures were irradiated (10 to 80 Gy and subjected to in vitro selection by exposure of irradiated callus to NaCl (0, 50, 100, 150, 200, and 250 mmol L− 1. Increasing NaCl concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+ and K+ concentration. Higher accumulation of proline and glycine betaine was observed in NaCl stressed callus irradiated at 20 Gy. Na+ concentration increased and K+ concentration decreased with increasing salt level. Irradiated callus showed 50–60% regeneration under NaCl stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%, number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

  6. Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

    Science.gov (United States)

    Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

    2015-01-01

    The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

  7. Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis.

    Science.gov (United States)

    Pepinsky, R Blake; Silvian, Laura; Berkowitz, Steven A; Farrington, Graham; Lugovskoy, Alexey; Walus, Lee; Eldredge, John; Capili, Allan; Mi, Sha; Graff, Christilyn; Garber, Ellen

    2010-05-01

    Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.

  8. Universal randomness

    Energy Technology Data Exchange (ETDEWEB)

    Dotsenko, Viktor S [Landau Institute for Theoretical Physics, Russian Academy of Sciences, Moscow (Russian Federation)

    2011-03-31

    In the last two decades, it has been established that a single universal probability distribution function, known as the Tracy-Widom (TW) distribution, in many cases provides a macroscopic-level description of the statistical properties of microscopically different systems, including both purely mathematical ones, such as increasing subsequences in random permutations, and quite physical ones, such as directed polymers in random media or polynuclear crystal growth. In the first part of this review, we use a number of models to examine this phenomenon at a simple qualitative level and then consider the exact solution for one-dimensional directed polymers in a random environment, showing that free energy fluctuations in such a system are described by the universal TW distribution. The second part provides detailed appendix material containing the necessary mathematical background for the first part. (reviews of topical problems)

  9. Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway.

    Science.gov (United States)

    Cooksley, Clare M; Zhang, Ying; Wang, Hengzheng; Redl, Stephanie; Winzer, Klaus; Minton, Nigel P

    2012-11-01

    The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to

  10. Random triangles

    OpenAIRE

    Matula, Dominik

    2013-01-01

    The author summarizes some previous results concerning random triangles. He describes the Gaussian triangle and random triangles whose vertices lie in a unit n-dimensional ball, in a rectangle or in a general bounded convex set. In the second part, the author deals with an inscribed triangle in a triangle - let ABC be an equilateral triangle and let M, N, O be three points, each laying on one side of the ABC. We call MNO inscribed triangle (in an equi- laterral triangle). The median triangle ...

  11. Random matrices

    CERN Document Server

    Mehta, Madan Lal

    1990-01-01

    Since the publication of Random Matrices (Academic Press, 1967) so many new results have emerged both in theory and in applications, that this edition is almost completely revised to reflect the developments. For example, the theory of matrices with quaternion elements was developed to compute certain multiple integrals, and the inverse scattering theory was used to derive asymptotic results. The discovery of Selberg's 1944 paper on a multiple integral also gave rise to hundreds of recent publications. This book presents a coherent and detailed analytical treatment of random matrices, leading

  12. Structure-activity investigation on laccases by computational and site directed mutagenesis studies

    OpenAIRE

    Delavari, Azar

    2016-01-01

    Laccases belong to multi copper oxidase enzyme family (EC 1.10.3.2). Their capacity to oxidíze a wide range of substrates makes them very attractive for the industry and are growing in importance for environmentally-friendly synthesis. Laccases have three different copper sites including, type 1 (T1), type 2 (T2) and type 3 (T3). The function of the T1 site is shuttling electrons from the substrate to the trinuclear copper cluster. During the catalytic cycle of laccase, four electrons are re...

  13. Electron Microscopy.

    Science.gov (United States)

    Beer, Michael

    1980-01-01

    Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

  14. Electronic Commerce

    OpenAIRE

    Slavko Đerić

    2016-01-01

    Electronic commerce can be defined in different ways. Any definition helps to understand and explain that concept as better as possible.. Electronic commerce is a set of procedures and technologies that automate the tasks of financial transactions using electronic means. Also, according to some authors, electronic commerce is defined as a new concept, which is being developed and which includes process of buying and selling or exchanging products, services or information via computer networks...

  15. Understand electronics

    CERN Document Server

    Bishop, Owen

    2013-01-01

    Understand Electronics provides a readable introduction to the exciting world of electronics for the student or enthusiast with little previous knowledge. The subject is treated with the minimum of mathematics and the book is extensively illustrated.This is an essential guide for the newcomer to electronics, and replaces the author's best-selling Beginner's Guide to Electronics.The step-by-step approach makes this book ideal for introductory courses such as the Intermediate GNVQ.

  16. Vacuum electronics

    CERN Document Server

    Eichmeier, Joseph A

    2008-01-01

    Nineteen experts from the electronics industry, research institutes and universities have joined forces to prepare this book. ""Vacuum Electronics"" covers the electrophysical fundamentals, the present state of the art and applications, as well as the future prospects of microwave tubes and systems, optoelectronics vacuum devices, electron and ion beam devices, light and X-ray emitters, particle accelerators and vacuum interrupters. These topics are supplemented by useful information about the materials and technologies of vacuum electronics and vacuum technology.

  17. Electronic components

    CERN Document Server

    Colwell, Morris A

    1976-01-01

    Electronic Components provides a basic grounding in the practical aspects of using and selecting electronics components. The book describes the basic requirements needed to start practical work on electronic equipment, resistors and potentiometers, capacitance, and inductors and transformers. The text discusses semiconductor devices such as diodes, thyristors and triacs, transistors and heat sinks, logic and linear integrated circuits (I.C.s) and electromechanical devices. Common abbreviations applied to components are provided. Constructors and electronics engineers will find the book useful

  18. ELECTRONIC SIGNATURES

    African Journals Online (AJOL)

    10332324

    'electronic signature' means data attached to, incorporated in, or logically associated with other data and which is intended by the user to serve as a signature;. The suggested new definition for an electronic signature reads as follows: 'electronic signature' means a sound, symbol or process that is (i) uniquely linked to the ...

  19. ELECTRONIC SIGNATURES

    African Journals Online (AJOL)

    10332324

    (a) facilitate ecommerce;2. (b) remove and prevent barriers to electronic communications in South Africa;3. (c) ensure that electronic transactions in the Republic conform to the highest international standards;4. (d) promote the development of electronic transactions services which are responsive to the needs of users and ...

  20. Sticker electronics

    KAUST Repository

    Hussain, Muhammad Mustafa

    2017-09-08

    Electronic stickers may be manufactured on flexible substrates (110, 120, 130) as layers and packaged together. The package may then have an adhesive applied to one side to provide capability for sticking the electronic devices to surfaces. The stickers can be wrappable, placed on surfaces, glued on walls or mirrors or wood or stone, and have electronics (112, 122, 132) which may or may not be ultrathin. Packaging for the electronic sticker can use polymer on cellulose manufacturing and/or three dimensional (3-D) printing. The electronic stickers may provide lighting capability, sensing capability, and/or recharging capabilities.

  1. Basic electronics

    CERN Document Server

    Holbrook, Harold D

    1971-01-01

    Basic Electronics is an elementary text designed for basic instruction in electricity and electronics. It gives emphasis on electronic emission and the vacuum tube and shows transistor circuits in parallel with electron tube circuits. This book also demonstrates how the transistor merely replaces the tube, with proper change of circuit constants as required. Many problems are presented at the end of each chapter. This book is comprised of 17 chapters and opens with an overview of electron theory, followed by a discussion on resistance, inductance, and capacitance, along with their effects on t

  2. Electronic Government and Electronic Participation

    NARCIS (Netherlands)

    Tambouris, E.; Scholl, H.J.; Janssen, M.F.W.H.A.; Wimmer, M.A.; Tarabanis, K.; Gascó, M.; Klievink, A.J.; Lindgren, I.; Milano, M.; Panagiotopoulos, P.; Pardo, T.A.; Parycek, P.; Sæbø, O.

    2015-01-01

    Electronic government and electronic participation continue to transform the public sector and society worldwide and are constantly being transformed themselves by emerging information and communication technologies. This book presents papers from the 14th International Federation for Information

  3. Electronic Government and Electronic Participation

    NARCIS (Netherlands)

    Tambouris, E; Scholl, H.J.; Janssen, M.F.W.H.A.; Wimmer, M.A.; Tarabanis, K; Gascó, M; Klievink, A.J.; Lindgren, I; Milano, M; Panagiotopoulos, P; Pardo, T.A.; Parycek, P; Sæbø, Ø

    2016-01-01

    Electronic government and electronic participation continue to transform the public sector and society worldwide and are constantly being transformed themselves by emerging information and communication technologies.This book presents papers from the 14th International Federation for Information

  4. Electron Tree

    DEFF Research Database (Denmark)

    Appelt, Ane L; Rønde, Heidi S

    2013-01-01

    The photo shows a close-up of a Lichtenberg figure – popularly called an “electron tree” – produced in a cylinder of polymethyl methacrylate (PMMA). Electron trees are created by irradiating a suitable insulating material, in this case PMMA, with an intense high energy electron beam. Upon discharge......, during dielectric breakdown in the material, the electrons generate branching chains of fractures on leaving the PMMA, producing the tree pattern seen. To be able to create electron trees with a clinical linear accelerator, one needs to access the primary electron beam used for photon treatments. We...... appropriated a linac that was being decommissioned in our department and dismantled the head to circumvent the target and ion chambers. This is one of 24 electron trees produced before we had to stop the fun and allow the rest of the accelerator to be disassembled....

  5. Ultra-wide band electromagnetic radiation does not affect UV-induced recombination and mutagenesis in yeast.

    Science.gov (United States)

    Pakhomova, O N; Belt, M L; Mathur, S P; Lee, J C; Akyel, Y

    1998-01-01

    Cell samples of the yeast Saccharomyces cerevisiae were exposed to 100 J/m2 of 254 nm ultraviolet (UV) radiation followed by a 30 min treatment with ultra-wide band (UWB) electromagnetic pulses. The UWB pulses (101-104 kV/m, 1.0 ns width, 165 ps rise time) were applied at the repetition rates of 0 Hz (sham), 16 Hz, or 600 Hz. The effect of exposures was evaluated from the colony-forming ability of the cells on complete and selective media and the number of aberrant colonies. The experiments established no effect of UWB exposure on the UV-induced reciprocal and non-reciprocal recombination, mutagenesis, or cell survival.

  6. The antimutagenic effect of monoterpenes against UV-irradiation-, 4NQO- and t-BOOH-induced mutagenesis in coli

    Directory of Open Access Journals (Sweden)

    Nikolić Biljana

    2011-01-01

    Full Text Available The aim of this work was to investigate the antimutagenic potential of monoterpenes from sage and basil in Escherichia coli. The mutagenic potential of monoterpenes was pre-screened with Salmonella/microsome reversion assay in strain TA100 and no mutagenic effect was detected. The antimutagenic potential against UV- 4NQO- and t-BOOH induced mutagenesis was evaluated in E. coli K12 and E. coli WP2 by reversion assays. The obtained results indicate that camphor and thujone reduce UV- and 4NQO-induced mutations; myrcene reduces t-BOOH-induced mutations, while eucalyptol and linalool reduce mutagenicity by all tested mutagens. Considering evolutionary conservation of DNA repair and antioxidative protection, the obtained results indicate that further antigenotoxicity studies should be undertaken in eukaryotes.

  7. Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Kok, Kin-Hang; Jin, Dong-Yan

    2017-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

  8. Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens.

    Science.gov (United States)

    Harper, Marina; Boyce, John D; Wilkie, Ian W; Adler, Ben

    2003-09-01

    Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.

  9. Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival

    OpenAIRE

    Sage, Evelyne; Harrison, Lynn

    2010-01-01

    A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1–2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990’s, and work has progresse...

  10. Overproduction of delta-endotoxins by sporeless Bacillus thuringiensis mutants obtained by nitrous acid mutagenesis.

    Science.gov (United States)

    Ben Khedher, Saoussen; Zouari, Nabil; Messaddeq, Nadia; Schultz, Patrick; Jaoua, Samir

    2011-01-01

    Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.

  11. Mutagenesis of tGCN5 core region reveals two critical surface residues F90 and R140

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, Kinjal Rajesh; Chan, Yan M.; Lee, Man X.; Yang, Ching Yao; Voloshchuk, Natalya [Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201 (United States); Montclare, Jin Kim, E-mail: jmontcla@poly.edu [Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201 (United States); Department of Biochemistry, SUNY-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203 (United States)

    2010-09-24

    Research highlights: {yields} Mutagenesis of the tGCN5 core region reveals two residues important for function. {yields} Developed a fluorescent lysate-based activity assay to assess mutants. {yields} Surface-exposed residues F90 and R140 of tGCN5 are critical for H3 acetylation. -- Abstract: Tetrahymena General Control Non-Derepressor 5 (tGCN5) is a critical regulator of gene transcription via acetylation of histones. Since the acetylation ability has been attributed to the 'core region', we perform mutagenesis of residues within the tGCN5 'core region' in order to identify those critical for function and stability. Residues that do not participate in catalysis are identified, mutated and characterized for activity, structure and thermodynamic stability. Variants I107V, Q114L, A121T and A130S maintain the acetylation function relative to wild-type tGCN5, while variants F90Y, F112R and R140H completely abolish function. Of the three non-functional variants, since F112 is mutated into a non-homologous charged residue, a loss in function is expected. However, the remaining two variants are mutated into homologous residues, suggesting that F90 and R140 are critical for the activity of tGCN5. While mutation to homologous residue maintains acetylation of histone H3 for the majority of the variants, the two surface-exposed residues, F90 and R140, appear to be essential for tGCN5 function, structure or stability.

  12. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.

    Science.gov (United States)

    Cai, Yupeng; Chen, Li; Liu, Xiujie; Guo, Chen; Sun, Shi; Wu, Cunxiang; Jiang, Bingjun; Han, Tianfu; Hou, Wensheng

    2017-05-16

    Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens-mediated transformation. Site-directed mutations were observed at all targeted sites by DNA sequencing analysis. T1-generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1-bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58', E116°20'). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long-day and short-day conditions. We identified some 'transgene-clean' soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These 'transgene-clean' mutants of GmFT2a may provide materials for more in-depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  13. Generation of peanut drought tolerant plants by pingyangmycin-mediated in vitro mutagenesis and hydroxyproline-resistance screening.

    Science.gov (United States)

    Sui, Jiongming; Wang, Ya; Wang, Peng; Qiao, Lixian; Sun, Shimeng; Hu, Xiaohui; Chen, Jing; Wang, Jingshan

    2015-01-01

    In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

  14. CRISPR/Cas9 Mutagenesis Reveals Versatile Roles of Hox Genes in Crustacean Limb Specification and Evolution.

    Science.gov (United States)

    Martin, Arnaud; Serano, Julia M; Jarvis, Erin; Bruce, Heather S; Wang, Jennifer; Ray, Shagnik; Barker, Carryn A; O'Connell, Liam C; Patel, Nipam H

    2016-01-11

    Crustaceans possess a diverse array of specialized limbs. Although shifts in Hox gene expression domains have been postulated to play a role in generating this limb diversity, little functional data have been provided to understand the precise roles of Hox genes during crustacean development. We used a combination of CRISPR/Cas9-targeted mutagenesis and RNAi knockdown to decipher the function of the six Hox genes expressed in the developing mouth and trunk of the amphipod Parhyale hawaiensis. These experimentally manipulated animals display specific and striking homeotic transformations. We found that abdominal-A (abd-A) and Abdominal-B (Abd-B) are required for proper posterior patterning, with knockout of Abd-B resulting in an animal with thoracic type legs along what would have been an abdomen, and abd-A disruption generating a simplified body plan characterized by a loss of specialization in both abdominal and thoracic appendages. In the thorax, Ubx is necessary for gill development and for repression of gnathal fate, and Antp dictates claw morphology. In the mouth, Scr and Antp confer the part-gnathal, part-thoracic hybrid identity of the maxilliped, and Scr and Dfd prevent antennal identity in posterior head segments. Our results allow us to define the role Hox genes play in specifying each appendage type in Parhyale, including the modular nature by which some appendages are patterned by Hox gene inputs. In addition, we define how changes in Hox gene expression have generated morphological differences between crustacean species. Finally, we also highlight the utility of CRISPR/Cas9-based somatic mutagenesis in emerging model organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion.

    Science.gov (United States)

    Fulton, Benjamin O; Sachs, David; Schwarz, Megan C; Palese, Peter; Evans, Matthew J

    2017-08-01

    The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus.IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain

  16. Generation of peanut drought tolerant plants by pingyangmycin-mediated in vitro mutagenesis and hydroxyproline-resistance screening.

    Directory of Open Access Journals (Sweden)

    Jiongming Sui

    Full Text Available In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP. After being extracted from mature seeds (cv. Huayu 20, the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation. The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS and nine individuals with higher protein content (>30% and 21 individuals with higher oil content (>58% were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

  17. Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

    Science.gov (United States)

    Weng, MeiZhi; Zheng, ZhongLiang; Bao, Wei; Cai, YongJun; Yin, Yan; Zou, GuoLin; Zou, GouLin

    2009-11-01

    Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

  18. Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait

    Science.gov (United States)

    Abt, Tom Den; Souffriau, Ben; Foulquié-Moreno, Maria R.; Duitama, Jorge; Thevelein, Johan M.

    2016-01-01

    Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to

  19. Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis.

    Science.gov (United States)

    Luo, Quan; Chen, Yu; Xia, Jing; Wang, Kai-Qiang; Cai, Yu-Jie; Liao, Xiang-Ru; Guan, Zheng-Bing

    2018-02-01

    Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, November 1, 1979-October 31, 1980

    Energy Technology Data Exchange (ETDEWEB)

    Evans, H.H.

    1980-10-01

    The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. Use of the host-cell reactivation viral suicide enrichment procedure was initiated in the isolation of repair-deficient mutants. Lightly mutagenized BHK cells were infected with irradiated Herpes simplex virus (HSV); several radiation-sensitive strains were isolated among the survivors of the infection. The characterization of these strains is progressing and the enrichments are continuing. That alterations in the frequency of mutation of C3H/10T 1/2 cells, occurring as a result of holding the cells in a confluent state following treatment with ethylmethane sulfonate, parallel the alterations in the frequency of neoplastic transformation was found. The repair capabilities of BHK cells were found to be intermediate in comparison to repair-proficient and -deficient human cells with regard to the reactivation of HSV treated with various inactivating agents. The effect of confluency and of low serum levels on DNA synthesis, as well as the response to the cytotoxic effects of MNNG and acriflavin were determined in BHK cells in preparation for the investigation of the role of DNA repair in mutagenesis and transformation. It was also found that C3H/10T 1/2 cells partially recover from the toxic effects of 4-nitroquinoline-1-oxide if they are held in a confluent state for 6 to 22 hrs following treatment. Addition of catalase did not alleviate the toxic effects of 4-NQO. The cells contain a relatively high endogenous level of this enzyme. (ERB)

  1. Localization of Electrons in a One-Dimensional Disordered Crystal ...

    African Journals Online (AJOL)

    Localization of electrons in a one-dimensional disorder crystal and suppression of conduction were investigated in the research work. These were achieved by modeling the disorders within the crystal lattice with random potential barriers. The transmission coefficients of the electron (non-interacting electron) in the random ...

  2. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics.

    NARCIS (Netherlands)

    Smits, B.M.; Peters, T.A.; Mul, J.D.; Croes, H.J.E.; Fransen, J.A.M.; Beynon, A.J.; Guryev, V.; Plasterk, R.H.; Cuppen, E.P.J.G.

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis

  3. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

    NARCIS (Netherlands)

    Smits, B.M.; Peters, T.A.; Mul, J.D.; Croes, H.J.; Fransen, J.A.; Beynon, A.J.; Guryev, V.; Plasterk, R.; Cuppen, E.

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis

  4. Microspore induced doubled haploids production from ethyl methanesulfonate (EMS) soaked flower buds is an efficient strategy for mutagenesis in Chinese cabbage

    NARCIS (Netherlands)

    Lu, Yin; Dai, Shuangyan; Gu, Aixia; Liu, Mengyang; Wang, Yanhua; Luo, Shuangxia; Zhao, Yujing; Wang, Shan; Xuan, Shuxin; Chen, Xueping; Li, Xiaofeng; Bonnema, Guusje; Zhao, Jianjun; Shen, Shuxing

    2016-01-01

    Chinese cabbage buds were soaked with Ethyl methanesulfonate (EMS) to induce mutagenesis. The influence of different EMS concentrations and treatment durations on microspore development, embryo production rate and seedling rate were evaluated in five Chinese cabbage genotypes. Mutations in four

  5. Abstracts of the Conference on Mechanisms of DNA Repair and Mutagenesis on the 100. Anniversary of the Discovery of Polonium and Radium

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-12-31

    The conference covered various aspects of mutagenesis and mechanisms of DNA repair. UV and ionizing radiation were use to induce DNA lesions in bacteria, yeast and cell cultures of higher organisms. This allows study of influence of mutations on particular processes in the cell. Mechanisms of resistance were also investigated. Biological investigations were performed using labelled compounds.

  6. Wigner-like crystallization of Anderson-localized electron systems with low electron densities

    CERN Document Server

    Slutskin, A A; Pepper, M

    2002-01-01

    We consider an electron system under conditions of strong Anderson localization, taking into account interelectron long-range Coulomb repulsion. We establish that at sufficiently low electron densities and sufficiently low temperatures the Coulomb electron interaction brings about ordering of the Anderson-localized electrons into a structure that is close to an ideal (Wigner) crystal lattice, provided the dimension of the system is > 1. This Anderson-Wigner glass (AWG) is a new macroscopic electron state that, on the one hand, is beyond the conventional Fermi glass concept, and on the other hand, qualitatively differs from the known 'plain' Wigner glass (inherent in self-localized electron systems) in that the random slight electron displacements from the ideal crystal sites essentially depend on the electron density. With increasing electron density the AWG is found to turn into the plain Wigner glass or Fermi glass, depending on the width of the random spread of the electron levels. It is shown that the res...

  7. Random walks in a random environment

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    Abstract. Random walks as well as diffusions in random media are considered. Methods are developed that allow one to establish large deviation results for both the 'quenched' and the 'averaged' case. Keywords. Large deviations; random walks in a random environment. 1. Introduction. A random walk on Zd is a stochastic ...

  8. EDITORIAL: Synaptic electronics Synaptic electronics

    Science.gov (United States)

    Demming, Anna; Gimzewski, James K.; Vuillaume, Dominique

    2013-09-01

    neuromorphic circuit composed of a nanoscale 1-kbit resistive random-access memory (RRAM) cross-point array of synapses and complementary metal-oxide-semiconductor (CMOS) neuron circuits [13]; a WO3-x-based nanoionics device from Masakazu Aono's group with a wide scale of reprogrammable memorization functions [14]; a new spike-timing dependent plasticity scheme based on a MOS transistor as a selector and a RRAM as a variable resistance device [15]; a new hybrid memristor-CMOS neuromorphic circuit [16]; and a photo-assisted atomic switch [17]. Synaptic electronics evidently has many emerging facets, and Duygu Kuzum, Shimeng Yu, and H-S Philip Wong in the US provide a review of the field, including the materials, devices and applications [18]. In embracing the expertise acquired over thousands of years of evolution, biomimetics and bio-inspired design is a common, smart approach to technological innovation. Yet in successfully mimicking the physiological mechanisms of the human mind synaptic electronics research has a potential impact that is arguably unprecedented. That the quirks and eccentricities recently unearthed in the behaviour of nanomaterials should lend themselves so accommodatingly to emulating synaptic functions promises some very exciting developments in the field, as the articles in this special issue emphasize. References [1] von Neumann J (ed) 2012 The Computer and the Brain 3rd edn (Yale: Yale University Press) [2] Strukov D B, Snider G S, Stewart D R and Williams R S 2008 The missing memristor found Nature 453 80-3 [3] Chua L O 1971 Memristor—the missing circuit element IEEE Trans. Circuit Theory 18 507-19 [4] Chua L O 2013 Memristor, Hodgkin-Huxley, and Edge of Chaos Nanotechnology 24 383001 [5] Pickett M D and Williams R S 2013 Phase transitions enable computational universality in neuristor-based cellular automata Nanotechnology 24 384002 [6] Cruz-Albrecht J M, Derosier T and Srinivasa N 2013 Scalable neural chip with synaptic electronics using CMOS

  9. Probing the Paracoccus denitrificans cytochrome c1 / c552 interaction by mutagenesis and fast kinetics†

    Science.gov (United States)

    Janzon, Julia; Yuan, Quan; Malatesta, Francesco; Hellwig, Petra; Ludwig, Bernd; Durham, Bill; Millett, Francis

    2008-01-01

    Electron transfer (ET) between Paracoccus denitrificans cytochrome c1 and cytochrome c552 was studied using the soluble redox fragments cyt c1CF and cyt c552F. A new ruthenium cyt c552F derivative labeled at C23 (Ruz-23-c552F) was designed to measure rapid electron transfer with cyt c1CF in the physiological direction using flash photolysis. The bimolecular rate constant k12 decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected below 35 mM ionic strength with the ruthenium photoexcitation technique in which k12 became very rapid (3 × 109 M−1s−1) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ruz-23-c552F and c1CF was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was greater than 30 µM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ruz-39-Cc formed a tight 1:1 complex with cyt c1CF at ionic strengths below 60 mM with an intracomplex electron transfer rate constant of 50,000 s−1. A group of cyt c1CF variants in the presumed docking site were generated based on information from the yeast cyt bc1/cyt c co-crystal structure. Kinetic analysis of cyt c1CF mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c552F, and suggest that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions. PMID:19006325

  10. Electronic Commerce

    Directory of Open Access Journals (Sweden)

    Slavko Đerić

    2016-12-01

    Full Text Available Electronic commerce can be defined in different ways. Any definition helps to understand and explain that concept as better as possible.. Electronic commerce is a set of procedures and technologies that automate the tasks of financial transactions using electronic means. Also, according to some authors, electronic commerce is defined as a new concept, which is being developed and which includes process of buying and selling or exchanging products, services or information via computer networks, including the Internet. Electronic commerce is not limited just to buying and selling, but it also includes all pre-sales and after-sales ongoing activities along the supply chain. Introducing electronic commerce, using the Internet and Web services in business, realizes the way to a completely new type of economy - internet economy.

  11. Micropower electronics

    CERN Document Server

    Keonjian, Edward

    1964-01-01

    Micropower Electronics deals with the operation of modern electronic equipment at micropower levels and the problems associated with micropower electronics. Topics covered include the relations between minimum required power density and frequency response for semiconductor triode amplifiers; physical realization of digital logic circuits; micropower microelectronic subsystems; and metal-oxide-semiconductor field-effect devices for micropower logic circuitry. This book is comprised of 10 chapters and begins with an analysis of fundamental relationships and basic requirements pertinent to the ph

  12. Microfluidic electronics.

    Science.gov (United States)

    Cheng, Shi; Wu, Zhigang

    2012-08-21

    Microfluidics, a field that has been well-established for several decades, has seen extensive applications in the areas of biology, chemistry, and medicine. However, it might be very hard to imagine how such soft microfluidic devices would be used in other areas, such as electronics, in which stiff, solid metals, insulators, and semiconductors have previously dominated. Very recently, things have radically changed. Taking advantage of native properties of microfluidics, advances in microfluidics-based electronics have shown great potential in numerous new appealing applications, e.g. bio-inspired devices, body-worn healthcare and medical sensing systems, and ergonomic units, in which conventional rigid, bulky electronics are facing insurmountable obstacles to fulfil the demand on comfortable user experience. Not only would the birth of microfluidic electronics contribute to both the microfluidics and electronics fields, but it may also shape the future of our daily life. Nevertheless, microfluidic electronics are still at a very early stage, and significant efforts in research and development are needed to advance this emerging field. The intention of this article is to review recent research outcomes in the field of microfluidic electronics, and address current technical challenges and issues. The outlook of future development in microfluidic electronic devices and systems, as well as new fabrication techniques, is also discussed. Moreover, the authors would like to inspire both the microfluidics and electronics communities to further exploit this newly-established field.

  13. Electron Bifurcation

    Energy Technology Data Exchange (ETDEWEB)

    Peters, John W.; Miller, Anne-Frances; Jones, Anne K.; King, Paul W.; Adams, Michael W. W.

    2016-04-01

    Electron bifurcation is the recently recognized third mechanism of biological energy conservation. It simultaneously couples exergonic and endergonic oxidation-reduction reactions to circumvent thermodynamic barriers and minimize free energy loss. Little is known about the details of how electron bifurcating enzymes function, but specifics are beginning to emerge for several bifurcating enzymes. To date, those characterized contain a collection of redox cofactors including flavins and iron-sulfur clusters. Here we discuss the current understanding of bifurcating enzymes and the mechanistic features required to reversibly partition multiple electrons from a single redox site into exergonic and endergonic electron transfer paths.

  14. Polymer electronics

    CERN Document Server

    Hsin-Fei, Meng

    2013-01-01

    Polymer semiconductor is the only semiconductor that can be processed in solution. Electronics made by these flexible materials have many advantages such as large-area solution process, low cost, and high performance. Researchers and companies are increasingly dedicating time and money in polymer electronics. This book focuses on the fundamental materials and device physics of polymer electronics. It describes polymer light-emitting diodes, polymer field-effect transistors, organic vertical transistors, polymer solar cells, and many applications based on polymer electronics. The book also disc

  15. Electron holography

    CERN Document Server

    Tonomura, Akira

    1993-01-01

    Holography was devised for breaking through the resolution limit of electron microscopes The advent of a "coherent" field emission electron beam has enabled the use of Electron Holography in various areas of magnetic domain structures observation, fluxon observation in superconductors, and fundamental experiments in physics which have been inaccessible using other techniques After examining the fundamentals of electron holography and its applications to the afore mentioned fields, a detailed discussion of the Aharonov-Bohm effect and the related experiments is presented Many photographs and illustrations are included to elucidate the text

  16. Random tensors

    CERN Document Server

    Gurau, Razvan

    2017-01-01

    Written by the creator of the modern theory of random tensors, this book is the first self-contained introductory text to this rapidly developing theory. Starting from notions familiar to the average researcher or PhD student in mathematical or theoretical physics, the book presents in detail the theory and its applications to physics. The recent detections of the Higgs boson at the LHC and gravitational waves at LIGO mark new milestones in Physics confirming long standing predictions of Quantum Field Theory and General Relativity. These two experimental results only reinforce today the need to find an underlying common framework of the two: the elusive theory of Quantum Gravity. Over the past thirty years, several alternatives have been proposed as theories of Quantum Gravity, chief among them String Theory. While these theories are yet to be tested experimentally, key lessons have already been learned. Whatever the theory of Quantum Gravity may be, it must incorporate random geometry in one form or another....

  17. Random functions and turbulence

    CERN Document Server

    Panchev, S

    1971-01-01

    International Series of Monographs in Natural Philosophy, Volume 32: Random Functions and Turbulence focuses on the use of random functions as mathematical methods. The manuscript first offers information on the elements of the theory of random functions. Topics include determination of statistical moments by characteristic functions; functional transformations of random variables; multidimensional random variables with spherical symmetry; and random variables and distribution functions. The book then discusses random processes and random fields, including stationarity and ergodicity of random

  18. Printed Electronics

    Science.gov (United States)

    Crain, John M. (Inventor); Lettow, John S. (Inventor); Aksay, Ilhan A. (Inventor); Korkut, Sibel (Inventor); Chiang, Katherine S. (Inventor); Chen, Chuan-Hua (Inventor); Prud'Homme, Robert K. (Inventor)

    2016-01-01

    Printed electronic device comprising a substrate onto at least one surface of which has been applied a layer of an electrically conductive ink comprising functionalized graphene sheets and at least one binder. A method of preparing printed electronic devices is further disclosed.

  19. Electronic Cereal.

    Science.gov (United States)

    Frentrup, Julie R.; Phillips, Donald B.

    1996-01-01

    Describes activities that use Froot Loops breakfast cereal to help students master the concepts of valence electrons and chemical bonding and the implications of the duet and octet rules. Involves students working in groups to create electron dot structures for various compounds. (JRH)

  20. Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots.

    Science.gov (United States)

    Shen, Xinqian; Chen, Yunpeng; Liu, Tong; Hu, Xiaolu; Gu, Zhenfang

    2013-09-01

    A Tn5 transposition vector, pMOD-tet-egfp, was constructed and used for the random insertional mutagenesis of Bacillus pumilus. Various parameters were investigated to increase the transformation efficiency B. pumilus DX01 via Tn5 transposition complexes (transposome): bacterial growth phase, type of electroporation buffer, electric field strength, and recovery medium. Transformation efficiency was up to 3 × 10(4) transformants/μg of DNA under the optimized electroporation conditions, and a total of 1,467 gfp-tagged transformants were obtained. Fluorescence-activated cell sorting analysis showed that all gfp-tagged bacterial cells expressed GFP, indicating that foreign DNA has been successfully integrated into the genome of B. pumilus and expressed. Finally, flanking DNA sequences were isolated from several transformants and colonization of rice roots by B. pumilus DX01 was also studied. The method developed here will be useful for creating an insertion mutant library of gram-positive bacteria, thus facilitating their molecular genetic and cytological studies.

  1. Transposon Mutagenesis Screen Identifies Potential Lung Cancer Drivers and CUL3 as a Tumor Suppressor

    Science.gov (United States)

    Dorr, Casey; Janik, Callie; Weg, Madison; Been, Raha A.; Bader, Justin; Kang, Ryan; Ng, Brandon; Foran, Lindsey; Landman, Sean R.; O’Sullivan, M. Gerard; Steinbach, Michael; Sarver, Aaron L.; Silverstein, Kevin A. T.; Largaespada, David A.

    2015-01-01

    Non-small cell lung cancers (NSCLCs) harbor thousands of passenger events that hide genetic drivers. Even highly recurrent events in NSCLC, such as mutations in PTEN, EGFR, KRAS, and ALK, are only detected in, at most, 30% of patients. Thus, many unidentified low-penetrant events are causing a significant portion of lung cancers. To detect low-penetrance drivers of NSCLC a forward genetic screen was performed in mice using the Sleeping Beauty (SB) DNA transposon as a random mutagen to generate lung tumors in a Pten deficient background. SB mutations coupled with Pten deficiency were sufficient to produce lung tumors in 29% of mice. Pten deficiency alone, without SB mutations, resulted in lung tumors in 11% of mice, while the rate in control mice was ~3%. In addition, thyroid cancer and other carcinomas as well as the presence of bronchiolar and alveolar epithelialization in mice deficient for Pten were also identified. Analysis of common transposon insertion sites identified 76 candidate cancer driver genes. These genes are frequently dysregulated in human lung cancers and implicate several signaling pathways. Cullin3 (Cul3), a member of an ubiquitin ligase complex that plays a role in the oxidative stress response pathway, was identified in the screen and evidence demonstrates that Cul3 functions as a tumor suppressor. PMID:25995385

  2. Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.

    Science.gov (United States)

    Baliga, N S; DasSarma, S

    1999-04-01

    Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

  3. Mutagenesis Objective Search and Selection Tool (MOSST: an algorithm to predict structure-function related mutations in proteins

    Directory of Open Access Journals (Sweden)

    Asenjo Juan A

    2011-04-01

    Full Text Available Abstract Background Functionally relevant artificial or natural mutations are difficult to assess or predict if no structure-function information is available for a protein. This is especially important to correctly identify functionally significant non-synonymous single nucleotide polymorphisms (nsSNPs or to design a site-directed mutagenesis strategy for a target protein. A new and powerful methodology is proposed to guide these two decision strategies, based only on conservation rules of physicochemical properties of amino acids extracted from a multiple alignment of a protein family where the target protein belongs, with no need of explicit structure-function relationships. Results A statistical analysis is performed over each amino acid position in the multiple protein alignment, based on different amino acid physical or chemical characteristics, including hydrophobicity, side-chain volume, charge and protein conformational parameters. The variances of each of these properties at each position are combined to obtain a global statistical indicator of the conservation degree of each property. Different types of physicochemical conservation are defined to characterize relevant and irrelevant positions. The differences between statistical variances are taken together as the basis of hypothesis tests at each position to search for functionally significant mutable sites and to identify specific mutagenesis targets. The outcome is used to statistically predict physicochemical consensus sequences based on different properties and to calculate the amino acid propensities at each position in a given protein. Hence, amino acid positions are identified that are putatively responsible for function, specificity, stability or binding interactions in a family of proteins. Once these key functional positions are identified, position-specific statistical distributions are applied to divide the 20 common protein amino acids in each position of the protein

  4. Dietary flavonoids bind to mono-ubiquitinated annexin A1 in nuclei, and inhibit chemical induced mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Fusao, E-mail: fhirata@wayne.edu; Harada, Takasuke; Corcoran, George B.; Hirata, Aiko

    2014-01-15

    Highlight: • Nuclear mono-ubiquitinated annexin A1 is involved in DNA damage induced mutagenesis. • Dietary flavonoids bind to and inhibit purified mono-ubiquitinated annexin A1 helicase. • Dietary flavonoids show anti-mutagenic action. • Annexin A1 may serve as a putative target of cancer chemoprevention by flavonoids. - Abstract: In order to investigate the mechanisms of anti-mutagenic action by dietary flavonoids, we investigated if they inhibit mutation of the thymidine kinase (tk) gene in L5178Ytk(±) lymphoma cells. Silibinin, quercetin and genistein suppressed mutation of the tk gene induced in L5178Ytk(±) lymphoma cells by methyl methanesulfonate (MMS) and As{sup 3+}. Flavone and flavonol were less effective. To establish that mutation of the tk gene in L5178Ytk(±) lymphoma cells by MMS and As{sup 3+} is mediated through mono-ubiquitinated annexin A1, L5178Ytk(±) lymphoma cells were treated with annexin A1 anti-sense oligonucleotide. The treatment reduced mRNA as well as protein levels of annexin A1, and suppressed mutation of the tk gene. Nuclear extracts from L5178Ytk(±) lymphoma cells catalyzed translesion DNA synthesis with an oligonucleotide template containing 8-oxo-guanosine in an annexin A1 dependent manner. This translesion DNA synthesis was inhibited by the anti-mutagenic flavonoids, silibinin, quercetin and genistein, in a concentration dependent manner, but only slightly by flavone and flavonol. Because these observations implicate involvement of annexin A1 in mutagenesis, we examined if flavonoids suppress nuclear annexin A1 helicase activity. Silibinin, quercetin and genistein inhibited ssDNA binding, DNA chain annealing and DNA unwinding activities of purified nuclear mono-ubiquitinated annexin A1. Flavone and flavonol were ineffective. The apparent direct binding of anti-mutagenic flavonoids to the annexin A1 molecule was supported by fluorescence quenching. Taken together, these findings illustrate that nuclear annexin A1 may be

  5. Mutagenesis-mediated decrease of pathogenicity as a feature of the mutant spectrum of a viral population.

    Directory of Open Access Journals (Sweden)

    Marta Sanz-Ramos

    Full Text Available BACKGROUND: RNA virus populations are heterogeneous ensembles of closely related genomes termed quasispecies. This highly complex distribution of variants confers important properties to RNA viruses and influences their pathogenic behavior. It has been hypothesized that increased mutagenesis of viral populations, by treatment with mutagenic agents, can induce alterations in the pathogenic potential of a virus population. In this work we investigate whether mutagenized foot-and-mouth disease virus (FMDV populations display changes in their virulence in mice. METHODOLOGY AND PRINCIPAL FINDINGS: FMDV C-S8c1 was passaged in BHK cells in the presence of the mutagenic agent ribavirin. Decline in viral titer and viral RNA progeny was observed in the first passage, fluctuating around a constant value thereafter. Hence, the specific infectivity remained stable during the passages. The viral population harvested from passage 9 (P9 R showed decreased virulence in mice, with a lethal dose 50 (LD(50 >10(4 PFU, as compared with LD(50 of 50 PFU of the parental population FMDV C-S8c1. This decrease in virulence was associated to a 20-fold increase in the mutation frequency of the P9 R population with respect to C-S8c1. Interestingly, individual biological clones isolated from the attenuated population P9 R were as virulent as the parental virus C-S8c1. Furthermore, a mixed population of C-S8c1 and P9 R was inoculated into mice and showed decreased virulence as compared to C-S8c1, suggesting that population P9 R is able to suppress the virulent phenotype of C-S8c1. CONCLUSION: Ribavirin-mediated mutagenesis of an FMDV population resulted in attenuation in vivo, albeit a large proportion of its biological clones displayed a highly virulent phenotype. These results, together with the suppression of C-S8c1 by mutagenized P9 R population, document a suppressive effect of mutagenized viral quasispecies in vivo, and suggest novel approaches to the treatment and

  6. Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1.

    Science.gov (United States)

    Puthan Veetil, Vinod; Raj, Hans; Quax, Wim J; Janssen, Dick B; Poelarends, Gerrit J

    2009-06-01

    Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of L-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of L-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which L-aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of L-aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for L-aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326

  7. Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking.

    Science.gov (United States)

    Zhang, Yihong; Colenso, Charlotte K; El Harchi, Aziza; Cheng, Hongwei; Witchel, Harry J; Dempsey, Chris E; Hancox, Jules C

    2016-08-01

    The antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ∼45nM, whilst inward IhERG tails in a high K(+) external solution ([K(+)]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone's inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ∼20-fold that of WT IhERG, similar to the pore helical mutant S624A (∼22-fold WT control). The IC50 for F656A mutant IhERG was ∼17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A∼Y652A>F656A>V659A>G648A>T623A. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Digital electronics

    CERN Document Server

    Morris, John

    2013-01-01

    An essential companion to John C Morris's 'Analogue Electronics', this clear and accessible text is designed for electronics students, teachers and enthusiasts who already have a basic understanding of electronics, and who wish to develop their knowledge of digital techniques and applications. Employing a discovery-based approach, the author covers fundamental theory before going on to develop an appreciation of logic networks, integrated circuit applications and analogue-digital conversion. A section on digital fault finding and useful ic data sheets completes th

  9. Electronic diagrams

    CERN Document Server

    Colwell, Morris A

    1976-01-01

    Electronic Diagrams is a ready reference and general guide to systems and circuit planning and in the preparation of diagrams for both newcomers and the more experienced. This book presents guidelines and logical procedures that the reader can follow and then be equipped to tackle large complex diagrams by recognition of characteristic 'building blocks' or 'black boxes'. The goal is to break down many of the barriers that often seem to deter students and laymen in learning the art of electronics, especially when they take up electronics as a spare time occupation. This text is comprised of nin

  10. Electronic identity

    CERN Document Server

    de Andrade, Norberto Nuno Gomes; Argles, David

    2014-01-01

    With the increasing availability of electronic services, security and a reliable means by which identity is verified is essential.Written by Norberto Andrade the first chapter of this book provides an overview of the main legal and regulatory aspects regarding electronic identity in Europe and assesses the importance of electronic identity for administration (public), business (private) and, above all, citizens. It also highlights the role of eID as a key enabler of the economy.In the second chapter Lisha Chen-Wilson, David Argles, Michele Schiano di Zenise and Gary Wills discuss the user-cent

  11. Polymer electronics

    CERN Document Server

    Geoghegan, Mark

    2013-01-01

    Polymer electronics is the science behind many important new developments in technology, such as the flexible electronic display (e-ink) and many new developments in transistor technology. Solar cells, light-emitting diodes, and transistors are all areas where plastic electronics is likely to, or is already having, a serious impact on our daily lives. With polymer transistors and light-emitting diodes now being commercialised, there is a clear need for a pedagogic text thatdiscusses the subject in a clear and concise fashion suitable for senior undergraduate and graduate students. The content

  12. Stretchable electronics

    CERN Document Server

    Someya, Takao

    2012-01-01

    With its comprehensive coverage this handbook and ready reference brings together some of the most outstanding scientists in the field to lay down the undisputed knowledge on how to make electronics stretchable.As such, it focuses on gathering and evaluating the materials, designs, models and technologies that enable the fabrication of fully elastic electronic devices which can sustain high strain. Furthermore, it provides a review of those specific applications that directly benefit from highly compliant electronics, including transistors, photonic devices and sensors. In addition to stre

  13. Electron optics

    CERN Document Server

    Grivet, Pierre; Bertein, F; Castaing, R; Gauzit, M; Septier, Albert L

    1972-01-01

    Electron Optics, Second English Edition, Part I: Optics is a 10-chapter book that begins by elucidating the fundamental features and basic techniques of electron optics, as well as the distribution of potential and field in electrostatic lenses. This book then explains the field distribution in magnetic lenses; the optical properties of electrostatic and magnetic lenses; and the similarities and differences between glass optics and electron optics. Subsequent chapters focus on lens defects; some electrostatic lenses and triode guns; and magnetic lens models. The strong focusing lenses and pris

  14. Starting electronics

    CERN Document Server

    Brindley, Keith

    2005-01-01

    Starting Electronics is unrivalled as a highly practical introduction for hobbyists, students and technicians. Keith Brindley introduces readers to the functions of the main component types, their uses, and the basic principles of building and designing electronic circuits. Breadboard layouts make this very much a ready-to-run book for the experimenter; and the use of multimeter, but not oscilloscopes, puts this practical exploration of electronics within reach of every home enthusiast's pocket. The third edition has kept the simplicity and clarity of the original. New material

  15. A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

    Science.gov (United States)

    Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

  16. Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli.

    Science.gov (United States)

    Zhong, Chao; You, Chun; Wei, Ping; Zhang, Yi-Heng Percival

    2017-01-01

    We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3' and 5' overlapping termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning pr