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Sample records for saturation mutagenesis approach

  1. Promoter analysis by saturation mutagenesis

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    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.

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

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    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.

  3. 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

  4. Genomic approaches to DNA repair and mutagenesis.

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    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.

  5. 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.

  6. Saturation mutagenesis of lysine 12 leads to the identification of derivatives of nisin A with enhanced antimicrobial activity.

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    Evelyn M Molloy

    Full Text Available It is becoming increasingly apparent that innovations from the "golden age" of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12. A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.

  7. Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

    Science.gov (United States)

    Molloy, Evelyn M.; Field, Des; Connor, Paula M. O'.; Cotter, Paul D.; Hill, Colin; Ross, R. Paul

    2013-01-01

    It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus. PMID:23505531

  8. 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

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

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    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.

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

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    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

  11. 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

  12. One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

    Science.gov (United States)

    Acevedo, Juan Pablo; Reetz, Manfred T; Asenjo, Juan A; Parra, Loreto P

    2017-05-01

    Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T5015 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T5015. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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    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.

  14. 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...

  15. 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...

  16. 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%.

  17. Underlying role of mitochondrial mutagenesis in the pathogenesis of a disease and current approaches for translational research.

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    Paraskevaidi, Maria; Martin-Hirsch, Pierre L; Kyrgiou, Maria; Martin, Francis L

    2017-05-01

    Mitochondrial diseases have been extensively investigated over the last three decades, but many questions regarding their underlying aetiologies remain unanswered. Mitochondrial dysfunction is not only responsible for a range of neurological and myopathy diseases but also considered pivotal in a broader spectrum of common diseases such as epilepsy, autism and bipolar disorder. These disorders are a challenge to diagnose and treat, as their aetiology might be multifactorial. In this review, the focus is placed on potential mechanisms capable of introducing defects in mitochondria resulting in disease. Special attention is given to the influence of xenobiotics on mitochondria; environmental factors inducing mutations or epigenetic changes in the mitochondrial genome can alter its expression and impair the whole cell's functionality. Specifically, we suggest that environmental agents can cause damage in mitochondrial DNA and consequently lead to mutagenesis. Moreover, we describe current approaches for handling mitochondrial diseases, as well as available prenatal diagnostic tests, towards eliminating these maternally inherited diseases. Undoubtedly, more research is required, as current therapeutic approaches mostly employ palliative therapies rather than targeting primary mechanisms or prophylactic approaches. Much effort is needed into further unravelling the relationship between xenobiotics and mitochondria, as the extent of influence in mitochondrial pathogenesis is increasingly recognised. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Experimental study on distributed optical fiber-based approach monitoring saturation line in levee engineering

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    Su, Huaizhi; Li, Hao; Kang, Yeyuan; Wen, Zhiping

    2018-02-01

    Seepage is one of key factors which affect the levee engineering safety. The seepage danger without timely detection and rapid response may likely lead to severe accidents such as seepage failure, slope instability, and even levee break. More than 90 percent of levee break events are caused by the seepage. It is very important for seepage behavior identification to determine accurately saturation line in levee engineering. Furthermore, the location of saturation line has a major impact on slope stability in levee engineering. Considering the structure characteristics and service condition of levee engineering, the distributed optical fiber sensing technology is introduced to implement the real-time observation of saturation line in levee engineering. The distributed optical fiber temperature sensor system (DTS)-based monitoring principle of saturation line in levee engineering is investigated. An experimental platform, which consists of DTS, heating system, water-supply system, auxiliary analysis system and levee model, is designed and constructed. The monitoring experiment of saturation line in levee model is implemented on this platform. According to the experimental results, the numerical relationship between moisture content and thermal conductivity in porous medium is identified. A line heat source-based distributed optical fiber method obtaining the thermal conductivity in porous medium is developed. A DTS-based approach is proposed to monitor the saturation line in levee engineering. The embedment pattern of optical fiber for monitoring saturation line is presented.

  19. CGC/saturation approach for soft interactions at high energy: Inclusive production

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    E. Gotsman

    2015-06-01

    Full Text Available In this letter we demonstrate that our dipole model is successful in describing inclusive production within the same framework as diffractive physics. We believe that this achievement stems from the fact that our approach incorporates the positive features of the Reggeon approach and CGC/saturation effective theory, for high energy QCD.

  20. 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

  1. A model for strong interactions at high energy based on the CGC/saturation approach

    Energy Technology Data Exchange (ETDEWEB)

    Gotsman, E.; Maor, U. [Tel Aviv University, Department of Particle Physics, School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Levin, E. [Tel Aviv University, Department of Particle Physics, School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Universidad Tecnica Federico Santa Maria and Centro Cientifico-Tecnologico de Valparaiso, Departamento de Fisica, Valparaiso (Chile)

    2015-01-01

    We present our first attempt to develop a model for soft interactions at high energy, based on the BFKL Pomeron and the CGC/saturation approach. We construct an eikonal-type model, whose opacity is determined by the exchange of the dressed BFKL Pomeron. The Green function of the Pomeron is calculated in the framework of the CGC/saturation approach. Using five parameters we achieve a reasonable description of the experimental data at high energies (W ≥ 0.546TeV) with overall χ{sup 2}/d.o.f. ∼ 2. The model results in different behavior for the single- and double-diffraction cross sections at high energies.The singlediffraction cross section reaches a saturated value (about 10mb) at high energies, while the double-diffraction cross section continues growing slowly. (orig.)

  2. CGC/saturation approach for soft interactions at high energy: a two channel model

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    Gotsman, E.; Maor, U. [Tel Aviv University, Department of Particle Physics, School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Levin, E. [Tel Aviv University, Department of Particle Physics, School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Universidad Tecnica Federico Santa Maria, Departemento de Fisica, Centro Cientifico-Tecnologico de Valparaiso, Valparaiso (Chile)

    2015-05-15

    In this paper we continue the development of a model for strong interactions at high energy, based on two ingredients: the CGC/saturation approach and the BFKL Pomeron. In our approach, the unknown mechanism of confinement of quarks and gluons is characterized by several numerical parameters, which are extracted from the experimental data. We demonstrate that the two channel model successfully describes the experimental data, including both the value of the elastic slope and the energy behavior of the single diffraction cross section. We show that the disagreement with the experimental data of our previous single channel eikonal model (Gotsman et al., Eur Phys J C 75:1-18, 2015) stems from the simplified approach used for the hadron structure and is not related to our principal theoretical input, based on the CGC/saturation approach. (orig.)

  3. Description of the Radiological Decontamination by Electromigration in Saturated Concrete: A Multi Species Approach

    Energy Technology Data Exchange (ETDEWEB)

    Frizon, F.; Thouvenot, P.; Ollivier, J. P.; Lorente, S.

    2002-02-28

    A numerical model was developed in order to describe the electromigration of radioelements through contaminated concrete. The transport of several ionic species across a saturated sample of material, is described by the Nernst-Planck relation. The multi species approach provides a better understanding of the phenomena involved: it stresses the need to know, as precisely as possible, the material characteristics and the history of the contamination. The approach also sheds lights on the way the radionuclide (cesium 137) is carried away from a concrete sample during a decontamination simulation.

  4. Fuzzy Saturated Output Feedback Tracking Control for Robot Manipulators: A Singular Perturbation Theory Based Approach

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    Huashan Liu

    2011-09-01

    Full Text Available To deal with the problem of the output feedback tracking (OFT control with bounded torque inputs of robot manipulators, we propose a generalized fuzzy saturated OFT controller based on singular perturbation theory. First, considering the fact that the output toque of joint actuators is limited, a general expression for a class of saturation functions is given to be applied in the control law. Second, to carry out the whole closed-loop control with only position measurements, linear and nonlinear filters are optionally involved to generate a pseudo signal to surrogate the actual velocity tracking error. As a third contribution, a fuzzy regulator is added to obtain a self-tuning performance in tackling the disturbances. Moreover, an explicit but strict stability proof of the system based on the stability theory of singularly perturbed systems is presented. Finally, numerical simulations on several sample controllers are implemented to verify the effectiveness of the proposed approach.

  5. Fuzzy Saturated Output Feedback Tracking Control for Robot Manipulators: A Singular Perturbation Theory Based Approach

    Directory of Open Access Journals (Sweden)

    Huashan Liu

    2011-09-01

    Full Text Available To deal with the problem of the output feedback tracking (OFT control with bounded torque inputs of robot manipulators, we propose a generalized fuzzy saturated OFT controller based on singular perturbation theory. First, considering the fact that the output toque of joint actuators is limited, a general expression for a class of saturation functions is given to be applied in the control law. Second, to carry out the whole closed‐loop control with only position measurements, linear and nonlinear filters are optionally involved to generate a pseudo signal to surrogate the actual velocity tracking error. As a third contribution, a fuzzy regulator is added to obtain a self‐tuning performance in tackling the disturbances. Moreover, an explicit but strict stability proof of the system based on the stability theory of singularly perturbed systems is presented. Finally, numerical simulations on several sample controllers are implemented to verify the effectiveness of the proposed approach.

  6. CGC/saturation approach for soft interactions at high energy: survival probability of central exclusive production

    Energy Technology Data Exchange (ETDEWEB)

    Gotsman, E.; Maor, U. [Tel Aviv University, Department of Particle Physics, Raymond and Beverly Sackler Faculty of Exact Science, School of Physics and Astronomy, Tel Aviv (Israel); Levin, E. [Tel Aviv University, Department of Particle Physics, Raymond and Beverly Sackler Faculty of Exact Science, School of Physics and Astronomy, Tel Aviv (Israel); Universidad Tecnica Federico Santa Maria, Departemento de Fisica, Centro Cientifico-Tecnologico de Valparaiso, Valparaiso (Chile)

    2016-04-15

    We estimate the value of the survival probability for central exclusive production in a model which is based on the CGC/saturation approach. Hard and soft processes are described in the same framework. At LHC energies, we obtain a small value for the survival probability. The source of the small value is the impact parameter dependence of the hard amplitude. Our model has successfully described a large body of soft data: elastic, inelastic and diffractive cross sections, inclusive production and rapidity correlations, as well as the t-dependence of deep inelastic diffractive production of vector mesons. (orig.)

  7. Poromechanics Parameters of Fluid-Saturated Chemically Active Fibrous Media Derived from a Micromechanical Approach

    Science.gov (United States)

    Parthasarathy, Ranganathan; Singh, Viraj; Spencer, Paulette

    2014-01-01

    The authors have derived macroscale poromechanics parameters for chemically active saturated fibrous media by combining microstructure-based homogenization with Hill's volume averaging. The stress-strain relationship of the dry fibrous media is first obtained by considering the fiber behavior. The constitutive relationships applicable to saturated media are then derived in the poromechanics framework using Hill's Lemmas. The advantage of this approach is that the resultant continuum model assumes a form suited to study porous materials, while retaining the effect of discrete fiber deformation. As a result, the model is able to predict the influence of microscale phenomena such as fiber buckling on the overall behavior, and in particular, on the poromechanics constants. The significance of the approach is demonstrated using the effect of drainage and fiber nonlinearity on monotonic compressive stress-strain behavior. The model predictions conform to the experimental observations for articular cartilage. The method can potentially be extended to other porous materials such as bone, clays, foams, and concrete. PMID:25419475

  8. A novel approach to deterministic performance analysis of guidance loops with saturation

    NARCIS (Netherlands)

    Weiss, M.; Bucco, D.

    2011-01-01

    Since saturation often plays an important role in limiting the performance of guidance loops, performance analysis of guidance loops with saturation has been a popular subject of investigation. Most work so far has concentrated on the effect of saturation on stochastic performance of guidance loops,

  9. CGC/saturation approach for soft interactions at high energy: long range rapidity correlations

    Energy Technology Data Exchange (ETDEWEB)

    Gotsman, E.; Maor, U. [Tel Aviv University, Department of Particle Physics, School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Levin, E. [Tel Aviv University, Department of Particle Physics, School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Universidad Tecnica Federico Santa Maria and Centro Cientifico- Tecnologico de Valparaiso, Departemento de Fisica, Valparaiso (Chile)

    2015-11-15

    In this paper we continue our program to construct a model for high energy soft interactions that is based on the CGC/saturation approach. The main result of this paper is that we have discovered a mechanism that leads to large long range rapidity correlations and results in large values of the correlation function R(y{sub 1}, y{sub 2}) ≥ 1, which is independent of y{sub 1} and y{sub 2}. Such a behavior of the correlation function provides strong support for the idea that at high energies the system of partons that is produced is not only dense but also has strong attractive forces acting between the partons. (orig.)

  10. The Structure of Urease Activiation Complexes Examined by Flexibility Analysis, Mutagenesis, and Small-angle X-ray Scattering Approaches

    Energy Technology Data Exchange (ETDEWEB)

    Quiroz, Soledad [Michigan State University, East Lansing; Sukuru, Sai Chetan K. [Michigan State University, East Lansing; Hausinger, Robert P. [Michigan State University, East Lansing; Kuhn, Leslie A. [Michigan State University, East Lansing; Heller, William T [ORNL

    2008-01-01

    Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC){sub 3} induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle X-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complex that includes all of the accessory proteins. SAXS analyses of urease, (UreABC-UreD){sub 3}, and (UreABC-UreDF){sub 3} confirm that UreD and UreF bind near UreB at the periphery of the (UreAC){sub 3} structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF){sub 3} allows CO{sub 2} and nickel ions to gain access to the nascent active site.

  11. Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Mungalpara, J; Steen, A

    2014-01-01

    BACKGROUND AND PURPOSE: The cyclopentapeptide FC131 (cyclo(-L-Arg(1) -L-Arg(2) -L-2-Nal(3) -Gly(4) -D-Tyr(5) -)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4...... activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. KEY RESULTS: The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic......-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity. CONCLUSIONS AND IMPLICATIONS: Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes...

  12. Requirements for reliable determination of binding affinity constants by saturation analysis approach.

    Science.gov (United States)

    Borgna, Jean-Louis

    2004-12-01

    involving moderate underestimation) directly depends on the alpha value; (2) the magnitude of K underestimation mainly depends on the KN value; it is moderate (usually 100-fold), when KN > 10(2). In this case, the K underestimation is modulated by the alpha and kn values. Practical situations which afford high KN and thus might result in very marked underestimation of K are discussed. A single R dilution method is proposed to assess the validity of K determinations using the R saturation analysis approach.

  13. Multiphase fluid flow and subsequent geochemical transport invariably saturated fractured rocks: 1. Approaches

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Tianfu; Pruess, Karsten

    2000-08-08

    Reactive fluid flow and geochemical transport in unsaturated fractured rocks has received increasing attention for studies of contaminant transport, groundwater quality, waste disposal, acid mine drainage remediation, mineral deposits, sedimentary diagenesis, and fluid-rock interactions in hydrothermal systems. This paper presents methods for modeling geochemical systems that emphasize: (1) involvement of the gas phase in addition to liquid and solid phases in fluid flow, mass transport and chemical reactions, (2) treatment of physically and chemically heterogeneous and fractured rocks, (3) the effect of heat on fluid flow and reaction properties and processes, and (4) the kinetics of fluid-rock interaction. The physical and chemical process model is embodied in a system of partial differential equations for flow and transport, coupled to algebraic equations and ordinary differential equations for chemical interactions. For numerical solution, the continuum equations are discretized in space and time. Space discretization is based on a flexible integral finite difference approach that can use irregular gridding to model geologic structure; time is discretized fully implicitly as a first-order finite difference. Heterogeneous and fractured media are treated with a general multiple interacting continua method that includes double-porosity, dual-permeability, and multi-region models as special cases. A sequential iteration approach is used to treat the coupling between fluid flow and mass transport on the one hand, chemical reactions on the other. Applications of the methods developed here to variably saturated geochemical systems are presented in a companion paper (part 2, this issue).

  14. Stress transfer from pile group in saturated and unsaturated soil using theoretical and experimental approaches

    Directory of Open Access Journals (Sweden)

    al-Omari Raid R.

    2017-01-01

    Full Text Available Piles are often used in groups, and the behavior of pile groups under the applied loads is generally different from that of single pile due to the interaction of neighboring piles, therefore, one of the main objectives of this paper is to investigate the influence of pile group (bearing capacity, load transfer sharing for pile shaft and tip in comparison to that of single piles. Determination of the influence of load transfer from the pile group to the surrounding soil and the mechanism of this transfer with increasing the load increment on the tip and pile shaft for the soil in saturated and unsaturated state (when there is a negative pore water pressure. Different basic properties are used that is (S = 90%, γd = 15 kN / m3, S = 90%, γd = 17 kN / m3 and S = 60%, γd =15 kN / m3. Seven model piles were tested, these was: single pile (compression and pull out test, 2×1, 3×1, 2×2, 3×2 and 3×3 group. The stress was measured with 5 cm diameter soil pressure transducer positioned at a depth of 5 cm below the pile tip for all pile groups. The measured stresses below the pile tip using a soil pressure transducer positioned at a depth of 0.25L (where L is the pile length below the pile tip are compared with those calculated using theoretical and conventional approaches. These methods are: the conventional 2V:1H method and the method used the theory of elasticity. The results showed that the method of measuring the soil stresses with soil pressure transducer adopted in this study, gives in general, good results of stress transfer compared with the results obtained from the theoretical and conventional approaches.

  15. Fuzzy Saturated Output Feedback Tracking Control for Robot Manipulators: A Singular Perturbation Theory Based Approach

    National Research Council Canada - National Science Library

    Liu, Huashan; Hao, Kuangrong; Lai, Xiaobo

    2011-01-01

    To deal with the problem of the output feedback tracking (OFT) control with bounded torque inputs of robot manipulators, we propose a generalized fuzzy saturated OFT controller based on singular perturbation theory...

  16. Fuzzy Saturated Output Feedback Tracking Control for Robot Manipulators: A Singular Perturbation Theory Based Approach

    OpenAIRE

    Huashan Liu; Kuangrong Hao; Xiaobo Lai

    2011-01-01

    To deal with the problem of the output feedback tracking (OFT) control with bounded torque inputs of robot manipulators, we propose a generalized fuzzy saturated OFT controller based on singular perturbation theory. First, considering the fact that the output toque of joint actuators is limited, a general expression for a class of saturation functions is given to be applied in the control law. Second, to carry out the whole closed‐loop control with only position measurements, linear and...

  17. NDVI saturation adjustment: a new approach for improving cropland performance estimates in the Greater Platte River Basin, USA

    Science.gov (United States)

    Gu, Yingxin; Wylie, Bruce K.; Howard, Daniel M.; Phuyal, Khem P.; Ji, Lei

    2013-01-01

    In this study, we developed a new approach that adjusted normalized difference vegetation index (NDVI) pixel values that were near saturation to better characterize the cropland performance (CP) in the Greater Platte River Basin (GPRB), USA. The relationship between NDVI and the ratio vegetation index (RVI) at high NDVI values was investigated, and an empirical equation for estimating saturation-adjusted NDVI (NDVIsat_adjust) based on RVI was developed. A 10-year (2000–2009) NDVIsat_adjust data set was developed using 250-m 7-day composite historical eMODIS (expedited Moderate Resolution Imaging Spectroradiometer) NDVI data. The growing season averaged NDVI (GSN), which is a proxy for ecosystem performance, was estimated and long-term NDVI non-saturation- and saturation-adjusted cropland performance (CPnon_sat_adjust, CPsat_adjust) maps were produced over the GPRB. The final CP maps were validated using National Agricultural Statistics Service (NASS) crop yield data. The relationship between CPsat_adjust and the NASS average corn yield data (r = 0.78, 113 samples) is stronger than the relationship between CPnon_sat_adjust and the NASS average corn yield data (r = 0.67, 113 samples), indicating that the new CPsat_adjust map reduces the NDVI saturation effects and is in good agreement with the corn yield ground observations. Results demonstrate that the NDVI saturation adjustment approach improves the quality of the original GSN map and better depicts the actual vegetation conditions of the GPRB cropland systems.

  18. Applying of modern approach to evaluation of initial hydrocarbon reserves at the edge of saturation pressure

    Directory of Open Access Journals (Sweden)

    Віталій Ігорович Коваль

    2016-07-01

    Full Text Available The article deals the features for evaluation of initial saturated hydrocarbon reserves, which developed as the depletion of reservoir energy and the maintenance of reservoir pressure. A new method of material balance is used with accounting of the gravity distribution of reservoir system components, extraction of each component of reservoir fluids, change of their physical properties in the development of deposits

  19. 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.

  20. Processes, mechanisms, parameters, and modeling approaches for partially saturated flow in soil and rock media; Yucca Mountain Site Characterization Project

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.S.Y.; Narasimhan, T.N. [Lawrence Berkeley Lab., CA (United States)

    1993-06-01

    This report discusses conceptual models and mathematical equations, analyzes distributions and correlations among hydrological parameters of soils and tuff, introduces new path integration approaches, and outlines scaling procedures to model potential-driven fluid flow in heterogeneous media. To properly model the transition from fracture-dominated flow under saturated conditions to matrix-dominated flow under partially saturated conditions, characteristic curves and permeability functions for fractures and matrix need to be improved and validated. Couplings from two-phase flow, heat transfer, solute transport, and rock deformation to liquid flow are also important. For stochastic modeling of alternating units of welded and nonwelded tuff or formations bounded by fault zones, correlations and constraints on average values of saturated permeability and air entry scaling factor between different units need to be imposed to avoid unlikely combinations of parameters and predictions. Large-scale simulations require efficient and verifiable numerical algorithms. New path integration approaches based on postulates of minimum work and mass conservation to solve flow geometry and potential distribution simultaneously are introduced. This verifiable integral approach, together with fractal scaling procedures to generate statistical realizations with parameter distribution, correlation, and scaling taken into account, can be used to quantify uncertainties and generate the cumulative distribution function for groundwater travel times.

  1. The Saturation+ Approach to Behavior Change: Case Study of a Child Survival Radio Campaign in Burkina Faso

    Science.gov (United States)

    Murray, Joanna; Remes, Pieter; Ilboudo, Rita; Belem, Mireille; Salouka, Souleymane; Snell, Will; Wood, Cathryn; Lavoie, Matthew; Deboise, Laurent; Head, Roy

    2015-01-01

    A 35-month cluster randomized controlled trial was conducted in Burkina Faso to test whether a radio campaign focused on child health, broadcast between March 2012 and January 2015, could reduce under-5 mortality. This paper describes the design and implementation of the mass media intervention in detail, including the Saturation+ principles that underpinned the approach, the creative process, the lessons learned, and recommendations for implementing this intervention at scale. The Saturation+ approach focuses on the 3 core principles of saturation (ensuring high exposure to campaign messages), science (basing campaign design on data and modeling), and stories (focusing the dramatic climax on the target behavior) to maximize the impact of behavior change campaigns. In Burkina Faso, creative partnerships with local radio stations helped us obtain free airtime in exchange for training and investing in alternative energy supplies to solve frequent energy problems faced by the stations. The campaign used both short spots and longer drama formats, but we consider the short spots as a higher priority to retain during scale-up, as they are more cost-effective than longer formats and have the potential to ensure higher exposure of the population to the messages. The implementation research synthesized in this paper is designed to enable the effective adoption and integration of evidence-based behavior change communication interventions into health care policy and practice. PMID:26681703

  2. A new two-stage approach for predicting the soil water characteristic from saturation to oven-dryness

    DEFF Research Database (Denmark)

    Karup Jensen, Dan; Tuller, Markus; de Jonge, Lis Wollesen

    2015-01-01

    The soil water characteristic (SWC) is one of the most important properties required for understanding plant-soil relationships and is crucial for modeling gas and water flow in soils. Measuring the SWC is laborious, and until now the dry-region soil water retention has commonly been excluded due...... with clay and organic carbon contents ranging from 0.01 to 0.52 kg kg-1 and 0 to 0.07 kg kg-1, respectively, were used for the model development. Measuring the SWC from saturation to oven-dryness was accomplished with Tempe cells and a water vapor sorption analyzer. The model was subsequently tested...... to slow and inaccurate measurements. Hence, models applied to predict the SWC consequently exclude the dry region and are often only applicable for specific soil textural classifications. The present study proposes a new two-step approach to prediction of the continuous SWC from saturation to oven dryness...

  3. 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.

  4. 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

  5. Saturated hydraulic conductivity and biofilms: A theoretical approach linking pore and pedon scale

    Science.gov (United States)

    Richter, M.; Moenickes, S.; Richter, O.; Schröder, T.

    2012-04-01

    The fate of active substances in the soil environment is shaped by soil physical properties as well as microbial life. Microorganisms degrading those substances occur in soil pores either in suspension or as biofilms on grain surfaces. At the same scale, i.e. pore scale, the soil physical properties texture, density, porosity, and water content have an impact on transport behaviour of active substances. Macroscopic parameters describe these processes at pedon scale; e.g. hydraulic conductivity summarizes the effect of named pore scale parameters. Narsilio et al. [2009] derived a relationship between the saturated hydraulic conductivity and pore scale water velocity fields based on Navier-Stokes equation for incompressible fluids. However, they did not analyse the influence of heterogeneity and microbial activity, whereas microorganisms, especially biofilms, do have an impact on hydraulic conductivity [Vandevivere and Baveye, 1992]. Biofilms alter the pore geometry while growing. This alteration directly influences the soil water flow field and hence the convective transport of active substances. Here, we present a way to couple the saturated hydraulic conductivity at macro scale to biomass population dynamics and pore space. The hydraulic conductivity will be analysed with regard to heterogeneous soils. The model combining fluid flow, reactive transport, and biofilm dynamics is applied to investigate the degradation and transport behaviour of pesticides in heterogeneous soils.

  6. Escape from flatland: increasing saturation as an approach to improving clinical success.

    Science.gov (United States)

    Lovering, Frank; Bikker, Jack; Humblet, Christine

    2009-11-12

    The medicinal chemistry community has become increasingly aware of the value of tracking calculated physical properties such as molecular weight, topological polar surface area, rotatable bonds, and hydrogen bond donors and acceptors. We hypothesized that the shift to high-throughput synthetic practices over the past decade may be another factor that may predispose molecules to fail by steering discovery efforts toward achiral, aromatic compounds. We have proposed two simple and interpretable measures of the complexity of molecules prepared as potential drug candidates. The first is carbon bond saturation as defined by fraction sp(3) (Fsp(3)) where Fsp(3) = (number of sp(3) hybridized carbons/total carbon count). The second is simply whether a chiral carbon exists in the molecule. We demonstrate that both complexity (as measured by Fsp(3)) and the presence of chiral centers correlate with success as compounds transition from discovery, through clinical testing, to drugs. In an attempt to explain these observations, we further demonstrate that saturation correlates with solubility, an experimental physical property important to success in the drug discovery setting.

  7. Pyrite oxidation in saturated and Unsaturated Porous Media Flow: AComparison of alternative mathematical modeling approaches

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Tianfu; White, Stephen P.; Pruess, Karsten

    1998-02-15

    Pyrite (FeS{sub 2}) is one of the most common naturally occurring minerals that is present in many subsurface environments. It plays an important role in the genesis of enriched ore deposits through weathering reactions, is the most abundant sulfide mineral in many mine tailings, and is the primary source of acid drainage from mines and waste rock piles. The pyrite oxidation reaction serves as a prototype for oxidative weathering processes with broad significance for geoscientific, engineering, and environmental applications. Mathematical modeling of these processes is extremely challenging because aqueous concentrations of key species vary over an enormous range, oxygen inventory and supply are typically small in comparison to pyrite inventory, and chemical reactions are complex, involving kinetic control and microbial catalysis. We present the mathematical formulation of a general multi-phase advective-diffusive reactive transport model for redox processes. Two alternative implementations were made in the TOUGHREACT and TOUGH2-CHEM simulation codes which use sequential iteration and simultaneous solution, respectively. The simulators are applied to reactive consumption of pyrite in (1) saturated flow of oxidizing water, and (2) saturated-unsaturated flow in which oxygen transport occurs in both aqueous and gas phases. Geochemical evolutions predicted from different process models are compared, and issues of numerical accuracy and efficiency are discussed.

  8. Central venous oxygen saturation and thoracic admittance during dialysis: new approaches to hemodynamic monitoring

    DEFF Research Database (Denmark)

    Cordtz, J.; Olde, B.; Solem, K.

    2008-01-01

    events are reflected in the central venous oxygen saturation (ScO(2)) and thoracic admittance (TA) during dialysis. Twenty ambulatory HD patients, 11 hypotension prone (HP) and 9 hypotension resistant, with central vascular access, were monitored during 3 HD sessions each. ScO(2), TA, finger blood...... in ScO(2) and TA correlated much closer than did changes in ScO(2) and DeltaBV (r=0.43 and 0.18, respectively). Our results suggest that an intradialytic decrease in cardiac output, as reflected by a fall in ScO(2), is a common feature to HD patients prone to IDH. In patients using a central vascular...... access, ScO(2) and TA measurements may be more specific to the pathophysiologic events preceding IDH than DeltaBV-the current standard monitoring method Udgivelsesdato: 2008/7...

  9. Intelligent Approach for Analysis of Respiratory Signals and Oxygen Saturation in the Sleep Apnea/Hypopnea Syndrome

    Science.gov (United States)

    Moret-Bonillo, Vicente; Alvarez-Estévez, Diego; Fernández-Leal, Angel; Hernández-Pereira, Elena

    2014-01-01

    This work deals with the development of an intelligent approach for clinical decision making in the diagnosis of the Sleep Apnea/Hypopnea Syndrome, SAHS, from the analysis of respiratory signals and oxygen saturation in arterial blood, SaO2. In order to accomplish the task the proposed approach makes use of different artificial intelligence techniques and reasoning processes being able to deal with imprecise data. These reasoning processes are based on fuzzy logic and on temporal analysis of the information. The developed approach also takes into account the possibility of artifacts in the monitored signals. Detection and characterization of signal artifacts allows detection of false positives. Identification of relevant diagnostic patterns and temporal correlation of events is performed through the implementation of temporal constraints. PMID:25035712

  10. Development of theoretical oxygen saturation calibration curve based on optical density ratio and optical simulation approach

    Science.gov (United States)

    Jumadi, Nur Anida; Beng, Gan Kok; Ali, Mohd Alauddin Mohd; Zahedi, Edmond; Morsin, Marlia

    2017-09-01

    The implementation of surface-based Monte Carlo simulation technique for oxygen saturation (SaO2) calibration curve estimation is demonstrated in this paper. Generally, the calibration curve is estimated either from the empirical study using animals as the subject of experiment or is derived from mathematical equations. However, the determination of calibration curve using animal is time consuming and requires expertise to conduct the experiment. Alternatively, an optical simulation technique has been used widely in the biomedical optics field due to its capability to exhibit the real tissue behavior. The mathematical relationship between optical density (OD) and optical density ratios (ODR) associated with SaO2 during systole and diastole is used as the basis of obtaining the theoretical calibration curve. The optical properties correspond to systolic and diastolic behaviors were applied to the tissue model to mimic the optical properties of the tissues. Based on the absorbed ray flux at detectors, the OD and ODR were successfully calculated. The simulation results of optical density ratio occurred at every 20 % interval of SaO2 is presented with maximum error of 2.17 % when comparing it with previous numerical simulation technique (MC model). The findings reveal the potential of the proposed method to be used for extended calibration curve study using other wavelength pair.

  11. Hydromechanical behavior of a quasi-saturated compacted soils on drying-wetting paths-experimental and numerical approaches

    Directory of Open Access Journals (Sweden)

    Andriantrehina Soanarivo Rinah

    2016-01-01

    Full Text Available This paper presents an experimental and numerical investigation funded by the French National Project “Terredurable”, which is devoted to the study of soils in quasi-saturated state. The experimental study is focused on the behavior of compacted soils on drying-wetting paths and the macroscopic effect of the drying path on shrinkage and cracking. Furthermore, a protocol for image analysis of crack in drying tests was developed. Two approaches are used for the measurement of surface strains and identification of the ultimate stress before the formation of the first crack, using VIC-2D software, and for the monitoring of crack evolution, using ImageJ software. The aim of the numerical approach is to reproduce the drying experiments with a finite difference code (FLAC 3D, in order to understand the stress conditions that can explain crack initiation, without modeling the crack formation itself.

  12. 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

  13. Simulation of saturated and unsaturated flow in karst systems at catchment scale using a double continuum approach

    Directory of Open Access Journals (Sweden)

    J. Kordilla

    2012-10-01

    Full Text Available The objective of this work is the simulation of saturated and unsaturated flow in a karstified aquifer using a double continuum approach. The HydroGeoSphere code (Therrien et al., 2006 is employed to simulate spring discharge with the Richards equations and van Genuchten parameters to represent flow in the (1 fractured matrix and (2 conduit continuum coupled by a linear exchange term. Rapid vertical small-scale flow processes in the unsaturated conduit continuum are accounted for by applying recharge boundary conditions at the bottom of the saturated model domain. An extensive sensitivity analysis is performed on single parameters as well as parameter combinations. The transient hydraulic response of the karst spring is strongly controlled by the matrix porosity as well as the van Genuchten parameters of the unsaturated matrix, which determine the head dependent inter-continuum water transfer when the conduits are draining the matrix. Sensitivities of parameter combinations partially reveal a non-linear dependence over the parameter space. This can be observed for parameters not belonging to the same continuum as well as combinations, which involve the exchange parameter, showing that results of the double continuum model may depict a certain degree of ambiguity. The application of van Genuchten parameters for simulation of unsaturated flow in karst systems is critically discussed.

  14. Saturated Switching Systems

    CERN Document Server

    Benzaouia, Abdellah

    2012-01-01

    Saturated Switching Systems treats the problem of actuator saturation, inherent in all dynamical systems by using two approaches: positive invariance in which the controller is designed to work within a region of non-saturating linear behaviour; and saturation technique which allows saturation but guarantees asymptotic stability. The results obtained are extended from the linear systems in which they were first developed to switching systems with uncertainties, 2D switching systems, switching systems with Markovian jumping and switching systems of the Takagi-Sugeno type. The text represents a thoroughly referenced distillation of results obtained in this field during the last decade. The selected tool for analysis and design of stabilizing controllers is based on multiple Lyapunov functions and linear matrix inequalities. All the results are illustrated with numerical examples and figures many of them being modelled using MATLAB®. Saturated Switching Systems will be of interest to academic researchers in con...

  15. 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....

  16. A new stochastic hydraulic conductivity approach for modeling one-dimensional vertical flow in variably saturated porous media.

    Science.gov (United States)

    Vrettas, M. D.; Fung, I. Y.

    2014-12-01

    The degree of carbon climate feedback by terrestrial ecosystems is intimately tied to the availability of moisture for photosynthesis, transpiration and decomposition. The vertical distribution of subsurface moisture and its accessibility for evapotranspiration is a key determinant of the fate of ecosystems and their feedback on the climate system. A time series of five years of high frequency (every 30 min) observations of water table at a research site in Northern California shows that the water tables, 18 meters below the surface, can respond in less than 8 hours to the first winter rains, suggesting very fast flow through micro-pores and fractured bedrock. Not quite as quickly as the water table rises after a heavy rain, the elevated water level recedes, contributing to down-slope flow and stream flow. The governing equation of our model uses the well-known Richards' equation, which is a non-linear PDE, derived by applying the continuity requirement to Darcy's law. The most crucial parameter of this PDE is the hydraulic conductivity K(θ), which describes the speed at which water can move in the underground. We specify a saturation profile as a function of depth (i.e. Ksat(z)) and allow K(θ) to vary not only with the soil moisture saturation but also include a stochastic component which mimics the effects of fracture flow and other naturally occurring heterogeneity, that is evident in the subsurface. A large number of Monte Carlo simulation are performed in order to identify optimal settings for the new model, as well as analyze the results of this new approach on the available data. Initial findings from this exploratory work are encouraging and the next steps include testing this new stochastic approach on data from other sites and also apply ensemble based data assimilation algorithms in order to estimate model parameters with the available measurements.

  17. 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.

  18. Experimental and numerical approaches of the hydro-mechanical behaviour of a quasi-saturated compacted clayey soil

    Directory of Open Access Journals (Sweden)

    Li Zhong-Sen

    2016-01-01

    Full Text Available The present research is funded by the French National Project « TerreDurable », which is dedicated to the study of soils in quasi-saturated conditions (close to saturation for the analysis of stability and settlement of earth structures such as embankment, dams. A global presentation of the drying-wetting test shows the volume change, air entry and soil-water characteristics of the soil at slurry and oven-dried conditions. Unsaturated undrained triaxial test was carried out in order to investigate the variation of pore-water pressure from quasi-saturated domain to saturation. The experimental results of the triaxial test are then modeled using a two-dimensional explicit finite difference program (Flac 2D. A constitutive law developed in the TerreDurable project allows better understanding the behaviour of quasi-saturated soils using the water retention curve of quasi-saturated domain proposed by Boutonnier (2007, 2010. A simple effective stress model is used (Cam Clay by taking into account both the suction and the compressibility of equivalent fluid (water + air. The results from numerical calculation and experimental measurements are compared.

  19. Direct comparison of the hygroscopic properties of ammonium sulfate and sodium chloride aerosol at relative humidities approaching saturation.

    Science.gov (United States)

    Walker, Jim S; Wills, Jon B; Reid, Jonathan P; Wang, Liangyu; Topping, David O; Butler, Jason R; Zhang, Yun-Hong

    2010-12-09

    Holographic optical tweezers are used to make comparative measurements of the hygroscopic properties of single component aqueous aerosol containing sodium chloride and ammonium sulfate over a range of relative humidity from 84% to 96%. The change in RH over the course of the experiment is monitored precisely using a sodium chloride probe droplet with accuracy better than ±0.09%. The measurements are used to assess the accuracy of thermodynamic treatments of the relationship between water activity and solute mass fraction with particular attention focused on the dilute solute limit approaching saturation vapor pressure. The consistency of the frequently used Clegg-Brimblecombe-Wexler (CBW) treatment for predicting the hygroscopic properties of sodium chloride and ammonium sulfate aerosol is confirmed. Measurements of the equilibrium size of ammonium sulfate aerosol are found to agree with predictions to within an uncertainty of ±0.2%. Given the accuracy of treating equilibrium composition, the inconsistencies highlighted in recent calibration measurements of critical supersaturations of sodium chloride and ammonium sulfate aerosol cannot be attributed to uncertainties associated with the thermodynamic predictions and must have an alternative origin. It is concluded that the CBW treatment can allow the critical supersaturation to be estimated for sodium chloride and ammonium sulfate aerosol with an accuracy of better than ±0.002% in RH. This corresponds to an uncertainty of ≤1% in the critical supersaturation for typical supersaturations of 0.2% and above. This supports the view that these systems can be used to accurately calibrate instruments that measure cloud condensation nuclei concentrations at selected supersaturations. These measurements represent the first study in which the equilibrium properties of two particles of chemically distinct composition have been compared simultaneously and directly alongside each other in the same environment.

  20. 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.

  1. 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.

  2. Genomic Approaches to DNA repair and Mutagenesis

    OpenAIRE

    Wyrick, John J.; Roberts, Steven A.

    2015-01-01

    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 processin...

  3. Facts about saturated fats

    Science.gov (United States)

    ... fat diary with low-fat or nonfat milk, yogurt, and cheese. Eat more fruits, vegetables, whole grains, and other foods with low or no saturated fat. Alternative Names Cholesterol - saturated fat; Atherosclerosis - saturated fat; Hardening of the ...

  4. Saturated fat (image)

    Science.gov (United States)

    Saturated fat can raise blood cholesterol and can put you at risk for heart disease and stroke. You should ... limit any foods that are high in saturated fat. Sources of saturated fat include whole-milk dairy ...

  5. Deriving rock transport properties from NMR relaxation experiments on partially de-saturated rocks using an inverse modelling approach based on the triangular pore model

    Science.gov (United States)

    Nordlund, C. L.; Mohnke, O.; Klitzsch, N.

    2013-12-01

    Nuclear magnetic resonance (NMR) is a method used over a wide field of geophysical applications e.g. petrophysics, logging or hydrogeology, to non-destructively and quickly determine transport and storage properties of rocks and soils. Measured NMR amplitudes indicate the rock's fluid content, whereas the NMR relaxation rates are used to derive pore size distributions and permeability as they are related to physiochemical properties of the rock-fluid interface, i.e. surface relaxivity. The aim of this work is to utilize triangular pore shapes in the inverse modeling of NMR relaxation data in order to better predict the water retention curves. Therefore, an inverse modeling scheme (Mohnke et al.; this conference) to derive NMR surface relaxivity along with pore radii distributions are determined has been adapted on the basis of triangular pore bundle models. . This approach then offers the advantage of having the triangular pores be partially saturated, thus accounting for a residual water content trapped in pore corners during drainage and imbibition (Tuller and Or, 2001). Additionally, besides the trapped water the presence of a water film at the pore walls is accounted for in the modeled and inverted NMR relaxation time distributions and thus allows to more accurately describe laboratory measurement results. Numerical parameter studies were carried out to assess the influence of pore shapes and contact angles on both, transport and NMR properties. Inverse models derived from laboratory NMR data on partially saturated reservoir rocks are in a good agreement with measured water retention data.

  6. Failure of fluid-saturated granular materials: a unified approach to capture diffuse and localized instability mechanisms

    Science.gov (United States)

    Mihalache, Constance; Buscarnera, Giuseppe

    2013-04-01

    possible occurrence of diffuse and localized failure mechanisms. While the mathematical strategy to calculate the failure indices is discussed in a general manner, a particular application is presented, focused on the simulation of the mechanical response of loose, saturated sands. Failure mechanisms are simulated under axisymmetric, plane-strain and simple shear conditions. For each of these cases, the indices are used to infer whether a failure mode is more likely to occur in a localized or homogeneous manner. At this reference, the role of drainage conditions is specifically explored. The results of numerical analyses are compared to data from classical experiments available in the literature. Finally, the theory is used to explain the mechanisms of failure that may have provoked a series of underwater flow failures in the shallow sand veneers located along the banks of the Jamuna River, in Bangladesh [Hight et al, 1998] The analyses conducted in this work elucidate the remarkable dependence of geomaterial stability on both kinematic conditions and pore-fluid constraints. In particular, the links between generalized failure mechanisms and the onset of landslides has been elucidated through numerical simulations. The results obtained by our analyses therefore provide a simple and consistent strategy to unify the mathematical description of the material instabilities that are involved in a variety of failure mechanisms of geological settings. Buscarnera G., Dattola G., Di Prisco C. (2011) " Controllability, uniqueness and existence of the incremental response: A mathematical criterion for elastoplastic constitutive laws", International Journal of Solids and Structures, 48 (13), pp. 1867-1878. Hight, D. W., Georgiannou, V. N., Martin, P. L., and Mundegar, A. K. (1998) "Flow slides in micaceous sand." Problematic soils, Yanagisawa, E., Moroto, N., and Mitachi, T., eds., Baklema, Rotterdam, Sendai, Japan, pp. 945-958.

  7. 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.

  8. An Accurate CT Saturation Classification Using a Deep Learning Approach Based on Unsupervised Feature Extraction and Supervised Fine-Tuning Strategy

    Directory of Open Access Journals (Sweden)

    Muhammad Ali

    2017-11-01

    Full Text Available Current transformer (CT saturation is one of the significant problems for protection engineers. If CT saturation is not tackled properly, it can cause a disastrous effect on the stability of the power system, and may even create a complete blackout. To cope with CT saturation properly, an accurate detection or classification should be preceded. Recently, deep learning (DL methods have brought a subversive revolution in the field of artificial intelligence (AI. This paper presents a new DL classification method based on unsupervised feature extraction and supervised fine-tuning strategy to classify the saturated and unsaturated regions in case of CT saturation. In other words, if protection system is subjected to a CT saturation, proposed method will correctly classify the different levels of saturation with a high accuracy. Traditional AI methods are mostly based on supervised learning and rely heavily on human crafted features. This paper contributes to an unsupervised feature extraction, using autoencoders and deep neural networks (DNNs to extract features automatically without prior knowledge of optimal features. To validate the effectiveness of proposed method, a variety of simulation tests are conducted, and classification results are analyzed using standard classification metrics. Simulation results confirm that proposed method classifies the different levels of CT saturation with a remarkable accuracy and has unique feature extraction capabilities. Lastly, we provided a potential future research direction to conclude this paper.

  9. 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

  10. 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.

  11. Prediction of gas hydrate saturation throughout the seismic section in Krishna Godavari basin using multivariate linear regression and multi-layer feed forward neural network approach

    Digital Repository Service at National Institute of Oceanography (India)

    Singh, Y.; Nair, R.R.; Singh, H.; Datta, P.; Jaiswal, P.; Dewangan, P.; Ramprasad, T.

    Stepwise linear regression, multi-layer feed forward neural (MLFN) network method was used to predict the 2D distribution of P-wave velocity, resistivity, porosity, and gas hydrate saturation throughout seismic section NGHP-01 in the Krishna...

  12. 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.

  13. Theory of graphene saturable absorption

    Science.gov (United States)

    Marini, A.; Cox, J. D.; García de Abajo, F. J.

    2017-03-01

    Saturable absorption is a nonperturbative nonlinear optical phenomenon that plays a pivotal role in the generation of ultrafast light pulses. Here we show that this effect emerges in graphene at unprecedentedly low light intensities, thus opening avenues to new nonlinear physics and applications in optical technology. Specifically, we theoretically investigate saturable absorption in extended graphene by developing a semianalytical nonperturbative single-particle approach, describing electron dynamics in the atomically-thin material using the two-dimensional Dirac equation for massless Dirac fermions, which is recast in the form of generalized Bloch equations. By solving the electron dynamics nonperturbatively, we account for both interband and intraband contributions to the intensity-dependent saturated conductivity and conclude that the former dominates regardless of the intrinsic doping state of the material. We obtain results in qualitative agreement with atomistic quantum-mechanical simulations of graphene nanoribbons including electron-electron interactions, finite-size, and higher-band effects. Remarkably, such effects are found to affect mainly the linear absorption, while the predicted saturation intensities are in good quantitative agreement in the limit of extended graphene. Additionally, we find that the modulation depth of saturable absorption in graphene can be electrically manipulated through an externally applied gate voltage. Our results are relevant for the development of graphene-based optoelectronic devices, as well as for applications in mode-locking and random lasers.

  14. 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...

  15. 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.

  16. 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.

  17. On the role of fluids in stick-slip dynamics of saturated granular fault gouge using a coupled computational fluid dynamics-discrete element approach

    Science.gov (United States)

    Dorostkar, Omid; Guyer, Robert A.; Johnson, Paul A.; Marone, Chris; Carmeliet, Jan

    2017-05-01

    The presence of fault gouge has considerable influence on slip properties of tectonic faults and the physics of earthquake rupture. The presence of fluids within faults also plays a significant role in faulting and earthquake processes. In this paper, we present 3-D discrete element simulations of dry and fluid-saturated granular fault gouge and analyze the effect of fluids on stick-slip behavior. Fluid flow is modeled using computational fluid dynamics based on the Navier-Stokes equations for an incompressible fluid and modified to take into account the presence of particles. Analysis of a long time train of slip events shows that the (1) drop in shear stress, (2) compaction of granular layer, and (3) the kinetic energy release during slip all increase in magnitude in the presence of an incompressible fluid, compared to dry conditions. We also observe that on average, the recurrence interval between slip events is longer for fluid-saturated granular fault gouge compared to the dry case. This observation is consistent with the occurrence of larger events in the presence of fluid. It is found that the increase in kinetic energy during slip events for saturated conditions can be attributed to the increased fluid flow during slip. Our observations emphasize the important role that fluid flow and fluid-particle interactions play in tectonic fault zones and show in particular how discrete element method (DEM) models can help understand the hydromechanical processes that dictate fault slip.

  18. 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.

  19. 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.

  20. 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.

  1. A new approach to assess the dependency of extant half-saturation coefficients on maximum process rates and estimate intrinsic coefficients.

    Science.gov (United States)

    Shaw, A; Takács, I; Pagilla, K R; Murthy, S

    2013-10-15

    The Monod equation is often used to describe biological treatment processes and is the foundation for many activated sludge models. The Monod equation includes a "half-saturation coefficient" to describe the effect of substrate limitations on the process rate and it is customary to consider this parameter to be a constant for a given system. The purpose of this study was to develop a methodology, and its use to show that the half-saturation coefficient for denitrification is not constant but is in fact a function of the maximum denitrification rate. A 4-step procedure is developed to investigate the dependency of half-saturation coefficients on the maximum rate and two different models are used to describe this dependency: (a) an empirical linear model and (b) a deterministic model based on Fick's law of diffusion. Both models are proved better for describing denitrification kinetics than assuming a fixed K(NO3) at low nitrate concentrations. The empirical model is more utilitarian whereas the model based on Fick's law has a fundamental basis that enables the intrinsic K(NO3) to be estimated. In this study data was analyzed from 56 denitrification rate tests and it was found that the extant K(NO3) varied between 0.07 mgN/L and 1.47 mgN/L (5th and 95th percentile respectively) with an average of 0.47 mgN/L. In contrast to this, the intrinsic K(NO3) estimated for the diffusion model was 0.01 mgN/L which indicates that the extant K(NO3) is greatly influenced by, and mostly describes, diffusion limitations. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Venous oxygen saturation.

    Science.gov (United States)

    Hartog, Christiane; Bloos, Frank

    2014-12-01

    Early detection and rapid treatment of tissue hypoxia are important goals. Venous oxygen saturation is an indirect index of global oxygen supply-to-demand ratio. Central venous oxygen saturation (ScvO2) measurement has become a surrogate for mixed venous oxygen saturation (SvO2). ScvO2 is measured by a catheter placed in the superior vena cava. After results from a single-center study suggested that maintaining ScvO2 values >70% might improve survival rates in septic patients, international practice guidelines included this target in a bundle strategy to treat early sepsis. However, a recent multicenter study with >1500 patients found that the use of central hemodynamic and ScvO2 monitoring did not improve long-term survival when compared to the clinical assessment of the adequacy of circulation. It seems that if sepsis is recognized early, a rapid initiation of antibiotics and adequate fluid resuscitation are more important than measuring venous oxygen saturation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Metamaterial saturable absorber mirror.

    Science.gov (United States)

    Dayal, Govind; Ramakrishna, S Anantha

    2013-02-01

    We propose a metamaterial saturable absorber mirror at midinfrared wavelengths that can show a saturation of absorption with intensity of incident light and switch to a reflecting state. The design consists of an array of circular metallic disks separated by a thin film of vanadium dioxide (VO(2)) from a continuous metallic film. The heating due to the absorption in the absorptive state causes the VO(2) to transit to a metallic phase from the low temperature insulating phase. The metamaterial switches from an absorptive state (R≃0.1%) to a reflective state (R>95%) for a specific threshold intensity of the incident radiation corresponding to the phase transition of VO(2), resulting in the saturation of absorption in the metamaterial. The computer simulations show over 99.9% peak absorbance, a resonant bandwidth of about 0.8 μm at 10.22 μm wavelengths, and saturation intensity of 140 mW cm(-2) for undoped VO(2) at room temperature. We also carried out numerical simulations to investigate the effects of localized heating and temperature distribution by solving the heat diffusion problem.

  5. 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 ...

  6. 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.

  7. High saturation magnetization of γ-Fe2O3 nano-particles by a facile one-step synthesis approach.

    Science.gov (United States)

    Cao, Derang; Li, Hao; Pan, Lining; Li, Jianan; Wang, Xicheng; Jing, Panpan; Cheng, Xiaohong; Wang, Wenjie; Wang, Jianbo; Liu, Qingfang

    2016-09-01

    We have demonstrated the synthesis of γ-Fe2O3 nano-particles through a facile and novel calcination process in the air. There is no pH regulation, gas atmosphere, additive, centrifugation or other complicated procedures during the preparing process. A detailed formation process of the nano-particles is proposed, and DMF as a polar solvent may slower the reaction process of calcination. The structures, morphologies, and magnetic properties of γ-Fe2O3 nano-particles were investigated systematically, and the pure γ-Fe2O3 nano-particles obtained at 200 °C display uniform morphology good magnetic property. The saturation magnetization of obtained pure γ-Fe2O3 is about 74 emu/g, which is comparable with bulk material (76 emu/g) and larger than other results. In addition, the photocatalytic activity for degradation of methylene blue is also studied, which shows proper photocatalytic activity.

  8. High saturation magnetization of γ-Fe2O3 nano-particles by a facile one-step synthesis approach

    Science.gov (United States)

    Cao, Derang; Li, Hao; Pan, Lining; Li, Jianan; Wang, Xicheng; Jing, Panpan; Cheng, Xiaohong; Wang, Wenjie; Wang, Jianbo; Liu, Qingfang

    2016-01-01

    We have demonstrated the synthesis of γ-Fe2O3 nano-particles through a facile and novel calcination process in the air. There is no pH regulation, gas atmosphere, additive, centrifugation or other complicated procedures during the preparing process. A detailed formation process of the nano-particles is proposed, and DMF as a polar solvent may slower the reaction process of calcination. The structures, morphologies, and magnetic properties of γ-Fe2O3 nano-particles were investigated systematically, and the pure γ-Fe2O3 nano-particles obtained at 200 °C display uniform morphology good magnetic property. The saturation magnetization of obtained pure γ-Fe2O3 is about 74 emu/g, which is comparable with bulk material (76 emu/g) and larger than other results. In addition, the photocatalytic activity for degradation of methylene blue is also studied, which shows proper photocatalytic activity. PMID:27581732

  9. Toward the analysis of the Petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D Floral organ identity functions require SEPALLATA-Like MADS box genes in Petunia

    NARCIS (Netherlands)

    Vandenbussche, M.; Zethof, J.; Souer, E.; Koes, R.; Tornielli, G.B.; Pezzotti, M.; Ferrario, S.I.T.; Angenent, G.C.; Gerats, T.

    2003-01-01

    We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and

  10. Saturated and trans fats

    National Research Council Canada - National Science Library

    Shader, Richard I

    2014-01-01

    ... Original Pancake Mix plus ingredients suggested by the recipe: 2 g saturated fat (SF) and no trans fatty acids or trans fat (TFA); bacon, Oscar Mayer Lower Sodium Bacon: 2.5 g SF and no TFA; sausages, Jimmy Dean Original Pork Sausage Links: 8 g SF and no TFA; potatoes, Ore-Ida Mini Tater Tots: 2 g SF and no TFA; and nondairy creamer, Nestlé Coffee-...

  11. 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.

  12. 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.

  13. Saturation in nuclei

    CERN Document Server

    Lappi, T

    2010-01-01

    This talk discusses some recent studies of gluon saturation in nuclei. We stress the connection between the initial condition in heavy ion collisions and observables in deep inelastic scattering (DIS). The dominant degree of freedom in the small x nuclear wavefunction is a nonperturbatively strong classical gluon field, which determines the initial condition for the glasma fields in the initial stages of a heavy ion collision. A correlator of Wilson lines from the same classical fields, known as the dipole cross section, can be used to compute many inclusive and exclusive observables in DIS.

  14. CGC/saturation approach for high energy soft interactions: 'soft' Pomeron structure and v{sub n} in hadron and nucleus collisions from Bose-Einstein correlations

    Energy Technology Data Exchange (ETDEWEB)

    Gotsman, E.; Maor, U. [Tel Aviv University, Department of Particle Physics School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Levin, E. [Tel Aviv University, Department of Particle Physics School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Science, Tel Aviv (Israel); Universidad Tecnica Federico Santa Maria and Centro Cientifico-Tecnologico de Valparaiso, Departemento de Fisica, Valparaiso (Chile)

    2016-11-15

    In the framework of our model of soft interactions at high energy based on the CGC/saturation approach, we show that Bose-Einstein correlations of identical gluons lead to large values of v{sub n}. We demonstrate how three dimensional scales of high energy interactions, hadron radius, typical size of the wave function in diffractive production of small masses (size of the constituent quark), and the saturation momentum, influence the values of BE correlations, and in particular, the values of v{sub n}. Our calculation shows that the structure of the 'dressed' Pomeron leads to values of v{sub n} which are close to experimental values for proton-proton scattering, 20 % smaller than the observed values for proton-lead collisions and close to lead-lead collisions for 0-10 % centrality. Bearing this result in mind, we conclude that it is premature to consider that the appearance of long range rapidity azimuthal correlations are due only to the hydrodynamical behaviour of the quark-gluon plasma. (orig.)

  15. 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.

  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. 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.

  18. 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.

  19. 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)

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Current and Noise Saturation in Graphene Superlattice

    Science.gov (United States)

    Yang, Wei; Lu, Xiaobo; Berthou, Simon; Wilmart, Quentin; Boukhicha, Mohamed; Voisin, Christophe; Zhang, Guangyu; Placais, Bernard

    One of the merits of graphene is that the Fermi level can be easily tuned by electrical gating, which render charge carriers n type or p type, or even insulating around the Dirac point (DP). By aligning graphene on top of Boron Nitride (BN), the presence of graphene superlattice makes transport properties even more versatile owning to the emergence of secondary Dirac points (SDPs). Here we present a study of high electric field performance of graphene superlattice obtained from epitaxial approach. By using microwave cavity, noise produced from graphene by joule heating is recorded up to 5GHz. Current and noise saturation are observed and investigated. Depending on Fermi energy, saturation can be attributed to intrinsic optical or remote surface polar phonon scattering at a doping far away from DP, while no saturation are found around DP. Moreover, noise saturation is identified around Fermi energy between DP and SDP, which can be attributed to the influence of van Hove singularity arising from the superlattice. Lastly, saturation due to the bias induced shift of DP, or so called Dirac fermion pinch-off, is well observed by local top gate technique. EU Graphene flagship project (Contract No. 604391).

  6. Saturated Zone Colloid Transport

    Energy Technology Data Exchange (ETDEWEB)

    H. S. Viswanathan

    2004-10-07

    This scientific analysis provides retardation factors for colloids transporting in the saturated zone (SZ) and the unsaturated zone (UZ). These retardation factors represent the reversible chemical and physical filtration of colloids in the SZ. The value of the colloid retardation factor, R{sub col} is dependent on several factors, such as colloid size, colloid type, and geochemical conditions (e.g., pH, Eh, and ionic strength). These factors are folded into the distributions of R{sub col} that have been developed from field and experimental data collected under varying geochemical conditions with different colloid types and sizes. Attachment rate constants, k{sub att}, and detachment rate constants, k{sub det}, of colloids to the fracture surface have been measured for the fractured volcanics, and separate R{sub col} uncertainty distributions have been developed for attachment and detachment to clastic material and mineral grains in the alluvium. Radionuclides such as plutonium and americium sorb mostly (90 to 99 percent) irreversibly to colloids (BSC 2004 [DIRS 170025], Section 6.3.3.2). The colloid retardation factors developed in this analysis are needed to simulate the transport of radionuclides that are irreversibly sorbed onto colloids; this transport is discussed in the model report ''Site-Scale Saturated Zone Transport'' (BSC 2004 [DIRS 170036]). Although it is not exclusive to any particular radionuclide release scenario, this scientific analysis especially addresses those scenarios pertaining to evidence from waste-degradation experiments, which indicate that plutonium and americium may be irreversibly attached to colloids for the time scales of interest. A section of this report will also discuss the validity of using microspheres as analogs to colloids in some of the lab and field experiments used to obtain the colloid retardation factors. In addition, a small fraction of colloids travels with the groundwater without any significant

  7. 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.

  8. Stability and stabilization of linear systems with saturating actuators

    CERN Document Server

    Tarbouriech, Sophie; Gomes da Silva Jr, João Manoel; Queinnec, Isabelle

    2011-01-01

    Gives the reader an in-depth understanding of the phenomena caused by the more-or-less ubiquitous problem of actuator saturation. Proposes methods and algorithms designed to avoid, manage or overcome the effects of actuator saturation. Uses a state-space approach to ensure local and global stability of the systems considered. Compilation of fifteen years' worth of research results.

  9. Serum albumin--a non-saturable carrier

    DEFF Research Database (Denmark)

    Brodersen, R; Honoré, B; Larsen, F G

    1984-01-01

    The shape of binding isotherms for sixteen ligands to human serum albumin showed no signs of approaching saturation at high ligand concentrations. It is suggested that ligand binding to serum albumin is essentially different from saturable binding of substrates to enzymes, of oxygen to haemoglobi...

  10. WAter Saturation Shift Referencing (WASSR) for chemical exchange saturation transfer experiments

    Science.gov (United States)

    Kim, Mina; Gillen, Joseph; Landman, Bennett. A.; Zhou, Jinyuan; van Zijl, Peter C.M.

    2010-01-01

    Chemical exchange saturation transfer (CEST) is a contrast mechanism exploiting exchange-based magnetization transfer (MT) between solute and water protons. CEST effects compete with direct water saturation and conventional MT processes and generally can only be quantified through an asymmetry analysis of the water saturation spectrum (Z-spectrum) with respect to the water frequency, a process that is exquisitely sensitive to magnetic field inhomogeneities. Here, it is shown that direct water saturation imaging allows measurement of the absolute water frequency in each voxel, allowing proper centering of Z-spectra on a voxel-by-voxel basis independent of spatial B0 field variations. Optimal acquisition parameters for this “water saturation shift referencing” or “WASSR” approach were estimated using Monte Carlo simulations and later confirmed experimentally. The optimal ratio of the WASSR sweep width to the linewidth of the direct saturation curve was found to be 3.3–4.0, requiring a sampling of 16–32 points. The frequency error was smaller than 1 Hz at signal to noise ratios of 40 or higher. The WASSR method was applied to study glycogen, where the chemical shift difference between the hydroxyl (OH) protons and bulk water protons at 3T is so small (0.75–1.25 ppm) that the CEST spectrum is inconclusive without proper referencing. PMID:19358232

  11. Water saturation shift referencing (WASSR) for chemical exchange saturation transfer (CEST) experiments.

    Science.gov (United States)

    Kim, Mina; Gillen, Joseph; Landman, Bennett A; Zhou, Jinyuan; van Zijl, Peter C M

    2009-06-01

    Chemical exchange saturation transfer (CEST) is a contrast mechanism that exploits exchange-based magnetization transfer (MT) between solute and water protons. CEST effects compete with direct water saturation and conventional MT processes, and generally can only be quantified through an asymmetry analysis of the water saturation spectrum (Z-spectrum) with respect to the water frequency, a process that is exquisitely sensitive to magnetic field inhomogeneities. Here it is shown that direct water saturation imaging allows measurement of the absolute water frequency in each voxel, allowing proper centering of Z-spectra on a voxel-by-voxel basis independently of spatial B(0) field variations. Optimal acquisition parameters for this "water saturation shift referencing" (WASSR) approach were estimated using Monte Carlo simulations and later confirmed experimentally. The optimal ratio of the WASSR sweep width to the linewidth of the direct saturation curve was found to be 3.3-4.0, requiring a sampling of 16-32 points. The frequency error was smaller than 1 Hz at signal-to-noise ratios of 40 or higher. The WASSR method was applied to study glycogen, where the chemical shift difference between the hydroxyl (OH) protons and bulk water protons at 3T is so small (0.75-1.25 ppm) that the CEST spectrum is inconclusive without proper referencing.

  12. Brine Distribution after Vacuum Saturation

    DEFF Research Database (Denmark)

    Hedegaard, Kathrine; Andersen, Bertel Lohmann

    1999-01-01

    Experiments with the vacuum saturation method for brine in plugs of chalk showed that a homogeneous distribution of brine cannot be ensured at saturations below 20% volume. Instead of a homogeneous volume distribution the brine becomes concentrated close to the surfaces of the plugs...

  13. Misconceptions in Reporting Oxygen Saturation

    NARCIS (Netherlands)

    Toffaletti, John; Zijlstra, Willem G.

    2007-01-01

    BACKGROUND: We describe some misconceptions that have become common practice in reporting blood gas and cooximetry results. In 1980, oxygen saturation was incorrectly redefined in a report of a new instrument for analysis of hemoglobin (Hb) derivatives. Oxygen saturation (sO(2)) was redefined as the

  14. 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.

  15. 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.

  16. 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

  17. Saturation current spikes eliminated in saturable core transformers

    Science.gov (United States)

    Schwarz, F. C.

    1971-01-01

    Unsaturating composite magnetic core transformer, consisting of two separate parallel cores designed so impending core saturation causes signal generation, terminates high current spike in converter primary circuit. Simplified waveform, demonstrates transformer effectiveness in eliminating current spikes.

  18. 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.

  19. 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 ...

  20. 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

  1. 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...

  2. 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

  3. 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 ...

  4. 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...

  5. 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

  6. 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

  7. 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.

  8. Pichia stipitis OYE 2.6 variants with improved catalytic efficiencies from site-saturation mutagenesis libraries.

    Science.gov (United States)

    Patterson-Orazem, Athéna; Sullivan, Bradford; Stewart, Jon D

    2014-10-15

    An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes. Several substrates were sterically demanding, and as hoped, creating additional active site volume yielded better conversions for these alkenes. The most impressive improvement was found for 2-butylidenecyclohexanone. The wild-type provided less than 20% conversion after 24h; a triple mutant afforded more than 60% conversion in the same time period. Moreover, even wild-type OYE 2.6 can reduce cyclohexenones with very bulky 4-substituents efficiently. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. 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.

  16. 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

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

    Science.gov (United States)

    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.

  18. 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.

  19. 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.

  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. Saturated fats: a perspective from lactation and milk composition.

    Science.gov (United States)

    German, J Bruce; Dillard, Cora J

    2010-10-01

    For recommendations of specific targets for the absolute amount of saturated fat intake, we need to know what dietary intake is most appropriate? Changing agricultural production and processing to lower the relative quantities of macronutrients requires years to accomplish. Changes can have unintended consequences on diets and the health of subsets of the population. Hence, what are the appropriate absolute amounts of saturated fat in our diets? Is the scientific evidence consistent with an optimal intake of zero? If not, is it also possible that a finite intake of saturated fats is beneficial to overall health, at least to a subset of the population? Conclusive evidence from prospective human trials is not available, hence other sources of information must be considered. One approach is to examine the evolution of lactation, and the composition of milks that developed through millennia of natural selective pressure and natural selection processes. Mammalian milks, including human milk, contain 50% of their total fatty acids as saturated fatty acids. The biochemical formation of a single double bond converting a saturated to a monounsaturated fatty acid is a pathway that exists in all eukaryotic organisms and is active within the mammary gland. In the face of selective pressure, mammary lipid synthesis in all mammals continues to release a significant content of saturated fatty acids into milk. Is it possible that evolution of the mammary gland reveals benefits to saturated fatty acids that current recommendations do not consider?

  2. 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

  3. Saturation of the turbulent dynamo.

    Science.gov (United States)

    Schober, J; Schleicher, D R G; Federrath, C; Bovino, S; Klessen, R S

    2015-08-01

    The origin of strong magnetic fields in the Universe can be explained by amplifying weak seed fields via turbulent motions on small spatial scales and subsequently transporting the magnetic energy to larger scales. This process is known as the turbulent dynamo and depends on the properties of turbulence, i.e., on the hydrodynamical Reynolds number and the compressibility of the gas, and on the magnetic diffusivity. While we know the growth rate of the magnetic energy in the linear regime, the saturation level, i.e., the ratio of magnetic energy to turbulent kinetic energy that can be reached, is not known from analytical calculations. In this paper we present a scale-dependent saturation model based on an effective turbulent resistivity which is determined by the turnover time scale of turbulent eddies and the magnetic energy density. The magnetic resistivity increases compared to the Spitzer value and the effective scale on which the magnetic energy spectrum is at its maximum moves to larger spatial scales. This process ends when the peak reaches a characteristic wave number k☆ which is determined by the critical magnetic Reynolds number. The saturation level of the dynamo also depends on the type of turbulence and differs for the limits of large and small magnetic Prandtl numbers Pm. With our model we find saturation levels between 43.8% and 1.3% for Pm≫1 and between 2.43% and 0.135% for Pm≪1, where the higher values refer to incompressible turbulence and the lower ones to highly compressible turbulence.

  4. Slow light in saturable absorbers

    OpenAIRE

    Macke, Bruno; Ségard, Bernard

    2008-01-01

    International audience; In connection with the experiments recently achieved on doped crystals, biological samples, doped optical fibers and semiconductor heterostructures, we revisit the theory of the propagation of a pulse-modulated light in a saturable absorber. Explicit analytical expressions of the transmitted pulse are obtained, enabling us to determine the parameters optimizing the time-delay of the transmitted pulse with respect to the incident pulse. We finally compare the maximum fr...

  5. Saturation of Van Allen's belts

    CERN Document Server

    Le Bel, E

    2002-01-01

    The maximum number of electrons that can be trapped in van Allen's belts has been evaluated at CEA-DAM more precisely than that commonly used in the space community. The modelization that we have developed allows to understand the disagreement (factor 50) observed between the measured and predicted electrons flux by US satellites and theory. This saturation level allows sizing-up of the protection on a satellite in case of energetic events. (authors)

  6. 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...

  7. Transition to Turbulent Dynamo Saturation

    Science.gov (United States)

    Seshasayanan, Kannabiran; Gallet, Basile; Alexakis, Alexandros

    2017-11-01

    While the saturated magnetic energy is independent of viscosity in dynamo experiments, it remains viscosity dependent in state-of-the-art 3D direct numerical simulations (DNS). Extrapolating such viscous scaling laws to realistic parameter values leads to an underestimation of the magnetic energy by several orders of magnitude. The origin of this discrepancy is that fully 3D DNS cannot reach low enough values of the magnetic Prandtl number Pm. To bypass this limitation and investigate dynamo saturation at very low Pm, we focus on the vicinity of the dynamo threshold in a rapidly rotating flow: the velocity field then depends on two spatial coordinates only, while the magnetic field consists of a single Fourier mode in the third direction. We perform numerical simulations of the resulting set of reduced equations for Pm down to 2 ×10-5. This parameter regime is currently out of reach to fully 3D DNS. We show that the magnetic energy transitions from a high-Pm viscous scaling regime to a low-Pm turbulent scaling regime, the latter being independent of viscosity. The transition to the turbulent saturation regime occurs at a low value of the magnetic Prandtl number, Pm ≃10-3 , which explains why it has been overlooked by numerical studies so far.

  8. SATURATED PICRIC ACID PREVENTS AUTOPHAGIA

    Directory of Open Access Journals (Sweden)

    V Rahimi-Movaghar

    2008-08-01

    Full Text Available "nThe dysesthesia and paresthesia that occurs in laboratory rats after spinal cord injury (SCI results in autophagia. This self-destructive behavior interferes with functional assessments in designed studies and jeopardizes the health of the injured rat. In this study, we evaluated role of saturated picric acid in the prevention of autophagia and self-mutilation. All rats were anesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg/kg and xylazine (10 mg/kg for the SCI procedures. In the first 39 rats, no solution applied to the hind limbs, but in the next 26 cases, we smeared the saturated picric acid on the tail, lower extremities, pelvic, and abdomen of the rats immediately after SCI. In the rats without picric acid, 23 rats died following autophagia, but in the 26 rats with picric acid, there was no autophagia (P < 0.001. Picric acid side effects in skin and gastrointestinal signs such as irritation, redness and diarrhea were not seen in any rat. Saturated picric acid is a topical solution that if used appropriately and carefully, might be safe and effectively prevents autophagia and self-mutilation. When the solution is applied to the lower abdomen and limbs, we presume that its bitterness effectively prevents the rat from licking and biting the limb.

  9. 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...

  10. 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.

  11. 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...

  12. 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.

  13. 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...

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

    Directory of Open Access Journals (Sweden)

    Ihssen Julian

    2012-09-01

    subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three. Conclusions This study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.

  15. 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.

  16. Semiconductor saturable absorbers for ultrafast terahertz signals

    OpenAIRE

    Hoffmann, Matthias C.; Turchinovich, Dmitry

    2010-01-01

    We demonstrate saturable absorber behavior of n-type semiconductors GaAs, GaP, and Ge in the terahertz THz frequency range at room temperature using nonlinear THz spectroscopy. The saturation mechanism is based on a decrease in electron conductivity of semiconductors at high electron momentum states, due to conduction band onparabolicity and scattering into satellite valleys in strong THz fields. Saturable absorber parameters, such as linear and nonsaturable transmission, and saturation fluen...

  17. Semiconductor saturable absorbers for ultrafast terahertz signals

    OpenAIRE

    Hoffmann, Matthias C.; Turchinovich, Dmitry

    2010-01-01

    We demonstrate saturable absorber behavior of n-type semiconductorsGaAs,GaP, and Ge in the terahertz (THz) frequency range at room temperature using nonlinear THz spectroscopy. The saturation mechanism is based on a decrease in electron conductivity of semiconductors at high electron momentum states, due to conduction band nonparabolicity and scattering into satellite valleys in strong THz fields. Saturable absorber parameters, such as linear and nonsaturable transmission, and saturation flue...

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Poole-Frenkel (PF) effect high field saturation

    OpenAIRE

    Ongaro, R.; Pillonnet, A.

    1989-01-01

    An improved Poole Frenkel (PF) effect, based upon a rigorous methodological approach, is proposed. The chosen model is stated precisely, emphasis being made explicitly on the subtending hypotheses. A systematic reference to Fermi-Dirac function, allows to establish quantitatively the concept of PF saturation. The resulting general theory integrates, as particular applications, the previous one-dimensional PF theories.

  20. From QCD to nuclear matter saturation

    Energy Technology Data Exchange (ETDEWEB)

    Ericson, Magda [Universite de Lyon, Univ. Lyon 1, CNRS/IN2P3, IPN Lyon, F-69622 Villeurbanne Cedex (France)]|[Theory division, CERN, CH-12111 Geneva (Switzerland); Chanfray, Guy [Universite de Lyon, Univ. Lyon 1, CNRS/IN2P3, IPN Lyon, F-69622 Villeurbanne Cedex (France)

    2007-03-15

    We discuss a relativistic chiral theory of nuclear matter with {sigma} and {omega} exchange using a formulation of the {sigma} model in which all the chiral constraints are automatically fulfilled. We establish a relation between the nuclear response to the scalar field and the QCD one which includes the nucleonic parts. It allows a comparison between nuclear and QCD information. Going beyond the mean field approach we introduce the effects of the pion loops supplemented by the short-range interaction. The corresponding Landau-Migdal parameters are taken from spin-isospin physics results. The parameters linked to the scalar meson exchange are extracted from lattice QCD results. These inputs lead to a reasonable description of the saturation properties, illustrating the link between QCD and nuclear physics. We also derive from the corresponding equation of state the density dependence of the quark condensate and of the QCD susceptibilities. (authors)

  1. 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...

  2. The use of saturation in qualitative research.

    Science.gov (United States)

    Walker, Janiece L

    2012-01-01

    Understanding qualitative research is an important component of cardiovascular nurses' practice and allows them to understand the experiences, stories, and perceptions of patients with cardiovascular conditions. In understanding qualitative research methods, it is essential that the cardiovascular nurse understands the process of saturation within qualitative methods. Saturation is a tool used for ensuring that adequate and quality data are collected to support the study. Saturation is frequently reported in qualitative research and may be the gold standard. However, the use of saturation within methods has varied. Hence, the purpose of this column is to provide insight for the cardiovascular nurse regarding the use of saturation by reviewing the recommendations for which qualitative research methods it is appropriate to use and how to know when saturation is achieved. In understanding saturation, the cardiovascular nurse can be a better consumer of qualitative research.

  3. 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

  4. 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 ...

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Symbolic Computation of Strongly Connected Components Using Saturation

    Science.gov (United States)

    Zhao, Yang; Ciardo, Gianfranco

    2010-01-01

    Finding strongly connected components (SCCs) in the state-space of discrete-state models is a critical task in formal verification of LTL and fair CTL properties, but the potentially huge number of reachable states and SCCs constitutes a formidable challenge. This paper is concerned with computing the sets of states in SCCs or terminal SCCs of asynchronous systems. Because of its advantages in many applications, we employ saturation on two previously proposed approaches: the Xie-Beerel algorithm and transitive closure. First, saturation speeds up state-space exploration when computing each SCC in the Xie-Beerel algorithm. Then, our main contribution is a novel algorithm to compute the transitive closure using saturation. Experimental results indicate that our improved algorithms achieve a clear speedup over previous algorithms in some cases. With the help of the new transitive closure computation algorithm, up to 10(exp 150) SCCs can be explored within a few seconds.

  12. An analysis of the saturation of a high gain FEL

    Energy Technology Data Exchange (ETDEWEB)

    Gluckstern, R.L.; Okamoto, Hiromi (Maryland Univ., College Park, MD (United States). Dept. of Physics); Krinsky, S. (Brookhaven National Lab., Upton, NY (United States))

    1992-12-01

    We study the saturated state of an untapered free electron laser in the Compton regime, arising after exponential amplification of an initial low level of radiation by an initially monoenergetic, unbunched electron beam. The saturated state of the FEL is described by oscillations about an equilibrium state. Using the two invariants of the motion, and certain assumptions motivated by computer simulations, we provide approximate analytic descriptions of the radiation field and electron distribution in the saturation regime. We first consider a one-dimensional approximation, and later extend our approach to treat an electron beam of finite radial extent. Of note is a result on the radiated power in the case of an electron beam with small radius.

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

    Science.gov (United States)

    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.

  14. Power flow control using distributed saturable reactors

    Science.gov (United States)

    Dimitrovski, Aleksandar D.

    2016-02-13

    A magnetic amplifier includes a saturable core having a plurality of legs. Control windings wound around separate legs are spaced apart from each other and connected in series in an anti-symmetric relation. The control windings are configured in such a way that a biasing magnetic flux arising from a control current flowing through one of the plurality of control windings is substantially equal to the biasing magnetic flux flowing into a second of the plurality of control windings. The flow of the control current through each of the plurality of control windings changes the reactance of the saturable core reactor by driving those portions of the saturable core that convey the biasing magnetic flux in the saturable core into saturation. The phasing of the control winding limits a voltage induced in the plurality of control windings caused by a magnetic flux passing around a portion of the saturable core.

  15. Response of an anisotropic liquid-saturated porous medium due to ...

    Indian Academy of Sciences (India)

    Eigenvalue approach, following Laplace and Fourier transforms, has been employed to find the general solution to the field equations in an anisotropic liquid-saturated porous medium, in the transformed domain. The results of isotropic liquid-saturated porous medium can be derived as a special case. A numerical inversion ...

  16. Saturated Fats and Cardiovascular Disease: Interpretations Not as Simple as They Once Were.

    Science.gov (United States)

    Bier, Dennis M

    2016-09-09

    Historically, the so-called "lipid hypothesis" has focused on the detrimental role of saturated fats per se in enhancing the risks of cardiovascular disease. Recently, a body of new information and systematic analyses of available data have questioned simple interpretation of the relationship of dietary saturated fats and of individual saturated fatty acids to CVD risk. Thus, current assessments of risks due to dietary fat consumption that emphasize the confounding nature of the dietary macronutrients substituted for dietary saturated fats and give broader recognition to the effect of patterns of food intake as a whole are the most productive approach to an overall healthy diet.

  17. [Sampling in qualitative research: a proposal for procedures to detect theoretical saturation].

    Science.gov (United States)

    Fontanella, Bruno Jose Barcellos; Luchesi, Bruna Moretti; Saidel, Maria Giovana Borges; Ricas, Janete; Turato, Egberto Ribeiro; Melo, Débora Gusmão

    2011-02-01

    A qualitative study's methodological transparency is considered a key factor for achieving its reliability and should be guaranteed by the researchers. Closing the sampling process by saturation is a common approach, but it is rarely made explicit in research reports. Qualitative researchers also commonly experience technical difficulties in objectively identifying saturation. This article proposes a method to organize sample closing by saturation, with a sequence of eight procedural steps for treating and analyzing data collected through open or semi-structured interviews or focus groups. The article aims to help researchers objectively explain how and when saturation occurred and to allow their readers to know how this process occurred.

  18. Using X-ray computed tomography to evaluate the initial saturation resulting from different saturation procedures

    DEFF Research Database (Denmark)

    Christensen, Britt Stenhøj Baun; Wildenschild, D; Jensen, K.H.

    2006-01-01

    for saturation. Evaluation of the different enhanced saturation techniques was done with Xray computed tomography (CT) and gravimetrically. The use of CT scanning makes it possible to observe the spatial distribution of wetting and non-wetting phases in the porous medium in a non-destructive way. In this case...... with pressurized nitrogen between each saturation and allowed to saturate for the same length of time for all the different procedures. Both gravimetric measurements and CT attenuation levels showed that venting the sample with carbon dioxide prior to saturation clearly improved initial saturation whereas the use...

  19. 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

  20. 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.

  1. Sensorial saturation and neonatal pain: a review.

    Science.gov (United States)

    Locatelli, Chiara; Bellieni, Carlo Valerio

    2017-08-23

    Sensorial saturation (SS) is an analgesic approach to babies' pain that includes three types of stimulations: oral sugar, massage and caregivers' voice. The aim of this review is to assess its efficacy. We performed an analysis of scientific literature from 2001 to 2017, retrieving those clinical trials where SS had been compared with other analgesic treatments during procedural pain in babies. We retrieved 14 studies. Pain sources were heel-prick in nine, eye examination and intramuscular shots in two each, and endotracheal aspiration in one. SS was the most effective treatment in all cases, except in endotracheal suctioning. No drawbacks were reported in any study using SS. SS is a safe and effective approach to neonatal pain due to heel-prick, more effective than oral sucrose or glucose in both term and preterm babies; it seems also effective in other types of acute procedural pain like eye examination or intramuscular injections, but more studies are needed to confirm these preliminary data. More studies are also needed to test SS efficacy for other procedures, and for older infants.

  2. Stochastic Modeling of Macrodispersion in Variably Saturated, Spatially Heterogeneous Formations

    Science.gov (United States)

    Russo, David

    2015-04-01

    The macrodispersion tensor, D, plays an important role in solute transport on the field scale. A key problem is how to relate D to the properties of the spatially heterogeneous formation. Under unsaturated flow conditions, the problem is further complicated inasmuch as the relevant flow parameters, the hydraulic conductivity and the water capacity, which depend on the formation properties, depend also on flow-controlled attributes in a highly nonlinear fashion. Consequently, under variably saturated conditions, quantification of D requires several simplifying assumptions regarding the constitutive relationships for unsaturated flow, the flow regime, and the spatial structure of the formation heterogeneity. The present talk focuses on the quantification of D in a variably saturated, spatially heterogeneous formation, accomplished by using a two-stage approach. The approach combines a stochastic, continuum description of a steady-state unsaturated flow, based on small-perturbation, first-order approximation of Darcy's law and the continuity equation for unsaturated flow, with a general Lagrangian description of the motion of an indivisible particle of a passive solute that is carried by the steady-state flow. The resultant, time-dependent D depends on the covariances of the water saturation and the components of the water flux vector, and their cross-covariances, which, in turn, depend on the (cross-)covariances of the relevant formation properties and the pressure head. The effect of few characteristics of the spatially heterogeneous, variably saturated flow system, on D is analyzed and discussed. Main findings reveal that under variably saturated flow conditions, the travel distance required for the principal components of D to approach their asymptotic values may be exceedingly large, particularly in relatively wet formations with significant stratification and with coarse-textured soil material associated with small capillary forces. Hence, in many practical

  3. 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

  4. ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus by treating mature sperm.

    Directory of Open Access Journals (Sweden)

    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.

  5. Saturated phosphatidic acids mediate saturated fatty acid–induced vascular calcification and lipotoxicity

    Science.gov (United States)

    Masuda, Masashi; Miyazaki-Anzai, Shinobu; Keenan, Audrey L.; Okamura, Kayo; Kendrick, Jessica; Chonchol, Michel; Offermanns, Stefan; Ntambi, James M.; Kuro-o, Makoto; Miyazaki, Makoto

    2015-01-01

    Recent evidence indicates that saturated fatty acid–induced (SFA-induced) lipotoxicity contributes to the pathogenesis of cardiovascular and metabolic diseases; however, the molecular mechanisms that underlie SFA-induced lipotoxicity remain unclear. Here, we have shown that repression of stearoyl-CoA desaturase (SCD) enzymes, which regulate the intracellular balance of SFAs and unsaturated FAs, and the subsequent accumulation of SFAs in vascular smooth muscle cells (VSMCs), are characteristic events in the development of vascular calcification. We evaluated whether SMC-specific inhibition of SCD and the resulting SFA accumulation plays a causative role in the pathogenesis of vascular calcification and generated mice with SMC-specific deletion of both Scd1 and Scd2. Mice lacking both SCD1 and SCD2 in SMCs displayed severe vascular calcification with increased ER stress. Moreover, we employed shRNA library screening and radiolabeling approaches, as well as in vitro and in vivo lipidomic analysis, and determined that fully saturated phosphatidic acids such as 1,2-distearoyl-PA (18:0/18:0-PA) mediate SFA-induced lipotoxicity and vascular calcification. Together, these results identify a key lipogenic pathway in SMCs that mediates vascular calcification. PMID:26517697

  6. Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity.

    Science.gov (United States)

    Masuda, Masashi; Miyazaki-Anzai, Shinobu; Keenan, Audrey L; Okamura, Kayo; Kendrick, Jessica; Chonchol, Michel; Offermanns, Stefan; Ntambi, James M; Kuro-O, Makoto; Miyazaki, Makoto

    2015-10-26

    Recent evidence indicates that saturated fatty acid-induced (SFA-induced) lipotoxicity contributes to the pathogenesis of cardiovascular and metabolic diseases; however, the molecular mechanisms that underlie SFA-induced lipotoxicity remain unclear. Here, we have shown that repression of stearoyl-CoA desaturase (SCD) enzymes, which regulate the intracellular balance of SFAs and unsaturated FAs, and the subsequent accumulation of SFAs in vascular smooth muscle cells (VSMCs), are characteristic events in the development of vascular calcification. We evaluated whether SMC-specific inhibition of SCD and the resulting SFA accumulation plays a causative role in the pathogenesis of vascular calcification and generated mice with SMC-specific deletion of both Scd1 and Scd2. Mice lacking both SCD1 and SCD2 in SMCs displayed severe vascular calcification with increased ER stress. Moreover, we employed shRNA library screening and radiolabeling approaches, as well as in vitro and in vivo lipidomic analysis, and determined that fully saturated phosphatidic acids such as 1,2-distearoyl-PA (18:0/18:0-PA) mediate SFA-induced lipotoxicity and vascular calcification. Together, these results identify a key lipogenic pathway in SMCs that mediates vascular calcification.

  7. Synthesis and characterization of saturated polyester and ...

    Indian Academy of Sciences (India)

    and nanocomposites derived from glycolyzed PET waste with varied compositions. SUNAIN KATOCH. ∗ ... Water vapour transmission (WVT) was determined for saturated polyester nanocomposite sheets according to ... ing the synthesis of saturated polyester (from GPET waste). This has been done for the estimation of the ...

  8. Ultrafast THz Saturable Absorption in Semiconductors

    DEFF Research Database (Denmark)

    Turchinovich, Dmitry; Hoffmann, Matthias C.

    2011-01-01

    We demonstrate THz saturable absorption in n-doped semiconductors GaAs, GaP, and Ge in a nonlinear THz time-domain spectroscopy experiment. Saturable absorption is caused by sample conductivity modulation due to electron heating and satellite valley scattering in the field of a strong THz pulse....

  9. Fault tolerant control of systems with saturations

    DEFF Research Database (Denmark)

    Niemann, Hans Henrik

    2013-01-01

    This paper presents framework for fault tolerant controllers (FTC) that includes input saturation. The controller architecture known from FTC is based on the Youla-Jabr-Bongiorno-Kucera (YJBK) parameterization is extended to handle input saturation. Applying this controller architecture in connec...

  10. Semiconductor saturable absorbers for ultrafast terahertz signals

    DEFF Research Database (Denmark)

    Hoffmann, Matthias C.; Turchinovich, Dmitry

    2010-01-01

    We demonstrate saturable absorber behavior of n-type semiconductors GaAs, GaP, and Ge in the terahertz THz frequency range at room temperature using nonlinear THz spectroscopy. The saturation mechanism is based on a decrease in electron conductivity of semiconductors at high electron momentum...

  11. Determination of saturation functions and wettability for chalk based on measured fluid saturations

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, D.; Bech, N.; Moeller Nielsen, C.

    1998-08-01

    The end effect of displacement experiments on low permeable porous media is used for determination of relative permeability functions and capillary pressure functions. Saturation functions for a drainage process are determined from a primary drainage experiment. A reversal of the flooding direction creates an intrinsic imbibition process in the sample, which enables determination if imbibition saturation functions. The saturation functions are determined by a parameter estimation technique. Scanning effects are modelled by the method of Killough. Saturation profiles are determined by NMR. (au)

  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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  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. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

    biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides. PMID:27913416

  5. 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

    instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides. Copyright © 2017 American Society for Microbiology.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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...

  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. Combinatorics of saturated secondary structures of RNA.

    Science.gov (United States)

    Clote, P

    2006-11-01

    Following Zuker (1986), a saturated secondary structure for a given RNA sequence is a secondary structure such that no base pair can be added without violating the definition of secondary structure, e.g., without introducing a pseudoknot. In the Nussinov-Jacobson energy model (Nussinov and Jacobson, 1980), where the energy of a secondary structure is -1 times the number of base pairs, saturated secondary structures are local minima in the energy landscape, hence form kinetic traps during the folding process. Here we present recurrence relations and closed form asymptotic limits for combinatorial problems related to the number of saturated secondary structures. In addition, Python source code to compute the number of saturated secondary structures having k base pairs can be found at the web servers link of bioinformatics.bc.edu/clotelab/.

  13. Saturated fat, carbohydrate, and cardiovascular disease

    National Research Council Canada - National Science Library

    Siri-Tarino, Patty W; Sun, Qi; Hu, Frank B; Krauss, Ronald M

    2010-01-01

    A focus of dietary recommendations for cardiovascular disease (CVD) prevention and treatment has been a reduction in saturated fat intake, primarily as a means of lowering LDL-cholesterol concentrations...

  14. Saturated thickness, High Plains aquifer, 2009

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — This raster data set represents the saturated thickness of the High Plains aquifer of the United States, 2009, in feet. The High Plains aquifer underlies...

  15. 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

  16. Semiconductor saturable absorbers for ultrafast THz signals

    DEFF Research Database (Denmark)

    Hoffmann, Matthias C.; Turchinovich, Dmitry

    We demonstrate saturable absorber behavior of n-type semiconductors in the THz frequency range using nonlinear THz spectroscopy. Further, we observe THz pulse shortening and increase of the group refractive index at high field strengths.......We demonstrate saturable absorber behavior of n-type semiconductors in the THz frequency range using nonlinear THz spectroscopy. Further, we observe THz pulse shortening and increase of the group refractive index at high field strengths....

  17. Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing

    Directory of Open Access Journals (Sweden)

    Zhang Jingqing

    2007-11-01

    Full Text Available Abstract Background The NAD(PH-dependent Pichia stipitis xylose reductase (PsXR is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. Results Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(PH in PsXR and altered using a semi-rational mutagenesis approach (CASTing. Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 U·mg-1, which is highest among PsXR mutants reported. Conclusion A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(PH preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(PH preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for

  18. Sensorless position estimation and control of permanent-magnet synchronous motors using a saturation model

    Science.gov (United States)

    Kassem Jebai, Al; Malrait, François; Martin, Philippe; Rouchon, Pierre

    2016-03-01

    Sensorless control of permanent-magnet synchronous motors at low velocity remains a challenging task. A now well-established method consists of injecting a high-frequency signal and using the rotor saliency, both geometric and magnetic-saturation induced. This paper proposes a clear and original analysis based on second-order averaging of how to recover the position information from signal injection; this analysis blends well with a general model of magnetic saturation. It also proposes a simple parametric model of the saturated motor, based on an energy function which simply encompasses saturation and cross-saturation effects. Experimental results on a surface-mounted motor and an interior magnet motor illustrate the relevance of the approach.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Complex geometrical optics of inhomogeneous and nonlinear saturable media

    Science.gov (United States)

    Berczynski, Pawel

    2013-05-01

    The method of complex geometrical optics (CGO) is presented, which describes Gaussian beam (GB) diffraction and self-focusing along curvilinear trajectory in smoothly inhomogeneous and nonlinear saturable media. CGO method reduces the problem of Gaussian beam propagation in inhomogeneous and nonlinear media to the system of the first order ordinary differential equations for the complex curvature of the wave front and for GB amplitude, which can be readily solved both analytically and numerically. As a result, CGO radically simplifies the description of Gaussian beam diffraction and self-focusing effects as compared to the other methods of nonlinear optics such as: variational method approach, method of moments and beam propagation method. The power of CGO method is presented on the example of the evolution of beam intensity and wave front cross-section along curvilinear central ray with torsion in weakly absorptive and nonlinear saturable graded-index fiber, where the effect of initial beam ellipticity is included into our description.

  1. Modeling saturable absorption for ultra short X-ray pulses

    Energy Technology Data Exchange (ETDEWEB)

    Hatada, Keisuke, E-mail: keisuke.hatada@unicam.it [CNISM, Sezione di Fisica, Scuola di Scienze e Tecnologie, Universit‘a di Camerino, via Madonna delle Carceri 9, I-62032 Camerino (Italy); INFN, Laboratori Nazionali di Frascati, 00044 Frascati (Italy); Di Cicco, Andrea [CNISM, Sezione di Fisica, Scuola di Scienze e Tecnologie, Universit‘a di Camerino, via Madonna delle Carceri 9, I-62032 Camerino (Italy)

    2014-10-15

    Saturable absorption was recently observed in transmission measurements above the L{sub II,III} edge of pure Al thin films using ultra short X-ray pulses at a free-electron-laser (FEL) facility. The high fluence reachable by FEL pulses, the shortness of the pulse duration, and the typical lifetime of the excited state are all important factors enabling observation of the phenomenon. We devised a simplified theoretical approach describing the saturation phenomenon using a three-channel model containing ground, excited and relaxed states. This phenomenological model explicitly includes the interaction between the solid and photon field in a semi-classical way, and the resulting non-linear coupled equation is solved numerically. We successfully applied this model to recent experimental results obtained using FEL radiation.

  2. SATURATED ZONE IN-SITU TESTING

    Energy Technology Data Exchange (ETDEWEB)

    P.W. REIMUS

    2004-11-08

    The purpose of this scientific analysis is to document the results and interpretations of field experiments that test and validate conceptual flow and radionuclide transport models in the saturated zone (SZ) near Yucca Mountain, Nevada. The test interpretations provide estimates of flow and transport parameters used in the development of parameter distributions for total system performance assessment (TSPA) calculations. These parameter distributions are documented in ''Site-Scale Saturated Zone Flow Model (BSC 2004 [DIRS 170037]), Site-Scale Saturated Zone Transport'' (BSC 2004 [DIRS 170036]), Saturated Zone Colloid Transport (BSC 2004 [DIRS 170006]), and ''Saturated Zone Flow and Transport Model Abstraction'' (BSC 2004 [DIRS 170042]). Specifically, this scientific analysis contributes the following to the assessment of the capability of the SZ to serve as part of a natural barrier for waste isolation for the Yucca Mountain repository system: (1) The bases for selection of conceptual flow and transport models in the saturated volcanics and the saturated alluvium located near Yucca Mountain. (2) Results and interpretations of hydraulic and tracer tests conducted in saturated fractured volcanics at the C-wells complex near Yucca Mountain. The test interpretations include estimates of hydraulic conductivities, anisotropy in hydraulic conductivity, storativities, total porosities, effective porosities, longitudinal dispersivities, matrix diffusion mass transfer coefficients, matrix diffusion coefficients, fracture apertures, and colloid transport parameters. (3) Results and interpretations of hydraulic and tracer tests conducted in saturated alluvium at the Alluvial Testing Complex (ATC) located at the southwestern corner of the Nevada Test Site (NTS). The test interpretations include estimates of hydraulic conductivities, storativities, total porosities, effective porosities, longitudinal dispersivities, matrix diffusion mass

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

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    mitochondrial genome mutagenesis, and antioxidants significantly rescue these events.

  4. 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

  5. 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.

  6. 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.

  7. 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...

  8. Saturated fats and cardiovascular disease risk: A review

    OpenAIRE

    Ishi Khosla; Gayatri C Khosla1

    2017-01-01

    Saturated fats have been in the line of fire for more than three decades. The major mistake in understanding fats was to equate all saturated fatty acids as one. The oversimplification of the relationship of saturated fats with cardiovascular disease (CVD) led to unwarranted removal of some valuable fats from our diets. Recently, the relationship of dietary saturated fats and that of individual saturated fatty acids (SFAs) to CVD risk has been reevaluated. All saturated fats are not equal and...

  9. Lipid order, saturation and surface property relationships: a study of human meibum saturation.

    Science.gov (United States)

    Mudgil, Poonam; Borchman, Douglas; Yappert, Marta C; Duran, Diana; Cox, Gregory W; Smith, Ryan J; Bhola, Rahul; Dennis, Gary R; Whitehall, John S

    2013-11-01

    Tear film stability decreases with age however the cause(s) of the instability are speculative. Perhaps the more saturated meibum from infants may contribute to tear film stability. The meibum lipid phase transition temperature and lipid hydrocarbon chain order at physiological temperature (33 °C) decrease with increasing age. It is reasonable that stronger lipid-lipid interactions could stabilize the tear film since these interactions must be broken for tear break up to occur. In this study, meibum from a pool of adult donors was saturated catalytically. The influence of saturation on meibum hydrocarbon chain order was determined by infrared spectroscopy. Meibum is in an anhydrous state in the meibomian glands and on the surface of the eyelid. The influence of saturation on the surface properties of meibum was determined using Langmuir trough technology. Saturation of native human meibum did not change the minimum or maximum values of hydrocarbon chain order so at temperatures far above or below the phase transition of human meibum, saturation does not play a role in ordering or disordering the lipid hydrocarbon chains. Saturation did increase the phase transition temperature in human meibum by over 20 °C, a relatively high amount. Surface pressure-area studies showing the late take off and higher maximum surface pressure of saturated meibum compared to native meibum suggest that the saturated meibum film is quite molecularly ordered (stiff molecular arrangement) and elastic (molecules are able to rearrange during compression and expansion) compared with native meibum films which are more fluid agreeing with the infrared spectroscopic results of this study. In saturated meibum, the formation of compacted ordered islands of lipids above the surfactant layer would be expected to decrease the rate of evaporation compared to fluid and more loosely packed native meibum. Higher surface pressure observed with films of saturated meibum compared to native meibum

  10. Soil Structure and Saturated Hydraulic Conductivity

    Science.gov (United States)

    Houskova, B.; Nagy, V.

    The role of soil structure on saturated hydraulic conductivity changes is studied in plough layers of texturally different soils. Three localities in western part of Slovakia in Zitny ostrov (Corn Island) were under investigation: locality Kalinkovo with light Calcaric Fluvisol (FAO 1970), Macov with medium heavy Calcari-mollic Fluvisol and Jurova with heavy Calcari-mollic Fluvisol. Soil structure was determined in dry as well as wet state and in size of macro and micro aggregates. Saturated hydraulic conductivity was measured by the help of double ring method. During the period of ring filling the soil surface was protected against aggregates damage by falling water drops. Spatial and temporal variability of studied parameters was evaluated. Cultivated crops were ensilage maize at medium heavy and heavy soil and colza at light soil. Textural composition of soil and actual water content at the beginning of measurement are one of major factor affecting aggregate stability and consequently also saturated hydraulic conductivity.

  11. Perturbative Saturation and the Soft Pomeron

    CERN Document Server

    Kovner, A; Kovner, Alex; Wiedemann, Urs Achim

    2002-01-01

    We show that perturbation theory provides two distinct mechanisms for the power like growth of hadronic cross sections at high energy. One, the leading BFKL effect is due to the growth of the parton density, and is characterized by the leading BFKL exponent. The other mechanism is due to the infrared diffusion, or the long range nature of the Coulomb field of perturbatively massless gluons. When perturbative saturation effects are taken into account, the first mechanism is rendered ineffective but the second one persists. We suggest that these two distinct mechanisms are responsible for the appearance of two pomerons. The density growth effects are responsible for the hard pomeron and manifest themselves in small systems (e.g. gamma^* or small size fluctuations in the proton wave function) where saturation effects are not important. The soft pomeron is the manifestation of the exponential growth of the black saturated regions which appear in typical hadronic systems. We point out that the nonlinear generaliza...

  12. Interger multiplication with overflow detection or saturation

    Energy Technology Data Exchange (ETDEWEB)

    Schulte, M.J.; Balzola, P.I.; Akkas, A.; Brocato, R.W.

    2000-01-11

    High-speed multiplication is frequently used in general-purpose and application-specific computer systems. These systems often support integer multiplication, where two n-bit integers are multiplied to produce a 2n-bit product. To prevent growth in word length, processors typically return the n least significant bits of the product and a flag that indicates whether or not overflow has occurred. Alternatively, some processors saturate results that overflow to the most positive or most negative representable number. This paper presents efficient methods for performing unsigned or two's complement integer multiplication with overflow detection or saturation. These methods have significantly less area and delay than conventional methods for integer multiplication with overflow detection and saturation.

  13. The Danish tax on saturated fat

    DEFF Research Database (Denmark)

    Vallgårda, Signild; Holm, Lotte; Jensen, Jørgen Dejgård

    2015-01-01

    BACKGROUND/OBJECTIVES: Health promoters have repeatedly proposed using economic policy tools, taxes and subsidies, as a means of changing consumer behaviour. As the first country in the world, Denmark introduced a tax on saturated fat in 2011. It was repealed in 2012. In this paper, we present...... on saturated fat had been suggested by two expert committees and was introduced with a majority in parliament, as a part of a larger economic reform package. Many actors, including representatives from the food industry and nutrition researchers, opposed the tax both before and after its introduction, claiming......, research was published showing that consumption of saturated fat had declined in Denmark. CONCLUSIONS: The analysis indicates that the Danish tax on fat was introduced mainly to increase public revenue. As the tax had no strong proponents and many influential adversaries, it was repealed. New research...

  14. 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

  15. 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.

  16. Evaluation of the Performance of Grouting Materials for Saturated Riprap

    Directory of Open Access Journals (Sweden)

    Daehyeon Kim

    2013-12-01

    Full Text Available In this study, four types of grout were developed to evaluate the effect of grouting of saturated riprap layers on ground water flow. The developed types of grout are divided into a quick-setting type and a general-type, and also into high and low viscosities. A number of grout tests were performed in a model acrylic chamber, 0.4 m in diameter and 2.0 m in length, for visual observation of injection. To reproduce the field flow condition of the saturated riprap layers (approach flow, the grout tests were carried out at 0 cm/s and 100 cm/s for the flow speed and 10 L/min for the grout injection speed after installing a flow injection opening on the lower part of the chamber. Based on the results of the grout tests, the injection of each grout in the saturated riprap layers was examined to find out the most effective grout.

  17. Tracking Controller for Intrinsic Output Saturated Systems in Presence of Amplitude and Rate Input Saturations

    DEFF Research Database (Denmark)

    Chater, E.; Giri, F.; Guerrero, Josep M.

    2014-01-01

    We consider the problem of controlling plants that are subject to multiple saturation constraints. Especially, we are interested in linear systems whose input is subject to amplitude and rate constraints of saturation type. Furthermore, the considered systems output is also subject to an intrinsi...

  18. Femoral venous oxygen saturation is no surrogate for central venous oxygen saturation

    NARCIS (Netherlands)

    van Beest, Paul A.; van der Schors, Alice; Liefers, Henriette; Coenen, Ludo G. J.; Braam, Richard L.; Habib, Najib; Braber, Annemarije; Scheeren, Thomas W. L.; Kuiper, Michael A.; Spronk, Peter E.

    2012-01-01

    Objective:  The purpose of our study was to determine if central venous oxygen saturation and femoral venous oxygen saturation can be used interchangeably during surgery and in critically ill patients. Design:  Prospective observational controlled study. Setting:  Nonacademic university-affiliated

  19. Saturated poroelastic actuators generated by topology optimization

    DEFF Research Database (Denmark)

    Andreasen, Casper Schousboe; Sigmund, Ole

    2011-01-01

    In this paper the fluid-structure interaction problem of a saturated porous media is considered. The pressure coupling properties of porous saturated materials change with the microstructure and this is utilized in the design of an actuator using a topology optimized porous material. By maximizing...... the coupling of internal fluid pressure and elastic shear stresses a slab of the optimized porous material deflects/deforms when a pressure is imposed and an actuator is created. Several phenomenologically based constraints are imposed in order to get a stable force transmitting actuator....

  20. On the saturation of astrophysical dynamos

    DEFF Research Database (Denmark)

    Dorch, Bertil; Archontis, Vasilis

    2004-01-01

    In the context of astrophysical dynamos we illustrate that the no-cosines flow, with zero mean helicity, can drive fast dynamo action and we study the dynamo's mode of operation during both the linear and non-linear saturation regimes. It turns out that in addition to a high growth rate in the li......In the context of astrophysical dynamos we illustrate that the no-cosines flow, with zero mean helicity, can drive fast dynamo action and we study the dynamo's mode of operation during both the linear and non-linear saturation regimes. It turns out that in addition to a high growth rate...

  1. Site-Scale Saturated Zone Flow Model

    Energy Technology Data Exchange (ETDEWEB)

    G. Zyvoloski

    2003-12-17

    The purpose of this model report is to document the components of the site-scale saturated-zone flow model at Yucca Mountain, Nevada, in accordance with administrative procedure (AP)-SIII.lOQ, ''Models''. This report provides validation and confidence in the flow model that was developed for site recommendation (SR) and will be used to provide flow fields in support of the Total Systems Performance Assessment (TSPA) for the License Application. The output from this report provides the flow model used in the ''Site-Scale Saturated Zone Transport'', MDL-NBS-HS-000010 Rev 01 (BSC 2003 [162419]). The Site-Scale Saturated Zone Transport model then provides output to the SZ Transport Abstraction Model (BSC 2003 [164870]). In particular, the output from the SZ site-scale flow model is used to simulate the groundwater flow pathways and radionuclide transport to the accessible environment for use in the TSPA calculations. Since the development and calibration of the saturated-zone flow model, more data have been gathered for use in model validation and confidence building, including new water-level data from Nye County wells, single- and multiple-well hydraulic testing data, and new hydrochemistry data. In addition, a new hydrogeologic framework model (HFM), which incorporates Nye County wells lithology, also provides geologic data for corroboration and confidence in the flow model. The intended use of this work is to provide a flow model that generates flow fields to simulate radionuclide transport in saturated porous rock and alluvium under natural or forced gradient flow conditions. The flow model simulations are completed using the three-dimensional (3-D), finite-element, flow, heat, and transport computer code, FEHM Version (V) 2.20 (software tracking number (STN): 10086-2.20-00; LANL 2003 [161725]). Concurrently, process-level transport model and methodology for calculating radionuclide transport in the saturated zone at Yucca

  2. Phase field modeling of partially saturated deformable porous media

    Science.gov (United States)

    Sciarra, Giulio

    2016-09-01

    A poromechanical model of partially saturated deformable porous media is proposed based on a phase field approach at modeling the behavior of the mixture of liquid water and wet air, which saturates the pore space, the phase field being the saturation (ratio). While the standard retention curve is expected still^ to provide the intrinsic retention properties of the porous skeleton, depending on the porous texture, an enhanced description of surface tension between the wetting (liquid water) and the non-wetting (wet air) fluid, occupying the pore space, is stated considering a regularization of the phase field model based on an additional contribution to the overall free energy depending on the saturation gradient. The aim is to provide a more refined description of surface tension interactions. An enhanced constitutive relation for the capillary pressure is established together with a suitable generalization of Darcy's law, in which the gradient of the capillary pressure is replaced by the gradient of the so-called generalized chemical potential, which also accounts for the "force", associated to the local free energy of the phase field model. A micro-scale heuristic interpretation of the novel constitutive law of capillary pressure is proposed, in order to compare the envisaged model with that one endowed with the concept of average interfacial area. The considered poromechanical model is formulated within the framework of strain gradient theory in order to account for possible effects, at laboratory scale, of the micro-scale hydro-mechanical couplings between highly localized flows (fingering) and localized deformations of the skeleton (fracturing).

  3. 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...

  4. 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.

  5. A New Robust Solver for Saturated-Unsaturated Richards' Equation

    Science.gov (United States)

    Barajas-Solano, D. A.; Tartakovsky, D. M.

    2012-12-01

    We present a novel approach for the numerical integration of the saturated-unsaturated Richards' equation, a degenerate parabolic partial differential equation that models flow in porous media. The method is based on the mixed (pore pressure-water content) form of RE, written as a set of differential algebraic equations (DAEs) of index-1 for the fully saturated case and index-2 for the partially saturated case. A DAE-based approach allows us to overcome the numerical challenges posed by the degenerate nature of the Richards' equation. The resulting set of DAEs is solved using the stiffly-accurate, single-step, 3-stage implicit Runge-Kutta method Radau IIA, chosen for its favorable accuracy and stability properties, and its ease of implementation. For each time step a nonlinear system of equations on the intermediate Runge-Kutta states of the pore pressure is solved, written so to ensure that the next step pore pressure and water content correspond to one another correctly. The implementation of our approach compares favorably to state-of-the-art DAE-based solvers in both one- and two-dimensional simulations. These solvers use multi-step backward difference formulas together with a pressure-based form of Richards' equation. To the best of our knowledge, our method is the first instance of a successful DAE-based solver that uses the mixed form of Richards' equation. We consider this a promising line of research, with future work to be done on the use of globally convergent methods for the solution of the occurring nonlinear systems of equations.

  6. 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.

  7. 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.

  8. Fullerene Transport in Saturated Porous Media

    Science.gov (United States)

    We investigated the effects of background solution chemistry and residence time within the soil column on the transport of aqu/C60 through saturated ultrapure quartz sand columns. Aqu/C60 breakthrough curves were obtained under different pore water velocities, solution pHs, and i...

  9. Oxygenation of saturated and unsaturated hydrocarbons with ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 118; Issue 5. Oxygenation of saturated and unsaturated hydrocarbons with sodium periodate catalyzed by manganese(III) tetra-arylporphyrins, to study the axial ligation of imidazole. Reza Tayebee. Volume 118 Issue 5 September 2006 pp 429-433 ...

  10. Nasal pulse oximetry overestimates oxygen saturation

    DEFF Research Database (Denmark)

    Rosenberg, J; Pedersen, M H

    1990-01-01

    Ten surgical patients were monitored with nasal and finger pulse oximetry (Nellcor N-200) for five study periods with alternating mouth and nasal breathing and switching of cables and sensors. Nasal pulse oximetry was found to overestimate arterial oxygen saturation by 4.7 (SD 1.4%) (bias...

  11. Determination of saturated and unsaturated hydraulic conductivity ...

    African Journals Online (AJOL)

    The estimation of hydraulic conductivity indicates how fluids flow through a substance and thus determine the water balance in the soil profile. In determining the saturated and unsaturated hydraulic conductivity of soil, five plots of 5.0 x 4.0 m were prepared with a PVC access tube installed in each plot. The plots were ...

  12. Understanding 'saturation' of radar signals over forests.

    Science.gov (United States)

    Joshi, Neha; Mitchard, Edward T A; Brolly, Matthew; Schumacher, Johannes; Fernández-Landa, Alfredo; Johannsen, Vivian Kvist; Marchamalo, Miguel; Fensholt, Rasmus

    2017-06-14

    There is an urgent need to quantify anthropogenic influence on forest carbon stocks. Using satellite-based radar imagery for such purposes has been challenged by the apparent loss of signal sensitivity to changes in forest aboveground volume (AGV) above a certain 'saturation' point. The causes of saturation are debated and often inadequately addressed, posing a major limitation to mapping AGV with the latest radar satellites. Using ground- and lidar-measurements across La Rioja province (Spain) and Denmark, we investigate how various properties of forest structure (average stem height, size and number density; proportion of canopy and understory cover) simultaneously influence radar backscatter. It is found that increases in backscatter due to changes in some properties (e.g. increasing stem sizes) are often compensated by equal magnitude decreases caused by other properties (e.g. decreasing stem numbers and increasing heights), contributing to the apparent saturation of the AGV-backscatter trend. Thus, knowledge of the impact of management practices and disturbances on forest structure may allow the use of radar imagery for forest biomass estimates beyond commonly reported saturation points.

  13. Wave propagation in thermoelastic saturated porous medium

    Indian Academy of Sciences (India)

    Biot 's theory for wave propagation in saturated porous solid is modified to study the propagation of thermoelastic waves in poroelastic medium. Propagation of plane harmonic waves is considered in isotropic poroelastic medium. Relations are derived among the wave-induced temperature in the medium and the ...

  14. Spontaneous emission from saturated parametric amplifiers

    DEFF Research Database (Denmark)

    Rottwitt, Karsten; Ott, Johan Raunkjær; Steffensen, Henrik

    2009-01-01

    Noise performance of parametric amplifiers is typically calculated assuming un-depleted operation. However, in many applications especially when applied as regenerative amplifiers in systems based on phase shift keyed modulation schemes, this assumption is not valid. Here we show the impact...... on accumulated spontaneous emission for a parametric amplifier operated in saturation....

  15. Elevated transferrin saturation and risk of diabetes

    DEFF Research Database (Denmark)

    Ellervik, Christina; Mandrup-Poulsen, Thomas; Andersen, Henrik Ullits

    2011-01-01

    OBJECTIVE We tested the hypothesis that elevated transferrin saturation is associated with an increased risk of any form of diabetes, as well as type 1 or type 2 diabetes separately. RESEARCH DESIGN AND METHODS We used two general population studies, The Copenhagen City Heart Study (CCHS, N = 9...

  16. Saturated fat, carbohydrates and cardiovascular disease

    NARCIS (Netherlands)

    Kuipers, R. S.; de Graaf, D. J.; Luxwolda, M. F.; Muskiet, M. H. A.; Dijck-Brouwer, D. A. J.; Muskiet, F. A. J.

    The dietary intake of saturated fatty acids (SAFA) is associated with a modest increase in serum total cholesterol, but not with cardiovascular disease (CVD). Replacing dietary SAFA with carbohydrates (CHO), notably those with a high glycaemic index, is associated with an increase in CVD risk in

  17. Two-beam interaction in saturable media

    DEFF Research Database (Denmark)

    Schjødt-Eriksen, Jens; Schmidt, Michel R.; Juul Rasmussen, Jens

    1998-01-01

    The dynamics of two coupled soliton solutions of the nonlinear Schrodinger equation with a saturable nonlinearity is investigated It is shown by means of a variational method and by direct numerical calculations that two well-separated solitons can orbit around each other, if their initial velocity...

  18. Synthesis and characterization of saturated polyester and ...

    Indian Academy of Sciences (India)

    Saturated polyester resin, derived from the glycolysis of polyethyleneterephthalate (PET) was examined as an effective way for PET recycling. The glycolyzed PET (GPET) was reacted with the mixture of phthalic anhydride and ethylene glycol (EG) with varied compositions and their reaction kinetic were studied. During ...

  19. Multi-spectral imaging of oxygen saturation

    Science.gov (United States)

    Savelieva, Tatiana A.; Stratonnikov, Aleksander A.; Loschenov, Victor B.

    2008-06-01

    The system of multi-spectral imaging of oxygen saturation is an instrument that can record both spectral and spatial information about a sample. In this project, the spectral imaging technique is used for monitoring of oxygen saturation of hemoglobin in human tissues. This system can be used for monitoring spatial distribution of oxygen saturation in photodynamic therapy, surgery or sports medicine. Diffuse reflectance spectroscopy in the visible range is an effective and extensively used technique for the non-invasive study and characterization of various biological tissues. In this article, a short review of modeling techniques being currently in use for diffuse reflection from semi-infinite turbid media is presented. A simple and practical model for use with a real-time imaging system is proposed. This model is based on linear approximation of the dependence of the diffuse reflectance coefficient on relation between absorbance and reduced scattering coefficient. This dependence was obtained with the Monte Carlo simulation of photon propagation in turbid media. Spectra of the oxygenated and deoxygenated forms of hemoglobin differ mostly in the red area (520 - 600 nm) and have several characteristic points there. Thus four band-pass filters were used for multi-spectral imaging. After having measured the reflectance, the data obtained are used for fitting the concentration of oxygenated and free hemoglobin, and hemoglobin oxygen saturation.

  20. 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.…

  1. 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.

  2. 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.

  3. Microscopic analysis of saturable absorbers: Semiconductor saturable absorber mirrors versus graphene

    Energy Technology Data Exchange (ETDEWEB)

    Hader, J.; Moloney, J. V. [Nonlinear Control Strategies, Inc., 3542 N. Geronimo Ave., Tucson, Arizona 85705 (United States); College of Optical Sciences, University of Arizona, Tucson, Arizona 85721 (United States); Yang, H.-J.; Scheller, M. [College of Optical Sciences, University of Arizona, Tucson, Arizona 85721 (United States); Koch, S. W. [Department of Physics and Materials Sciences Center, Philipps Universität Marburg, Renthof 5, 35032 Marburg (Germany)

    2016-02-07

    Fully microscopic many-body calculations are used to study the influence of strong sub-picosecond pulses on the carrier distributions and corresponding optical response in saturable absorbers used for mode-locking—semiconductor (quantum well) saturable absorber mirrors (SESAMs) and single layer graphene based saturable absorber mirrors (GSAMs). Unlike in GSAMs, the saturation fluence and recovery time in SESAMs show a strong spectral dependence. While the saturation fluence in the SESAM is minimal at the excitonic bandgap, the optimal recovery time and least pulse distortion due to group delay dispersion are found for excitation higher in the first subband. For excitation near the SESAM bandgap, the saturation fluence is about one tenth of that in the GSAM. At energies above the bandgap, the fluences in both systems become similar. A strong dependence of the saturation fluence on the pulse width in both systems is caused by carrier relaxation during the pulse. The recovery time in graphene is found to be about two to four times faster than that in the SESAMs. The occurrence of negative differential transmission in graphene is shown to be caused by dopant related carriers. In SESAMs, a negative differential transmission is found when exciting below the excitonic resonance where excitation induced dephasing leads to an enhancement of the absorption. Comparisons of the simulation data to the experiment show a very good quantitative agreement.

  4. Microscopic analysis of saturable absorbers: Semiconductor saturable absorber mirrors versus graphene

    Science.gov (United States)

    Hader, J.; Yang, H.-J.; Scheller, M.; Moloney, J. V.; Koch, S. W.

    2016-02-01

    Fully microscopic many-body calculations are used to study the influence of strong sub-picosecond pulses on the carrier distributions and corresponding optical response in saturable absorbers used for mode-locking—semiconductor (quantum well) saturable absorber mirrors (SESAMs) and single layer graphene based saturable absorber mirrors (GSAMs). Unlike in GSAMs, the saturation fluence and recovery time in SESAMs show a strong spectral dependence. While the saturation fluence in the SESAM is minimal at the excitonic bandgap, the optimal recovery time and least pulse distortion due to group delay dispersion are found for excitation higher in the first subband. For excitation near the SESAM bandgap, the saturation fluence is about one tenth of that in the GSAM. At energies above the bandgap, the fluences in both systems become similar. A strong dependence of the saturation fluence on the pulse width in both systems is caused by carrier relaxation during the pulse. The recovery time in graphene is found to be about two to four times faster than that in the SESAMs. The occurrence of negative differential transmission in graphene is shown to be caused by dopant related carriers. In SESAMs, a negative differential transmission is found when exciting below the excitonic resonance where excitation induced dephasing leads to an enhancement of the absorption. Comparisons of the simulation data to the experiment show a very good quantitative agreement.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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 ...

  10. 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 ...

  11. 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

  12. 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

  13. Saturation mutagenesis of a CepR binding site as a means to identify new quorum-regulated promoters in Burkholderia cenocepacia

    Science.gov (United States)

    Burkholderia cenocepacia, an opportunistic pathogen of humans, encodes the CepI and CepR proteins, which resemble the LuxI and LuxR quorum sensing proteins of Vibrio fischeri. CepI directs the synthesis of octanoylhomoserine lactone (OHL), while CepR is an OHL dependent transcription factor. In pr...

  14. Saturation mutagenesis reveals the importance of residues alpha R145 and alpha F146 of penicillin acylase in the synthesis of beta-lactam antibiotics

    NARCIS (Netherlands)

    Jager, Simon A. W.; Shapovalova, Irina V.; Jekel, Peter A.; Alkema, Wynand B. L.; Svedas, Vytas K.; Janssen, Dick B.; Švedas, Vytas K.

    2008-01-01

    Penicillin acylase (PA) from Escherichia coli can catalyze the coupling of an acyl group to penicillin- and cephalosporin-derived beta-lactam nuclei, a conversion that can be used for the industrial synthesis of beta-lactam. antibiotics. The modest synthetic properties of the wild-type enzyme make

  15. Engineering of the glycan-binding specificity of Agrocybe cylindracea galectin towards α(2,3)-linked sialic acid by saturation mutagenesis.

    Science.gov (United States)

    Imamura, Koji; Takeuchi, Hideaki; Yabe, Rikio; Tateno, Hiroaki; Hirabayashi, Jun

    2011-11-01

    Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.

  16. Replacing foods high in saturated fat by low-saturated fat alternatives: a computer simulation of the potential effects on reduction of saturated fat consumption

    NARCIS (Netherlands)

    Schickenberg, B.; Assema, P.; Brug, J.; Verkaik-Kloosterman, J.; Ocke, M.C.; Vries, de N.

    2009-01-01

    10 en%) increased from 23.3 % to 86.0 %. We conclude that the replacement of relatively few important high-saturated fat products by available lower-saturated fat alternatives can significantly reduce saturated fat intake and increase the proportion of individuals complying with recommended intake

  17. Saturated Zone In-Situ Testing

    Energy Technology Data Exchange (ETDEWEB)

    P. W. Reimus; M. J. Umari

    2003-12-23

    The purpose of this scientific analysis is to document the results and interpretations of field experiments that have been conducted to test and validate conceptual flow and radionuclide transport models in the saturated zone (SZ) near Yucca Mountain. The test interpretations provide estimates of flow and transport parameters that are used in the development of parameter distributions for Total System Performance Assessment (TSPA) calculations. These parameter distributions are documented in the revisions to the SZ flow model report (BSC 2003 [ 162649]), the SZ transport model report (BSC 2003 [ 162419]), the SZ colloid transport report (BSC 2003 [162729]), and the SZ transport model abstraction report (BSC 2003 [1648701]). Specifically, this scientific analysis report provides the following information that contributes to the assessment of the capability of the SZ to serve as a barrier for waste isolation for the Yucca Mountain repository system: (1) The bases for selection of conceptual flow and transport models in the saturated volcanics and the saturated alluvium located near Yucca Mountain. (2) Results and interpretations of hydraulic and tracer tests conducted in saturated fractured volcanics at the C-wells complex near Yucca Mountain. The test interpretations include estimates of hydraulic conductivities, anisotropy in hydraulic conductivity, storativities, total porosities, effective porosities, longitudinal dispersivities, matrix diffusion mass transfer coefficients, matrix diffusion coefficients, fracture apertures, and colloid transport parameters. (3) Results and interpretations of hydraulic and tracer tests conducted in saturated alluvium at the Alluvium Testing Complex (ATC), which is located at the southwestern corner of the Nevada Test Site (NTS). The test interpretations include estimates of hydraulic conductivities, storativities, total porosities, effective porosities, longitudinal dispersivities, matrix diffusion mass transfer coefficients, and

  18. Comparison of pulseoximetry oxygen saturation and arterial oxygen saturation in open heart intensive care unit

    Directory of Open Access Journals (Sweden)

    Alireza Mahoori

    2013-08-01

    Full Text Available Background: Pulseoximetry is widely used in the critical care setting, currently used to guide therapeutic interventions. Few studies have evaluated the accuracy of SPO2 (puls-eoximetry oxygen saturation in intensive care unit after cardiac surgery. Our objective was to compare pulseoximetry with arterial oxygen saturation (SaO2 during clinical routine in such patients, and to examine the effect of mild acidosis on this relationship.Methods: In an observational prospective study 80 patients were evaluated in intensive care unit after cardiac surgery. SPO2 was recorded and compared with SaO2 obtained by blood gas analysis. One or serial arterial blood gas analyses (ABGs were performed via a radial artery line while a reliable pulseoximeter signal was present. One hundred thirty seven samples were collected and for each blood gas analyses, SaO2 and SPO2 we recorded.Results: O2 saturation as a marker of peripheral perfusion was measured by Pulseoxim-etry (SPO2. The mean difference between arterial oxygen saturation and pulseoximetry oxygen saturation was 0.12%±1.6%. A total of 137 paired readings demonstrated good correlation (r=0.754; P<0.0001 between changes in SPO2 and those in SaO2 in samples with normal hemoglobin. Also in forty seven samples with mild acidosis, paired readings demonstrated good correlation (r=0.799; P<0.0001 and the mean difference between SaO2 and SPO2 was 0.05%±1.5%.Conclusion: Data showed that in patients with stable hemodynamic and good signal quality, changes in pulseoximetry oxygen saturation reliably predict equivalent changes in arterial oxygen saturation. Mild acidosis doesn’t alter the relation between SPO2 and SaO2 to any clinically important extent. In conclusion, the pulse oximeter is useful to monitor oxygen saturation in patients with stable hemodynamic.

  19. [Monitoring of jugular venous oxygen saturation].

    Science.gov (United States)

    Nakamura, Shunsuke

    2011-04-01

    The continuous monitoring of jugular venous oxygen saturation(SjO2) has become a practical method for monitoring global cerebral oxygenation and metabolism. SjO2 reflects the balance between the cerebral blood flow and the cerebral metabolic rate for oxygen (CMRO2), if arterial oxyhemoglobin saturation, hemoglobin concentration remain constant. Normal SjO2 values range between 55% and 75%. Low SjO2 indicates cerebral hypoperfusion or ischemia. Conversely, an increased SjO2 indicates either cerebral hyperemia or a disorder that decreases CMRO2. In minimizing secondary brain damage following resuscitation from cardiopulmonary arrest, SjO2 monitoring is thus considered to be an integral part of multimodality monitoring and can provide important information for the management of patients in neurointensive care.

  20. The danish tax on saturated fat

    DEFF Research Database (Denmark)

    Jensen, Jørgen Dejgård; Smed, Sinne

    Denmark introduced a new tax on saturated fat in food products with effect from October 2011. The objective of this paper is to make an effect assessment of this tax for some of the product categories most significantly affected by the new tax, namely fats such as butter, butter-blends, margarine...... on saturated fat in food products has had some effects on the market for the considered products, in that the level of consumption of fats dropped by 10 – 20%. Furthermore, the analysis points at shifts in demand from high-price supermarkets towards low-price discount stores – a shift that seems to have been...... – and broaden – the analysis at a later stage, when data are available for a longer period after the introduction of the fat tax....

  1. Nonlinear saturation of Weibel-type instabilities

    Science.gov (United States)

    Srinivasan, Bhuvana; Cagas, Petr; Hakim, Ammar

    2017-10-01

    Weibel-type instabilities, which grow in plasmas with anisotropic velocity distribution, have been studied for many years and drawn recent interest due to their broad applicability spanning from laboratory laser plasmas to origins of intergalactic magnetic fields in astrophysical plasmas. Magnetic particle trapping has been considered as the main mechanism of the nonlinear saturation of these instabilities. However, novel continuum kinetic and two-fluid five moment simulations show that there are additional effects - the transverse flow introduced by the magnetic field creates a secondary electrostatic two-stream instability which alters the saturation and is responsible for a quasi-periodic behavior in the nonlinear phase. This research was supported by the Air Force Office of Scientific Research under Grant Number FA9550-15-1-0193.

  2. Gluon saturation beyond (naive) leading logs

    Energy Technology Data Exchange (ETDEWEB)

    Beuf, Guillaume

    2014-12-15

    An improved version of the Balitsky–Kovchegov equation is presented, with a consistent treatment of kinematics. That improvement allows to resum the most severe of the large higher order corrections which plague the conventional versions of high-energy evolution equations, with approximate kinematics. This result represents a further step towards having high-energy QCD scattering processes under control beyond strict Leading Logarithmic accuracy and with gluon saturation effects.

  3. 2D Saturable Absorbers for Fibre Lasers

    Directory of Open Access Journals (Sweden)

    Robert I. Woodward

    2015-11-01

    Full Text Available Two-dimensional (2D nanomaterials are an emergent and promising platform for future photonic and optoelectronic applications. Here, we review recent progress demonstrating the application of 2D nanomaterials as versatile, wideband saturable absorbers for Q-switching and mode-locking fibre lasers. We focus specifically on the family of few-layer transition metal dichalcogenides, including MoS2, MoSe2 and WS2.

  4. Stabilization of Neutral Systems with Saturating Actuators

    Directory of Open Access Journals (Sweden)

    F. El Haoussi

    2012-01-01

    to determine stabilizing state-feedback controllers with large domain of attraction, expressed as linear matrix inequalities, readily implementable using available numerical tools and with tuning parameters that make possible to select the most adequate solution. These conditions are derived by using a Lyapunov-Krasovskii functional on the vertices of the polytopic description of the actuator saturations. Numerical examples demonstrate the effectiveness of the proposed technique.

  5. 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…

  6. Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe.

    Directory of Open Access Journals (Sweden)

    Roni F Rayes

    Full Text Available The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.

  7. Saturated Dispersive Extinction Theory of Red Shift

    Science.gov (United States)

    Wang, Ling Jun

    2012-03-01

    The Dispersive Extinction Theory (DET) proposed by WangfootnotetextWang, Ling Jun, Physics Essays, 18, No. 2, (2005). offers an alternative to the Big Bang. According to DET, the cosmic red shift is caused by the dispersive extinction of the star light during the propagation from the stars to the earth, instead of being caused by the Doppler shift due to the expansion of the universe.footnotetextHubble, E., Astrophys. J. 64, 321 (1926).^,footnotetextHubble, E., The Realm of the Nebulae, (Yale University Press, New Haven, 1936). DET allows an infinite, stable, non expanding universe, and is immune of the fundamental problems inherent to the Big Bang such as the horizon problem, the extreme violation of the conservation of mass, energy and charge, and the geocentric nature which violates the principle of relativity.footnotetextWang, Ling Jun, Physics Essays, 20, No. 2, (2007). The scenario dealt with in Reference (1) is a one in which the extinction by the space medium is not saturated. This work deals with a different scenario when the extinction is saturated. The saturated extinction causes limited energy loss, and the star light can travel a much greater distance than in the unsaturated scenario.

  8. Optimal oxygen saturation in premature infants

    Directory of Open Access Journals (Sweden)

    Meayoung Chang

    2011-09-01

    Full Text Available There is a delicate balance between too little and too much supplemental oxygen exposure in premature infants. Since underuse and overuse of supplemental oxygen can harm premature infants, oxygen saturation levels must be monitored and kept at less than 95% to prevent reactive oxygen species-related diseases, such as retinopathy of prematurity and bronchopulmonary dysplasia. At the same time, desaturation below 80 to 85% must be avoided to prevent adverse consequences, such as cerebral palsy. It is still unclear what range of oxygen saturation is appropriate for premature infants; however, until the results of further studies are available, a reasonable target for pulse oxygen saturation (SpO2 is 90 to 93% with an intermittent review of the correlation between SpO2 and the partial pressure of arterial oxygen tension (PaO2. Because optimal oxygenation depends on individuals at the bedside making ongoing adjustments, each unit must define an optimal target range and set alarm limits according to their own equipment or conditions. All staff must be aware of these values and adjust the concentration of supplemental oxygen frequently.

  9. Targeted mutagenesis in the malaria mosquito using TALE nucleases.

    Directory of Open Access Journals (Sweden)

    Andrea L Smidler

    Full Text Available Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs, and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.

  10. The influence of saturation on the cracking process in compacted desiccating clays

    Directory of Open Access Journals (Sweden)

    Noack Maria

    2016-01-01

    Full Text Available Tensile failure in unsaturated cohesive soils during desiccation is important for the design of geotechnical applications such as capping systems of landfills and sealing material of dikes. This study presents the results of different initial parameters of compacted clay samples such as gravimetric water content, dry density and degree of saturation. These parameters are varied systematically for each test to find the correlation between those parameters and the tensile failure. The tensile failure for all tests occurred by a comparable constant change of the degree of saturation. The soil specific saturation ratio Sr,s is defined as the quotient of saturation changes ΔSr to initial saturation Sr,0. This parameter related to the total suction shows an equal course for all results. All in all, the course of the soil specific saturation ratio Sr,s is independent of all initial parameters. These results provide a physical and hydraulic-mechanical description for modelling the desiccation process. To demonstrate the initiation and progress of tensile failure, the experimental results are modelled with a Discrete Element Method (DEM approach.

  11. Correcting reaction rates measured by saturation-transfer magnetic resonance spectroscopy

    Science.gov (United States)

    Gabr, Refaat E.; Weiss, Robert G.; Bottomley, Paul A.

    2008-04-01

    Off-resonance or spillover irradiation and incomplete saturation can introduce significant errors in the estimates of chemical rate constants measured by saturation-transfer magnetic resonance spectroscopy (MRS). Existing methods of correction are effective only over a limited parameter range. Here, a general approach of numerically solving the Bloch-McConnell equations to calculate exchange rates, relaxation times and concentrations for the saturation-transfer experiment is investigated, but found to require more measurements and higher signal-to-noise ratios than in vivo studies can practically afford. As an alternative, correction formulae for the reaction rate are provided which account for the expected parameter ranges and limited measurements available in vivo. The correction term is a quadratic function of experimental measurements. In computer simulations, the new formulae showed negligible bias and reduced the maximum error in the rate constants by about 3-fold compared to traditional formulae, and the error scatter by about 4-fold, over a wide range of parameters for conventional saturation transfer employing progressive saturation, and for the four-angle saturation-transfer method applied to the creatine kinase (CK) reaction in the human heart at 1.5 T. In normal in vivo spectra affected by spillover, the correction increases the mean calculated forward CK reaction rate by 6-16% over traditional and prior correction formulae.

  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. 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

  14. Improving ethanol fermentation performance of Saccharomyces cerevisiae in very high-gravity fermentation through chemical mutagenesis and meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing-Jing; Ding, Wen-Tao; Zhang, Guo-Chang; Wang, Jing-Yu [Tianjin Univ. (China). Dept. of Biochemical Engineering

    2011-08-15

    Genome shuffling is an efficient way to improve complex phenotypes under the control of multiple genes. For the improvement of strain's performance in very high-gravity (VHG) fermentation, we developed a new method of genome shuffling. A diploid ste2/ste2 strain was subjected to EMS (ethyl methanesulfonate) mutagenesis followed by meiotic recombination-mediated genome shuffling. The resulting haploid progenies were intrapopulation sterile and therefore haploid recombinant cells with improved phenotypes were directly selected under selection condition. In VHG fermentation, strain WS1D and WS5D obtained by this approach exhibited remarkably enhanced tolerance to ethanol and osmolarity, increased metabolic rate, and 15.12% and 15.59% increased ethanol yield compared to the starting strain W303D, respectively. These results verified the feasibility of the strain improvement strategy and suggested that it is a powerful and high throughput method for development of Saccharomyces cerevisiae strains with desired phenotypes that is complex and cannot be addressed with rational approaches. (orig.)

  15. PLD-grown thin film saturable absorbers

    Energy Technology Data Exchange (ETDEWEB)

    Tellkamp, Friedjof

    2012-11-01

    The subject of this thesis is the preparation and characterization of thin films made of oxidic dielectrics which may find their application as saturable absorber in passively Q-switched lasers. The solely process applied for fabrication of the thin films was the pulsed laser deposition (PLD) which stands out against other processes by its flexibility considering the composition of the systems to be investigated. Within the scope of this thesis the applied saturable absorbers can be divided into two fundamentally different kinds of functional principles: On the one hand, saturable absorption can be achieved by ions embedded in a host medium. Most commonly applied bulk crystals are certain garnets like YAG (Y{sub 3}Al{sub 5}O{sub 12}) or the spinel forsterite (Mg{sub 2}SiO{sub 4}), in each case with chromium as dopant. Either of these media was investigated in terms of their behavior as PLD-grown saturable absorber. Moreover, experiments with Mg{sub 2}GeO{sub 4}, Ca{sub 2}GeO{sub 4}, Sc{sub 2}O{sub 3}, and further garnets like YSAG or GSGG took place. The absorption coefficients of the grown films of Cr{sup 4+}:YAG were determined by spectroscopic investigations to be one to two orders of magnitude higher compared to commercially available saturable absorbers. For the first time, passive Q-switching of a Nd:YAG laser at 1064 nm with Cr{sup 4+}:YAG thin films could be realized as well as with Cr:Sc{sub 2}O{sub 3} thin films. On the other hand, the desirable effect of saturable absorption can also be generated by quantum well structures. For this purpose, several layer system like YAG/LuAG, Cu{sub 2}O/MgO, and ZnO/corumdum were investigated. It turned out that layer systems with indium oxide (In{sub 2}O{sub 3}) did not only grew in an excellent way but also showed up a behavior regarding their photo luminescence which cannot be explained by classical considerations. The observed luminescence at roughly 3 eV (410 nm) was assumed to be of excitonic nature and its

  16. Trans Fat Now Listed With Saturated Fat and Cholesterol

    Science.gov (United States)

    ... Trans Fat Now Listed With Saturated Fat and Cholesterol Share Tweet Linkedin Pin it More sharing options ... I Do About Saturated Fat, Trans Fat, and Cholesterol? When comparing foods, look at the Nutrition Facts ...

  17. Calcium phosphate saturation in the western Bay of Bengal

    Digital Repository Service at National Institute of Oceanography (India)

    Naik, S.; Reddy, C.V.G.

    Temperature, inorganic phosphate concentration and pH seem to be the major factors influencing the degree of saturation of calcium phosphate in sea water. Two water regions can be demarcated in the study area based on the saturation patterns...

  18. Nonmonotone Saturation Profiles for Hydrostatic Equilibrium in Homogeneous Porous Media

    NARCIS (Netherlands)

    Hilfer, R.; Doster, F.; Zegeling, P.A.|info:eu-repo/dai/nl/073634433

    2012-01-01

    Nonmonotonic saturation profiles (saturation overshoot) occur as travelling waves in gravity driven fingering. They seem important for preferential flow mechanisms and have found much attention recently. Here, we predict them even for hydrostatic equilibrium when all velocities vanish. We suggest

  19. Heavy Flavor Production in DGLAP improved Saturation Model

    CERN Document Server

    Sapeta, S

    2007-01-01

    The saturation model with DGLAP evolution is shown to give good description of the production of the charm and beauty quarks in deep inelastic scattering. The modifications of saturation properties caused by the presence of heavy quarks are also discussed.

  20. Rigid aleph_epsilon-saturated models of superstable theories

    OpenAIRE

    Shami, Ziv; Shelah, Saharon

    1999-01-01

    In a countable superstable NDOP theory, the existence of a rigid aleph_epsilon-saturated model implies the existence of 2^lambda rigid aleph_epsilon-saturated models of power lambda for every lambda>2^{aleph_0}.

  1. Water Flow in Karst Aquifer Considering Dynamically Variable Saturation Conduit

    Science.gov (United States)

    Tan, Chaoqun; Hu, Bill X.

    2017-04-01

    The karst system is generally conceptualized as dual-porosity system, which is characterized by low conductivity and high storage continuum matrix and high conductivity and quick flow conduit networks. And so far, a common numerical model for simulating flow in karst aquifer is MODFLOW2005-CFP, which is released by USGS in 2008. However, the steady-state approach for conduit flow in CFP is physically impractical when simulating very dynamic hydraulics with variable saturation conduit. So, we adopt the method proposed by Reimann et al. (2011) to improve current model, in which Saint-Venant equations are used to model the flow in conduit. Considering the actual background that the conduit is very big and varies along flow path and the Dirichlet boundary varies with rainfall in our study area in Southwest China, we further investigate the influence of conduit diameter and outflow boundary on numerical model. And we also analyze the hydraulic process in multi-precipitation events. We find that the numerical model here corresponds well with CFP for saturated conduit, and it could depict the interaction between matrix and conduit during very dynamic hydraulics pretty well compare with CFP.

  2. Analysis of a Heroin Epidemic Model with Saturated Treatment Function

    Directory of Open Access Journals (Sweden)

    Isaac Mwangi Wangari

    2017-01-01

    Full Text Available A mathematical model is developed that examines how heroin addiction spreads in society. The model is formulated to take into account the treatment of heroin users by incorporating a realistic functional form that “saturates” representing the limited availability of treatment. Bifurcation analysis reveals that the model has an intrinsic backward bifurcation whenever the saturation parameter is larger than a fixed threshold. We are particularly interested in studying the model’s global stability. In the absence of backward bifurcations, Lyapunov functions can often be found and used to prove global stability. However, in the presence of backward bifurcations, such Lyapunov functions may not exist or may be difficult to construct. We make use of the geometric approach to global stability to derive a condition that ensures that the system is globally asymptotically stable. Numerical simulations are also presented to give a more complete representation of the model dynamics. Sensitivity analysis performed by Latin hypercube sampling (LHS suggests that the effective contact rate in the population, the relapse rate of heroin users undergoing treatment, and the extent of saturation of heroin users are mechanisms fuelling heroin epidemic proliferation.

  3. Silica fractal atomic clusters saturated with OH

    CERN Document Server

    Olivi-Tran, N

    2003-01-01

    We constructed regular fractal SiOH atomic clusters which pending bonds are saturated with OH molecules. We calculated the binding energies of these clusters as well as for sp sup 2 hybridization as for sp sup 3 hybridizations. The result are the following: for the two hybridizations, the total binding energies have a linear dependence on the size of the fractal cluster, which comes directly from the scaling law of the fractal characteristic of the building of the cluster. We related by a scaling law, the number of electronic bonds and the total bonding energy.

  4. Chloride diffusion in partially saturated cementitious material

    DEFF Research Database (Denmark)

    Nielsen, Erik Pram; Geiker, Mette Rica

    2003-01-01

    The paper proposes a combined application of composite theory and Powers' model for microstructural development for the estimation of the diffusion coefficient as a function of the moisture content of a defect-free cementitious material. Measurements of chloride diffusion in mortar samples (440 kg....../m(3) rapid-hardening Portland cement, w/c = 0.5, maturity minimum 6 months) stored at 65% and 85% RH, as well as in vacuum-saturated mortar samples, illustrate the applicability of the method. (C) 2003 Elsevier Science Ltd. All rights reserved....

  5. Inheritance of reduced saturated fatty acid content in sunflower oil

    OpenAIRE

    Vick Brady A.; Jan C.C.; Miller Jerry F.

    2002-01-01

    In recent years, consumers have become concerned with reducing the saturated fat content of their diet. Studies have indicated that high levels of saturated fat consumption are correlated with increased risk of coronary heart disease. The total saturated fat content of oil from current sunflower hybrids averages about 130 g kg-1. To identify sunflower germplasm with reduced saturated fatty acid composition, a total of 884 cultivated sunflower accessions from the USDA-ARS North Central Regiona...

  6. 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.

  7. 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.

  8. 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.

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

    Science.gov (United States)

    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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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

  16. 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.

  17. Test of Scintillometer Saturation Correction Methods Using Field Experimental Data

    NARCIS (Netherlands)

    Kleissl, J.; Hartogensis, O.K.; Gomez, J.D.

    2010-01-01

    Saturation of large aperture scintillometer (LAS) signals can result in sensible heat flux measurements that are biased low. A field study with LASs of different aperture sizes and path lengths was performed to investigate the onset of, and corrections for, signal saturation. Saturation already

  18. A demonstration experiment for studying the properties of saturated vapor

    Science.gov (United States)

    Grebenev, Igor V.; Lebedeva, Olga V.; Polushkina, Svetlana V.

    2017-11-01

    The paper proposes an important demonstration experiment that can be used at secondary schools in physics. The described experiment helps students learn the main concepts of the topic ‘saturated vapor’, namely, evaporation, condensation, dynamic equilibrium, saturation vapor, partial pressure, and the dependence of saturated vapor pressure on temperature.

  19. 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...

  20. 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

  1. 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...

  2. 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 ...

  3. 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.

  4. High-Temperature Studies of Glass Dissolution Rates Close to Saturation

    Energy Technology Data Exchange (ETDEWEB)

    Zavarin, M; Roberts, S; Zhao, P; Williams, R; Rose, T; Rainer, A; Pawloski, G

    2004-06-14

    Most long-lived radionuclides associated with an underground nuclear test are incorporated into a melt glass and are released by glass dissolution to become part of the hydrologic source term (HST) (Pawloski et al., 2001). Although the rates of rhyolite glass dissolution are well known under conditions where the fluid is far from saturation with respect to glass, the rates are not well known under conditions where the fluid approaches saturation. These rates are commonly much lower than the far-fromsaturation rates, often by a factor greater than 100. In recent HST simulations (Pawloski et al., 2001; Pawloski et al., 2000; Tompson et al., 1999), we conservatively estimated steady-state release rates based on a far-from-saturation fluid conditions. In recent CHESHIRE near-field simulations (Pawloski et al., 2001), it was predicted that {approx}30% of the nuclear melt glass dissolved over 1000 years. Although the ''far-from-saturation rate'' approach provides a conservative estimate of glass dissolution, it may greatly overestimate the rates of melt glass dissolution. At CHESHIRE, less conservative estimates suggest that only {approx}1% of the nuclear melt glass will dissolve in 1000 years. Lower glass dissolution rates result in lower radionuclide release rates from nuclear melt glass. The following report documents glass dissolution experiments performed to measure glass dissolution rates close to saturation.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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...

  16. SITE-SCALE SATURATED ZONE TRANSPORT

    Energy Technology Data Exchange (ETDEWEB)

    S. KELLER

    2004-11-03

    This work provides a site-scale transport model for calculating radionuclide transport in the saturated zone (SZ) at Yucca Mountain, for use in the abstractions model in support of ''Total System Performance Assessment for License Application'' (TSPA-LA). The purpose of this model report is to provide documentation for the components of the site-scale SZ transport model in accordance with administrative procedure AP-SIII.10Q, Models. The initial documentation of this model report was conducted under the ''Technical Work Plan For: Saturated Zone Flow and Transport Modeling and Testing'' (BSC 2003 [DIRS 163965]). The model report has been revised in accordance with the ''Technical Work Plan For: Natural System--Saturated Zone Analysis and Model Report Integration'', Section 2.1.1.4 (BSC 2004 [DIRS 171421]) to incorporate Regulatory Integration Team comments. All activities listed in the technical work plan that are appropriate to the transport model are documented in this report and are described in Section 2.1.1.4 (BSC 2004 [DIRS 171421]). This report documents: (1) the advection-dispersion transport model including matrix diffusion (Sections 6.3 and 6.4); (2) a description and validation of the transport model (Sections 6.3 and 7); (3) the numerical methods for simulating radionuclide transport (Section 6.4); (4) the parameters (sorption coefficient, Kd ) and their uncertainty distributions used for modeling radionuclide sorption (Appendices A and C); (5) the parameters used for modeling colloid-facilitated radionuclide transport (Table 4-1, Section 6.4.2.6, and Appendix B); and (6) alternative conceptual models and their dispositions (Section 6.6). The intended use of this model is to simulate transport in saturated fractured porous rock (double porosity) and alluvium. The particle-tracking method of simulating radionuclide transport is incorporated in the finite-volume heat and mass transfer numerical

  17. 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.

  18. A large-scale mutant panel in wheat developed using heavy-ion beam mutagenesis and its application to genetic research

    Energy Technology Data Exchange (ETDEWEB)

    Murai, Koji, E-mail: murai@fpu.ac.jp [Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195 (Japan); Nishiura, Aiko [Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195 (Japan); Kazama, Yusuke [RIKEN, Innovation Center, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Abe, Tomoko [RIKEN, Innovation Center, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); RIKEN, Nishina Center, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2013-11-01

    Mutation analysis is a powerful tool for studying gene function. Heavy-ion beam mutagenesis is a comparatively new approach to inducing mutations in plants and is particularly efficient because of its high linear energy transfer (LET). High LET radiation induces a higher rate of DNA double-strand breaks than other mutagenic methods. Over the last 12 years, we have constructed a large-scale mutant panel in diploid einkorn wheat (Triticum monococcum) using heavy-ion beam mutagenesis. Einkorn wheat seeds were exposed to a heavy-ion beam and then sown in the field. Selfed seeds from each spike of M{sub 1} plants were used to generate M{sub 2} lines. Every year, we obtained approximately 1000 M{sub 2} lines and eventually developed a mutant panel with 10,000 M{sub 2} lines in total. This mutant panel is being systematically screened for mutations affecting reproductive growth, and especially for flowering-time mutants. To date, we have identified several flowering-time mutants of great interest: non-flowering mutants (mvp: maintained vegetative phase), late-flowering mutants, and early-flowering mutants. These novel mutations will be of value for investigations of the genetic mechanism of flowering in wheat.

  19. Saturated hydraulic conductivity of US soils grouped according textural class and bulk density

    Science.gov (United States)

    Importance of the saturated hydraulic conductivity as soil hydraulic property led to the development of multiple pedotransfer functions for estimating it. One approach to estimating Ksat was using textural classes rather than specific textural fraction contents as pedotransfer inputs. The objective...

  20. Saturated hydraulic conductivity of US soils grouped according to textural class and bulk density

    Science.gov (United States)

    Importance of the saturated hydraulic conductivity as soil hydraulic property led to the development of multiple pedotransfer functions for estimating it. One approach to estimating Ksat was using textural classes rather than specific textural fraction contents as pedotransfer inputs. The objective...

  1. Hot magnetized nuclear matter: Thermodynamic and saturation properties

    Energy Technology Data Exchange (ETDEWEB)

    Rezaei, Z. [Shiraz University, Physics Department and Biruni Observatory, College of Sciences, Shiraz (Iran, Islamic Republic of); Bordbar, G.H. [Shiraz University, Physics Department and Biruni Observatory, College of Sciences, Shiraz (Iran, Islamic Republic of); Center for Excellence in Astronomy and Astrophysics (CEAA-RIAAM)-Maragha, Maragha (Iran, Islamic Republic of)

    2017-03-15

    We have used a realistic nuclear potential, AV{sub 18}, and a many-body technique, the lowest-order constraint variational (LOCV) approach, to calculate the properties of hot magnetized nuclear matter. By investigating the free energy, spin polarization parameter, and symmetry energy, we have studied the temperature and magnetic field dependence of the saturation properties of magnetized nuclear matter. In addition, we have calculated the equation of state of magnetized nuclear matter at different temperatures and magnetic fields. It was found that the flashing temperature of nuclear matter decreases by increasing the magnetic field. In addition, we have studied the effect of the magnetic field on liquid gas phase transition of nuclear matter. The liquid gas coexistence curves, the order parameter of the liquid gas phase transition, and the properties of critical point at different magnetic fields have been calculated. (orig.)

  2. Standing torsional waves in a fully saturated, porous, circular cylinder

    CERN Document Server

    Solorza, S; 10.1111/j.1365-246X.2004.02198.x

    2004-01-01

    For dynamic measurement of the elastic moduli of a porous material saturated with viscous fluid using the resonance-bar technique, one also observes attenuation. In this article we have carried out the solution of the boundary-value problem associated with standing torsional oscillations of a finite, poroelastic, circular cylinder cast in the framework of volume-averaged theory of poroelasticity. Analysing this solution by eigenvalue perturbation approach we are able to develop expressions for torsional resonance and temporal attenuation frequencies in which the dependence upon the material properties are transparent. It shows how the attenuation is controlled by the permeability and the fluid properties, and how the resonance frequency drops over its value for the dry solid-frame due to the drag effect of fluid mass. Based upon this work we have a firm basis to determine solid-frame shear modulus, permeability, and tortuosity factor from torsional oscillation experiments.

  3. Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome

    Directory of Open Access Journals (Sweden)

    Berghman Luc R

    2007-09-01

    Full Text Available Abstract Background A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. Results The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2 of Influenza A virus, a domain of CD154 (CD154s or a combination of both into the outer membrane protein LamB of S. enteritidis. Conclusion We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when

  4. [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.

  5. Energy dependent saturable and reverse saturable absorption in cube-like polyaniline/polymethyl methacrylate film

    Energy Technology Data Exchange (ETDEWEB)

    Thekkayil, Remyamol [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695 547 (India); Philip, Reji [Light and Matter Physics Group, Raman Research Institute, C.V. Raman Avenue, Bangalore 560 080 (India); Gopinath, Pramod [Department of Physics, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695 547 (India); John, Honey, E-mail: honey@iist.ac.in [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695 547 (India)

    2014-08-01

    Solid films of cube-like polyaniline synthesized by inverse microemulsion polymerization method have been fabricated in a transparent PMMA host by an in situ free radical polymerization technique, and are characterized by spectroscopic and microscopic techniques. The nonlinear optical properties are studied by open aperture Z-scan technique employing 5 ns (532 nm) and 100 fs (800 nm) laser pulses. At the relatively lower laser pulse energy of 5 μJ, the film shows saturable absorption both in the nanosecond and femtosecond excitation domains. An interesting switchover from saturable absorption to reverse saturable absorption is observed at 532 nm when the energy of the nanosecond laser pulses is increased. The nonlinear absorption coefficient increases with increase in polyaniline concentration, with low optical limiting threshold, as required for a good optical limiter. - Highlights: • Synthesized cube-like polyaniline nanostructures. • Fabricated polyaniline/PMMA nanocomposite films. • At 5 μJ energy, saturable absorption is observed both at ns and fs regime. • Switchover from SA to RSA is observed as energy of laser beam increases. • Film (0.1 wt % polyaniline) shows high β{sub eff} (230 cm GW{sup −1}) and low limiting threshold at 150 μJ.

  6. Femoral venous oxygen saturation and central venous oxygen saturation in critically ill patients.

    Science.gov (United States)

    Zhang, Xiaohong; Wang, Jiandong; Dong, Yun; Chen, Youdai

    2015-08-01

    To investigate the relationship between central venous oxygen saturation (ScvO(2)) and femoral venous oxygen saturation (SfvO(2)) in a large group of critically ill patients. Observational study. A group of unselected critically ill patients with central line placed into superior vena cava were included. A 26-bed intensive care unit in a tertiary referral hospital. None. Venous blood samples of superior vena cava and femoral vein were collected within an interval of 5 to 15 minutes and analyzed with blood gas/electrolyte analyzer immediately. Although SfvO(2) was significantly correlated with ScvO(2) (r = 0.493, P 731 pairs of blood samples collected from 357 patients. The fit line of scatter diagram ScvO(2) vs SfvO(2) had a large intercept (48.68%) and a low slope (0.2978); ScvO(2) was still around 50% while SfvO(2) was nearing 0%. The distribution of blood flow, measured with Doppler ultrasound, had a similar trend in 237 patients and 412 measurements. The ratio of femoral artery flow over common carotid artery flow varied widely (from 0 to 7.13). Blood flow was not distributed in a fixed ratio to the superior vena cava-drained organs and tissues. Central venous oxygen saturation was not representative of the whole systemic circulation in critically ill patients. Central venous oxygen saturation alone might be misleading in goal-directed therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. 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

  8. 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

    's primary sequence into a group of functionally non-disruptive amino acids and a second group of functionally deleterious amino acids. Conclusions With this approach, not only conserved amino acid positions in a protein family can be labeled as functionally relevant, but also non-conserved amino acid positions can be identified to have a physicochemically meaningful functional effect. These results become a discriminative tool in the selection and elaboration of rational mutagenesis strategies for the protein. They can also be used to predict if a given nsSNP, identified, for instance, in a genomic-scale analysis, can have a functional implication for a particular protein and which nsSNPs are most likely to be functionally silent for a protein. This analytical tool could be used to rapidly and automatically discard any irrelevant nsSNP and guide the research focus toward functionally significant mutations. Based on preliminary results and applications, this technique shows promising performance as a valuable bioinformatics tool to aid in the development of new protein variants and in the understanding of function-structure relationships in proteins.

  9. A sulfide-saturated lunar mantle?

    Science.gov (United States)

    Brenan, James M.; Mungall, James E.

    2017-04-01

    Although much work has been done to understand the controls on the sulfur content at sulfide saturation (SCSS) for terrestrial melt compositions, little information exists to evaluate the SCSS for the high FeO compositions typical of lunar magmas, and at the reduced conditions of the Moon's interior. Experiments were done to measure the SCSS for a model low Ti mare basalt with 20 wt% FeO at 1400oC as a function of fO2 and pressure. Synthetic lunar basalt was encapsulated along with stoichiometric FeS in capsules made from Fe-Ir alloy. The fO2 of the experiment can be estimated by the heterogeneous equilibrium: Femetal + 1 /2 O2 = FeOsilicate Variation in the metal composition, by addition of Ir, serves to change the fO2 of the experiment. Capsule compositions spanning the range Fe25Ir75 to Fe96Ir4 (at%) were synthesized by sintering of pressed powders under reducing conditions. Fe100 capsules were fabricated from pure Fe rod. For a melt with 20 wt% FeO, this range in capsule composition spans the fO2 interval of ˜IW-1 (Fe100, Fe96Ir4) to IW+2.2 (Fe25Ir75). Experiments were done over the pressure interval of 0.1 MPa to 2 GPa. Results for experiments involving Fe100capsules indicate that the SCSS decreases from ˜2000 ppm (0.1 MPa) to 700 ppm (2 GPa). Experiments done thus far at 1 GPa, involving the range of capsule compositions indicated, show a marked decrease in SCSS as the Fe content of the capsule increases (fO2 decreases). Complementary to the decrease in SCSS is a drop in the sulfur content of the coexisting sulfide melt, from ˜50 at% at ΔIW = +2.2 to ˜20 at% at ΔIW-1. In fact, both the composition of the sulfide melt and the SCSS are essentially indistinguishable for Fe96Ir4 and Fe100 compositions. Results thus far indicate that at reduced conditions and high pressure, the SCSS for high FeO lunar compositions is low, and overlaps with Apollo 11 melt inclusion data. Importantly, such low SCSS does not require Fe metal saturation, and suggests that some

  10. The Danish tax on saturated fat

    DEFF Research Database (Denmark)

    Jensen, Jørgen Dejgård; Smed, Sinne

    2013-01-01

    Denmark introduced a new tax on saturated fat in food products with effect from October 2011. The objective of this paper is to make an effect assessment of this tax for some of the product categories most significantly affected by the new tax, namely fats such as butter, butter-blends, margarine...... fat in food products has had some effects on the market for the considered products, in that the level of consumption of fats dropped by 10 – 20%. Furthermore, the analysis points at shifts in demand from high-price supermarkets towards low-price discount stores – at least for some types of oils...... and fats, a shift that seems to have been utilized by discount chains to raise the prices of butter and margarine by more than the pure tax increase. Due to the relatively short data period with the tax being active, interpretation of these findings from a long-run perspective should be done...

  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. Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis.

    Directory of Open Access Journals (Sweden)

    Jonathan J Ipsaro

    Full Text Available Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.

  13. High-Throughput Sequencing and Mutagenesis to Accelerate the Domestication of Microlaena stipoides as a New Food Crop

    Science.gov (United States)

    Shapter, Frances M.; Cross, Michael; Ablett, Gary; Malory, Sylvia; Chivers, Ian H.; King, Graham J.; Henry, Robert J.

    2013-01-01

    Global food demand, climatic variability and reduced land availability are driving the need for domestication of new crop species. The accelerated domestication of a rice-like Australian dryland polyploid grass, Microlaena stipoides (Poaceae), was targeted using chemical mutagenesis in conjunction with high throughput sequencing of genes for key domestication traits. While M. stipoides has previously been identified as having potential as a new grain crop for human consumption, only a limited understanding of its genetic diversity and breeding system was available to aid the domestication process. Next generation sequencing of deeply-pooled target amplicons estimated allelic diversity of a selected base population at 14.3 SNP/Mb and identified novel, putatively mutation-induced polymorphisms at about 2.4 mutations/Mb. A 97% lethal dose (LD97) of ethyl methanesulfonate treatment was applied without inducing sterility in this polyploid species. Forward and reverse genetic screens identified beneficial alleles for the domestication trait, seed-shattering. Unique phenotypes observed in the M2 population suggest the potential for rapid accumulation of beneficial traits without recourse to a traditional cross-breeding strategy. This approach may be applicable to other wild species, unlocking their potential as new food, fibre and fuel crops. PMID:24367532

  14. High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop.

    Directory of Open Access Journals (Sweden)

    Frances M Shapter

    Full Text Available Global food demand, climatic variability and reduced land availability are driving the need for domestication of new crop species. The accelerated domestication of a rice-like Australian dryland polyploid grass, Microlaena stipoides (Poaceae, was targeted using chemical mutagenesis in conjunction with high throughput sequencing of genes for key domestication traits. While M. stipoides has previously been identified as having potential as a new grain crop for human consumption, only a limited understanding of its genetic diversity and breeding system was available to aid the domestication process. Next generation sequencing of deeply-pooled target amplicons estimated allelic diversity of a selected base population at 14.3 SNP/Mb and identified novel, putatively mutation-induced polymorphisms at about 2.4 mutations/Mb. A 97% lethal dose (LD₉₇ of ethyl methanesulfonate treatment was applied without inducing sterility in this polyploid species. Forward and reverse genetic screens identified beneficial alleles for the domestication trait, seed-shattering. Unique phenotypes observed in the M2 population suggest the potential for rapid accumulation of beneficial traits without recourse to a traditional cross-breeding strategy. This approach may be applicable to other wild species, unlocking their potential as new food, fibre and fuel crops.

  15. Inclusive hadron distributions in p+p collisions from saturation models of HERA DIS data.

    Energy Technology Data Exchange (ETDEWEB)

    Tribedy, P.; Venugopalan, R.

    2010-12-06

    Dipole models based on various saturation scenarios provide reasonable fits to small-x DIS inclusive, diffractive and exclusive data from HERA. Proton un-integrated gluon distributions extracted from such fits are employed in a k{sub {perpendicular}}-factorization framework to calculate inclusive gluon distributions at various energies. The n-particle multiplicity distribution predicted in the Glasma flux tube approach shows good agreement with data over a wide range of energies. Hadron inclusive transverse momentum distributions expressed in terms of the saturation scale demonstrate universal behavior over a wider kinematic range systematically with increasing center of mass energies.

  16. Using random forests to explore the effects of site attributes and soil properties on near-saturated and saturated hydraulic conductivity

    Science.gov (United States)

    Jorda, Helena; Koestel, John; Jarvis, Nicholas

    2014-05-01

    Knowledge of the near-saturated and saturated hydraulic conductivity of soil is fundamental for understanding important processes like groundwater contamination risks or runoff and soil erosion. Hydraulic conductivities are however difficult and time-consuming to determine by direct measurements, especially at the field scale or larger. So far, pedotransfer functions do not offer an especially reliable alternative since published approaches exhibit poor prediction performances. In our study we aimed at building pedotransfer functions by growing random forests (a statistical learning approach) on 486 datasets from the meta-database on tension-disk infiltrometer measurements collected from peer-reviewed literature and recently presented by Jarvis et al. (2013, Influence of soil, land use and climatic factors on the hydraulic conductivity of soil. Hydrol. Earth Syst. Sci. 17(12), 5185-5195). When some data from a specific source publication were allowed to enter the training set whereas others were used for validation, the results of a 10-fold cross-validation showed reasonable coefficients of determination of 0.53 for hydraulic conductivity at 10 cm tension, K10, and 0.41 for saturated conductivity, Ks. The estimated average annual temperature and precipitation at the site were the most important predictors for K10, while bulk density and estimated average annual temperature were most important for Ks prediction. The soil organic carbon content and the diameter of the disk infiltrometer were also important for the prediction of both K10 and Ks. However, coefficients of determination were around zero when all datasets of a specific source publication were excluded from the training set and exclusively used for validation. This may indicate experimenter bias, or that better predictors have to be found or that a larger dataset has to be used to infer meaningful pedotransfer functions for saturated and near-saturated hydraulic conductivities. More research is in progress

  17. Experimental Bullard-von Karman dynamo: MHD saturated regimes

    Science.gov (United States)

    Miralles, Sophie; Plihon, Nicolas; Pinton, Jean-François

    2014-05-01

    The dynamo instability, converting kinetic energy into magnetic energy, creates the magnetic fields of many astrophysical bodies for which the flows are highly turbulent. Those turbulent fluctuations restricts the range of parameters of numerical and theoretical predictions. As laboratory experiments are closer from natural parameters, this approach is favored in this work. In the past decades, dynamo action has been observed in experiments involving laminar flows [1] or fully turbulent flows [2] in liquid sodium. Nevertheless, the saturation of the velocity field by the Lorentz force due to the dynamo magnetic field is weak in those experiment because the control parameter is always close to the threshold of the instability (which is not the case in astrophysical situations). The details of the mechanism of the back reaction of Lorentz force on the flow are not known. We present here an experimental semi-synthetic dynamo, for which a fluid turbulent induction mechanism ('omega' effect) is associated to an external amplification applying a current into a pair of coils. The flow, called von-Karman, is produced by the counter rotation of two coaxial propellers in a cylindrical tank filled with liquid gallium. The resulting flow is highly turbulent (Re > 10 ^ 5). The amplification, mimicking a turbulent 'alpha' effect, allow to observe the dynamo instability at low magnetic Reynolds number (Rm ~ 2), far below the threshold of natural homogeneous dynamo. This experiment reaches non linear regimes, for which the saturation is a MHD process, at control parameter several times the critical value. The instability grows through an on-off intermittent regime evolving into a full MHD saturated regime for which the Lorentz force is in balance with the inertial one. The power budget is strongly modified by the dynamo magnetic field and we give an insight of the estimated rate of conversion of kinetic energy into magnetic one from experimental data. Very rich regimes such as

  18. Saturation of Van Allen's belts; Saturation des ceintures de Van Allen

    Energy Technology Data Exchange (ETDEWEB)

    Le Bel, E.; Simonet, F. [CEA Bruyeres-le-Chatel, 91 (France)

    2002-12-01

    The maximum number of electrons that can be trapped in van Allen's belts has been evaluated at CEA-DAM more precisely than that commonly used in the space community. The modelization that we have developed allows to understand the disagreement (factor 50) observed between the measured and predicted electrons flux by US satellites and theory. This saturation level allows sizing-up of the protection on a satellite in case of energetic events. (authors)

  19. Reaching saturation in patterned source vertical organic field effect transistors

    Science.gov (United States)

    Greenman, Michael; Sheleg, Gil; Keum, Chang-min; Zucker, Jonathan; Lussem, Bjorn; Tessler, Nir

    2017-05-01

    Like most of the vertical transistors, the Patterned Source Vertical Organic Field Effect Transistor (PS-VOFET) does not exhibit saturation in the output characteristics. The importance of achieving a good saturation is demonstrated in a vertical organic light emitting transistor; however, this is critical for any application requiring the transistor to act as a current source. Thereafter, a 2D simulation tool was used to explain the physical mechanisms that prevent saturation as well as to suggest ways to overcome them. We found that by isolating the source facet from the drain-source electric field, the PS-VOFET architecture exhibits saturation. The process used for fabricating such saturation-enhancing structure is then described. The new device demonstrated close to an ideal saturation with only 1% change in the drain-source current over a 10 V change in the drain-source voltage.

  20. Brain oxygen saturation assessment in neonates using T2-prepared blood imaging of oxygen saturation and near-infrared spectroscopy.

    Science.gov (United States)

    Alderliesten, Thomas; De Vis, Jill B; Lemmers, Petra Ma; Hendrikse, Jeroen; Groenendaal, Floris; van Bel, Frank; Benders, Manon Jnl; Petersen, Esben T

    2017-03-01

    Although near-infrared spectroscopy is increasingly being used to monitor cerebral oxygenation in neonates, it has a limited penetration depth. The T2-prepared Blood Imaging of Oxygen Saturation (T2-BIOS) magnetic resonance sequence provides an oxygen saturation estimate on a voxel-by-voxel basis, without needing a respiratory calibration experiment. In 15 neonates, oxygen saturation measured by T2-prepared blood imaging of oxygen saturation and near-infrared spectroscopy were compared. In addition, these measures were compared to cerebral blood flow and venous oxygen saturation in the sagittal sinus. A strong linear relation was found between the oxygen saturation measured by magnetic resonance imaging and the oxygen saturation measured by near-infrared spectroscopy ( R2 = 0.64, p infrared spectroscopy oxygen saturation, and magnetic resonance imaging measures of frontal cerebral blood flow, whole brain cerebral blood flow and venous oxygen saturation in the sagittal sinus ( R2 = 0.71, 0.50, 0.65; p infrared spectroscopy and T2-prepared blood imaging of oxygen saturation, confirming the validity of using of these techniques for determining cerebral oxygenation.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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...

  9. Quantitative 1D saturation profiles on chalk by NMR

    DEFF Research Database (Denmark)

    Olsen, Dan; Topp, Simon; Stensgaard, Anders

    1996-01-01

    Quantitative one-dimensional saturation profiles showing the distribution of water and oil in chalk core samples are calculated from NMR measurements utilizing a 1D CSI spectroscopy pulse sequence. Saturation profiles may be acquired under conditions of fluid flow through the sample. Results reveal...... that strong saturation gradients exist in chalk core samples after core floods, due to capillary effects. The method is useful in analysis of corefloods, e.g., for determination of capillary pressure functions...

  10. Saturated fat -a never ending story?

    Science.gov (United States)

    Svendsen, Karianne; Arnesen, Erik; Retterstøl, Kjetil

    2017-01-01

    Science has no clear message regarding health effects of saturated fats, it seems. Different RCTs, prospective cohort studies and meta-analysis have led to contrasting conclusions. The aim of the present commentary is to discuss some possible reasons for an apparently never-ending fat controversy. They are of a purely scientific nature, which is important to recognize, but unfortunately hard to overcome. First is the placebo problem. In pharmaceutical science, evidence-based medicine is often synonymous with data on verified medical events from long-lasting double-blind randomized placebo controlled trials. In nutritional science the lack of double-blind design and lack of placebo food generate less conclusive data than those achieved in pharmaceutical science. Some scientists may apply the same type of scientific criteria used to evaluate the effects of drugs for foods. This leaves an impression of insufficient data since in this respect the fundamental criteria for evidence based medicine are not present. The next scientific problem is the energy balance equation. In contrast to pharmaceuticals, nutrients contain energy. An increased intake of one nutrient will lead to a decreased intake of another. The effect of change in only one nutrient is then difficult to isolate. Lastly, in nutritional science, generalizability is difficult compared to pharmaceutical science. Food culture interferes with lifestyle and food habits change over time. In conclusion, all available knowledge, from molecular experiments to population studies, must be taken in to account, to convert scientific data into dietary recommendations.

  11. Assaying Carcinoembryonic Antigens by Normalized Saturation Magnetization

    Science.gov (United States)

    Huang, Kai-Wen; Chieh, Jen-Jie; Shi, Jin-Cheng; Chiang, Ming-Hsien

    2015-07-01

    Biofunctionalized magnetic nanoparticles (BMNs) that provide unique advantages have been extensively used to develop immunoassay methods. However, these developed magnetic methods have been used only for specific immunoassays and not in studies of magnetic characteristics of materials. In this study, a common vibration sample magnetometer (VSM) was used for the measurement of the hysteresis loop for different carcinoembryonic antigens (CEA) concentrations ( Φ CEA) based on the synthesized BMNs with anti-CEA coating. Additionally, magnetic parameters such as magnetization ( M), remanent magnetization ( M R), saturation magnetization ( M S), and normalized parameters (Δ M R/ M R and Δ M S/ M S) were studied. Here, Δ M R and Δ M s were defined as the difference between any ΦCEA and zero Φ CEA. The parameters M, Δ M R, and Δ M S increased with Φ CEA, and Δ M S showed the largest increase. Magnetic clusters produced by the conjugation of the BMNs to CEAs showed a Δ M S greater than that of BMNs. Furthermore, the relationship between Δ M S/ M S and Φ CEA could be described by a characteristic logistic function, which was appropriate for assaying the amount of CEAs. This analytic Δ M S/ M S and the BMNs used in general magnetic immunoassays can be used for upgrading the functions of the VSM and for studying the magnetic characteristics of materials.

  12. SATURATED ZONE FLOW AND TRANSPORT MODEL ABSTRACTION

    Energy Technology Data Exchange (ETDEWEB)

    B.W. ARNOLD

    2004-10-27

    The purpose of the saturated zone (SZ) flow and transport model abstraction task is to provide radionuclide-transport simulation results for use in the total system performance assessment (TSPA) for license application (LA) calculations. This task includes assessment of uncertainty in parameters that pertain to both groundwater flow and radionuclide transport in the models used for this purpose. This model report documents the following: (1) The SZ transport abstraction model, which consists of a set of radionuclide breakthrough curves at the accessible environment for use in the TSPA-LA simulations of radionuclide releases into the biosphere. These radionuclide breakthrough curves contain information on radionuclide-transport times through the SZ. (2) The SZ one-dimensional (I-D) transport model, which is incorporated in the TSPA-LA model to simulate the transport, decay, and ingrowth of radionuclide decay chains in the SZ. (3) The analysis of uncertainty in groundwater-flow and radionuclide-transport input parameters for the SZ transport abstraction model and the SZ 1-D transport model. (4) The analysis of the background concentration of alpha-emitting species in the groundwater of the SZ.

  13. Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection.

    Directory of Open Access Journals (Sweden)

    Irene Bianconi

    2011-02-01

    Full Text Available The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.

  14. Expanding the Lotus japonicus reverse genetics toolbox – Development of LORE1 retrotransposon mutagenesis and artificial miRNA-mediated silencing

    DEFF Research Database (Denmark)

    Urbanski, Dorian Fabian

    2011-01-01

    . The protocols developed in the current project are now the cornerstone of a new LORE1 reverse genetics resource characterized by efficient mutant line generation and accurate mutation annotation. In parallel, artificial microRNAs (amiRNAs) were designed based on both Arabidopsis and Lotus backbones......Currently, the most common approach to studying Lotus japonicus (Lotus) genes is forward genetics in which a gene responsible for the studied phenotype is identified through map-based cloning. In reverse genetics, the activity of a gene of interest is modified to discover its mutant phenotype....... Prior to this project, the only reverse genetics resource available in Lotus was the TILLING resource. In an attempt to advance Lotus genetic studies, present study is focused on the development of two additional resources. The first is based on insertional mutagenesis and the second on harnessing post...

  15. Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool.

    Directory of Open Access Journals (Sweden)

    Katherine F Donovan

    Full Text Available CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.

  16. 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

  17. Can Polyphosphate Biochemistry Affect Biological Apatite Saturation?

    Science.gov (United States)

    Omelon, S. J.; Matsuura, N.; Gorelikov, I.; Wynnyckyj, C.; Grynpas, M. D.

    2010-12-01

    Phosphorus (P) is an important and limiting element for life. One strategy for storing ortho phosphates (Pi) is polymerization. Polymerized Pi's (polyphosphates: (PO3-)n: polyPs) serve as a Pi bank, as well as a catiion chelator, energy source, & regulator of responses to stresses in the stationary phase of culture growth and development1. PolyP biochemistry has been investigated in yeasts, bacteria & plants2. Bigeochemical cycling of P includes the condensation of Pi into pyro (P2O7-4), & polyPs, & the release of Pi from these compounds by the hydrolytic degradation of Pi from phosphomonoester bonds. Alkaline phosphatase (ALP) is one of the predominate enzymes for regenerating Pi in aquatic systems3, & it cleaves Pi from polyPs. ALP is also the enzyme associated with apatite biomineralization in vertebrates4. PolyP was proposed to be the ALP substrate in bone mineralization5. Where calcium ions are plentiful in many aquatic environments, there is no requirement for aquatic life to generate Ca-stores. However, terrestrial vertebrates benefit from a bioavailable Ca-store such as apatite. The Pi storage strategy of polymerizing PO4-3 into polyPs dovetails well with Ca-banking, as polyPs sequester Ca, forming a neutral calcium polyphosphate (Ca-polyP: (Ca(PO3)2)n) complex. This neutral complex represents a high total [Ca+2] & [PO4-3], without the threat of inadvertent apatite precipitation, as the free [Ca+2] & [PO4-3], and therefore apatite saturation, are zero. Recent identification of polyP in regions of bone resorption & calcifying cartilage5 suggests that vertebrates may use polyP chemistry to bank Ca+2 and PO4-3. In vitro experiments with nanoparticulate Ca-polyP & ALP were undertaken to determine if carbonated apatite could precipitate from 1M Ca-polyP in Pi-free “physiological fluid” (0.1 M NaCl, 2 mM Ca+2, 0.8 mM Mg+2, pH ~8.0 ±0.5, 37 °C), as this is estimated to generate the [Ca+2] & [PO4-3] required to form the apatite content of bone tissue

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Effects of initial saturation on properties modification and displacement of tetrachloroethene with aqueous isobutanol.

    Science.gov (United States)

    Boyd, Glen R; Ocampo-Gómez, Ana M; Li, Minghua; Husserl, Johana

    2006-11-20

    Packed column experiments were conducted to study effects of initial saturation of tetrachloroethene (PCE) in the range of 1.0-14% pore volume (PV) on mobilization and downward migration of the non-aqueous phase liquid (NAPL) product upon contact with aqueous isobutanol ( approximately 10 vol.%). This study focused on the consequences of swelling beyond residual saturation. Columns were packed with mixtures of neat PCE, water and glass beads and waterflooded to establish a desired homogeneous residual saturation, and then flooded with aqueous isobutanol under controlled hydraulic conditions. Results showed a critical saturation of approximately 8% PV for these packed column experimental conditions. At low initial PCE saturations (8% PV), results showed NAPL-product mobilization and downward migration which was attributed to interfacial tension (IFT) reduction, swelling of the NAPL-product, and reduced density modification. Packed column results were compared with good agreement to theoretical predictions of NAPL-product mobilization using the total trapping number, N(T). In addition to the packed column study, preliminary batch experiments were conducted to study the effects of PCE volumetric fraction in the range of 0.5-20% on density, viscosity, and IFT modification as a function of time following contact with aqueous isobutanol ( approximately 10 vol.%). Modified NAPL-product fluid properties approached equilibrium within approximately 2 h of contact for density and viscosity. IFT reduction occurred immediately as expected. Measured fluid properties were compared with good agreement to theoretical equilibrium predictions based on UNIQUAC. Overall, this study demonstrates the importance of initial DNAPL saturation, and the associated risk of downward NAPL-product migration, in applying alcohol flooding for remediation of DNAPL contaminated ground water sites.

  11. Adaptive control of systems in cascade with saturation

    Science.gov (United States)

    Kannan, Suresh K.

    This thesis extends the use of neural-network-based model reference adaptive control to systems that occur as cascades. In general, these systems are not feedback linearizable. The approach taken is that of approximate feedback linearization of upper subsystems whilst treating the lower-subsystem states as virtual actuators. Similarly, lower-subsystems are also feedback linearized. Typically, approximate inverses are used for linearization purposes. Model error arising from the use of an approximate inverse is minimized using a neural-network as an adaptive element. Incorrect adaptation due to (virtual) actuator saturation and dynamics is avoided using the Pseudocontrol Hedging method. Using linear approximate inverses and linear reference models generally result in large desired pseudocontrol for large external commands. Even if the provided external command is feasible (null-controllable), there is no guarantee that the reference model trajectory is feasible. In order to mitigate this, nonlinear reference models based on nested-saturation methods are used to constrain the evolution of the reference model and thus the plant states. The method presented in this thesis lends itself to the inner-outer loop control of air vehicles, where the inner-loop controls attitude dynamics and the outer-loop controls the translational dynamics of the vehicle. The outer-loop treats the closed loop attitude dynamics as an actuator. Adaptation to uncertainty in the attitude, as well as the translational dynamics, is introduced, thus minimizing the effects of model error in all six degrees of freedom and leading to more accurate position tracking. A pole-placement approach is used to choose compensator gains for the tracking error dynamics. This alleviates timescale separation requirements, allowing the outer loop bandwidth to be closer to that of the inner loop, thus increasing position tracking performance. A poor model of the attitude dynamics and a basic kinematics model is

  12. Prioritized Control Allocation for Quadrotors Subject to Saturation

    NARCIS (Netherlands)

    Smeur, E.J.J.; de Wagter, C.; J.-M. Moschetta G. Hattenberger, H. de Plinval

    2017-01-01

    This paper deals with the problem of actuator saturation for INDI (Incremental Nonlinear Dynamic Inversion) controlled flying vehicles. The primary problem that arises from actuator saturation for quadrotors, is that of arbitrary control objective realization. We have integrated the weighted least

  13. Polar spots and stellar spindown: is dynamo saturation needed?

    NARCIS (Netherlands)

    Solanki, S. K.; Motamen, S.; Keppens, R.

    1997-01-01

    Dynamo saturation is often invoked when calculating the rotational evolution of cool stars. At rapid rotation rates a saturated dynamo reduces the angular momentum carried away by the stellar wind. This, in turn, may explain the high rotation rates present in the distribution of rotation periods in

  14. Polar spots and stellar spindown: Is dynamo saturation needed?

    NARCIS (Netherlands)

    Solanki, S. K.; Motamen, S.; Keppens, R.

    1997-01-01

    Dynamo saturation is often invoked when calculating the rotational evolution of cool stars. At rapid rotation rates a saturated dynamo reduces the angular momentum carried away by the stellar wind. This, in turn, may explain the high rotation rates present in the distribution of rotation periods in

  15. Gain characteristics of a saturated fiber optic parametric amplifier

    DEFF Research Database (Denmark)

    Rottwitt, Karsten; Lorenzen, Michael Rodas; Noordegraaf, Danny

    2008-01-01

    In this work we discuss saturation performance of a fiber optic parametric amplifier. A simple numerical model is described and applied to specific cases. A system experiment using a saturated amplifier illustrates a 4 dB improvement in required signal to noise ratio for a fixed bit error ratio....

  16. Renal vein oxygen saturation in renal artery stenosis

    DEFF Research Database (Denmark)

    Nielsen, K; Rehling, M; Henriksen, Jens Henrik Sahl

    1992-01-01

    Renal vein oxygen-saturation was measured in 56 patients with arterial hypertension and unilateral stenosis or occlusion of the renal artery. Oxygen-saturation in blood from the ischaemic kidney (84.4%, range 73-93%) was significantly higher than that from the 'normal' contralateral kidney (81...

  17. Comparison of empirical models and laboratory saturated hydraulic ...

    African Journals Online (AJOL)

    Numerous methods for estimating soil saturated hydraulic conductivity exist, which range from direct measurement in the laboratory to models that use only basic soil properties. A study was conducted to compare laboratory saturated hydraulic conductivity (Ksat) measurement and that estimated from empirical models.

  18. Excitable solitons in a semiconductor laser with a saturable absorber

    Science.gov (United States)

    Turconi, Margherita; Prati, Franco; Barland, Stéphane; Tissoni, Giovanna

    2015-11-01

    Self-pulsing cavity solitons may exist in a semiconductor laser with an intracavity saturable absorber. They show locally the passive Q -switching behavior that is typical of lasers with saturable absorbers in the plane-wave approximation. Here we show that excitable cavity solitons are also possible in a suitable parameter range and characterize their excitable dynamics and properties.

  19. Saturated hydraulic conductivity values of some forest soils of ...

    African Journals Online (AJOL)

    A simple falling-head method is presented for the laboratory determination of saturated hydraulic conductivity of some forest soils of Ghana. Using the procedure, it was found that saturated hydraulic conductivity was positively and negatively correlated with sand content and clay content, respectively, both at P = 0.05 level.

  20. Significance of saturation index of certain clay minerals in shallow ...

    Indian Academy of Sciences (India)

    In reality, a number of kinetic reasons exist that may ..... of SI with increase of pH. This increase of pH reduces the availability of H+ for ion exchange. Group B: The SI of Gibbsite ranges from near saturation to saturation. This may be due to the .... tion of ions in solution which emphasizes increased contribution of species with ...

  1. Retinal oxygen saturation in patients with systemic hypoxemia

    DEFF Research Database (Denmark)

    Traustason, Sindri; Jensen, Annette Schophuus; Arvidsson, Henrik Sven

    2011-01-01

    To assess spectrophotometric oximetry across a broad range of arterial saturation levels and to study the effect of chronic systemic hypoxemia on retinal oxygen extraction.......To assess spectrophotometric oximetry across a broad range of arterial saturation levels and to study the effect of chronic systemic hypoxemia on retinal oxygen extraction....

  2. Estimation of Saturation Flow Rates at Signalized Intersections

    Directory of Open Access Journals (Sweden)

    Chang-qiao Shao

    2012-01-01

    Full Text Available The saturation flow rate is a fundamental parameter to measure the intersection capacity and time the traffic signals. However, it is revealed that traditional methods which are mainly developed using the average value of observed queue discharge headways to estimate the saturation headway might lead to underestimate saturation flow rate. The goal of this paper is to study the stochastic nature of queue discharge headways and to develop a more accurate estimate method for saturation headway and saturation flow rate. Based on the surveyed data, the characteristics of queue discharge headways and the estimation method of saturated flow rate are studied. It is found that the average value of queue discharge headways is greater than the median value and that the skewness of the headways is positive. Normal distribution tests were conducted before and after a log transformation of the headways. The goodness-of-fit test showed that for some surveyed sites, the queue discharge headways can be fitted by the normal distribution and for other surveyed sites, the headways can be fitted by lognormal distribution. According to the queue discharge headway characteristics, the median value of queue discharge headways is suggested to estimate the saturation headway and a new method of estimation saturation flow rates is developed.

  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. Saturated porous continua in the frame of hybrid description

    Science.gov (United States)

    Brazgina, Olga V.; Ivanova, Elena A.; Vilchevskaya, Elena N.

    2016-09-01

    A method for modeling fluid-solid interactions in saturated porous media is proposed. The main challenge is the combination of the material and spatial descriptions. The deformation of the solid, which serves as a "container" to the fluid, is studied by following the motion of its material particles, i.e., in Lagrangian description. On the other hand, the motion of the fluid is described in spatial form, i.e., by using a Eulerian approach. However, the solid deforms and this implies a certain difference regarding the standard formulation used in spatial description of fluid mechanics where a fixed grid dissects space into elementary volumes. Here the grid is no longer fixed, and the elementary volumes will follow the deformation of the solid. Moreover, for the solid as well as for the fluid the balance equations are formulated in the current configuration, where interaction forces and couples are taken into account. By using Zhilin's approach, entropy and temperature are incorporated in the system of equations. Constitutive equations are constructed for both elastic and inelastic components of force and couple stress tensors and interaction force and couple. The constitutive equations for elastic components are found on the basis of the energy balance equation; the constitutive equations for the inelastic components are proposed in accordance with the second law of thermodynamics. Particular emphasis is placed on the constitutive equations of the interaction force and couple, which result in a symmetric form only because of the "hybrid" approach combining the Lagrangian with the Eulerian description. Three possible examples of application of the theory have been presented. For each example, all required assumptions were first stated and discussed and then the complete set of the corresponding equations was presented.

  5. Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

    Science.gov (United States)

    Wang, Shu-Yang; Jiang, Bo-Ling; Zhou, Xiang; Chen, Ji-Hong; Li, Wen-Jian; Liu, Jing; Hu, Wei; Xiao, Guo-Qing; Dong, Miao-Yin; Wang, Yu-Chen

    2015-01-01

    The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.

  6. Dynamics of saturated energy condensation in two-dimensional turbulence.

    Science.gov (United States)

    Chan, Chi-kwan; Mitra, Dhrubaditya; Brandenburg, Axel

    2012-03-01

    In two-dimensional forced Navier-Stokes turbulence, energy cascades to the largest scales in the system to form a pair of coherent vortices known as the Bose condensate. We show, both numerically and analytically, that the energy condensation saturates and the system reaches a statistically stationary state. The time scale of saturation is inversely proportional to the viscosity and the saturation energy level is determined by both the viscosity and the force. We further show that, without sufficient resolution to resolve the small-scale enstrophy spectrum, numerical simulations can give a spurious result for the saturation energy level. We also find that the movement of the condensate is similar to the motion of an inertial particle with an effective drag force. Furthermore, we show that the profile of the saturated coherent vortices can be described by a Gaussian core with exponential wings.

  7. 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.

  8. The distribution of saturated clusters in wetted granular materials

    Science.gov (United States)

    Li, Shuoqi; Hanaor, Dorian; Gan, Yixiang

    2017-06-01

    The hydro-mechanical behaviour of partially saturated granular materials is greatly influenced by the spatial and temporal distribution of liquid within the media. The aim of this paper is to characterise the distribution of saturated clusters in granular materials using an optical imaging method under different water drainage conditions. A saturated cluster is formed when a liquid phase fully occupies the pore space between solid grains in a localized region. The samples considered here were prepared by vibrating mono-sized glass beads to form closely packed assemblies in a rectangular container. A range of drainage conditions were applied to the specimen by tilting the container and employing different flow rates, and the liquid pressure was recorded at different positions in the experimental cell. The formation of saturated clusters during the liquid withdrawal processes is governed by three competing mechanisms arising from viscous, capillary, and gravitational forces. When the flow rate is sufficiently large and the gravity component is sufficiently small, the viscous force tends to destabilize the liquid front leading to the formation of narrow fingers of saturated material. As the water channels along these liquid fingers break, saturated clusters are formed inside the specimen. Subsequently, a spatial and temporal distribution of saturated clusters can be observed. We investigated the resulting saturated cluster distribution as a function of flow rate and gravity to achieve a fundamental understanding of the formation and evolution of such clusters in partially saturated granular materials. This study serves as a bridge between pore-scale behavior and the overall hydro-mechanical characteristics in partially saturated soils.

  9. Examining Spectral Reflectance Saturation in Landsat Imagery and Corresponding Solutions to Improve Forest Aboveground Biomass Estimation

    Directory of Open Access Journals (Sweden)

    Panpan Zhao

    2016-06-01

    Full Text Available The data saturation problem in Landsat imagery is well recognized and is regarded as an important factor resulting in inaccurate forest aboveground biomass (AGB estimation. However, no study has examined the saturation values for different vegetation types such as coniferous and broadleaf forests. The objective of this study is to estimate the saturation values in Landsat imagery for different vegetation types in a subtropical region and to explore approaches to improving forest AGB estimation. Landsat Thematic Mapper imagery, digital elevation model data, and field measurements in Zhejiang province of Eastern China were used. Correlation analysis and scatterplots were first used to examine specific spectral bands and their relationships with AGB. A spherical model was then used to quantitatively estimate the saturation value of AGB for each vegetation type. A stratification of vegetation types and/or slope aspects was used to determine the potential to improve AGB estimation performance by developing a specific AGB estimation model for each category. Stepwise regression analysis based on Landsat spectral signatures and textures using grey-level co-occurrence matrix (GLCM was used to develop AGB estimation models for different scenarios: non-stratification, stratification based on either vegetation types, slope aspects, or the combination of vegetation types and slope aspects. The results indicate that pine forest and mixed forest have the highest AGB saturation values (159 and 152 Mg/ha, respectively, Chinese fir and broadleaf forest have lower saturation values (143 and 123 Mg/ha, respectively, and bamboo forest and shrub have the lowest saturation values (75 and 55 Mg/ha, respectively. The stratification based on either vegetation types or slope aspects provided smaller root mean squared errors (RMSEs than non-stratification. The AGB estimation models based on stratification of both vegetation types and slope aspects provided the most

  10. Elastoplastic model for unsaturated, quasi-saturated and fully saturated fine soils

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    Lai Ba Tien

    2016-01-01

    Full Text Available In unsaturated soils, the gaseous phase is commonly assumed to be continuous. This assumption is no more valid at high saturation ratio. In that case, air bubbles and pockets can be trapped in the porous network by the liquid phase and the gas phase becomes discontinuous. This trapped air reduces the apparent compressibility of the pore fluid and affect the mechanical behavior of the soil. Although it is trapped in the pores, its dissolution can take place. Dissolved air can migrate through the pore space, either by following the flow of the fluid or by diffusion. In this context, this paper present a hydro mechanical model that separately considers the kinematics and the mechanical behavior of each fluid species (eg liquid water, dissolved air, gaseous air and the solid matrix. This new model was implemented in a C++ code. Some numerical simulations are performed to demonstrate the ability of this model to reproduce a continuous transition of unsaturated to saturated states.

  11. Changes in air saturation and air water interfacial area during surfactant-enhanced air sparging in saturated sand

    Science.gov (United States)

    Kim, Heonki; Choi, Kyong-Min; Moon, Ji-Won; Annable, Michael D.

    2006-11-01

    Reduction in the surface tension of groundwater, prior to air sparging for removal of volatile organic contaminant from aquifer, can greatly enhance the air content and the extent of influence when air sparging is implemented. However, detailed information on the functional relationship between water saturation, air-water contact area induced by air sparging and the surface tension of water has not been available. In this study, the influence of adding water-soluble anionic surfactant (sodium dodecyl benzene sulfonate) into groundwater before air sparging on the air-water interfacial area and water saturation was investigated using a laboratory-scale sand packed column. It was found that water saturation decreases with decreasing surface tension of water until it reaches a point where this trend is reversed so that water saturation increases with further decrease in the surface tension. The lowest water saturation of 0.58 was achieved at a surface tension of 45.4 dyn/cm, which is considered as the optimum surface tension for maximum de-saturation for the initially water-saturated sand used in this study. The air-water contact area generated in the sand column due to air sparging was measured using a gaseous interfacial tracer, n-decane, and was found to monotonically increase with decreasing water saturation. The results of this study provide useful design information for surfactant-enhanced air sparging removal of volatile contaminants from aquifers.

  12. Sub- and superluminal propagation of intense pulses in media with saturated and reverse absorption.

    Science.gov (United States)

    Agarwal, G S; Dey, Tarak Nath

    2004-05-21

    We develop models for the propagation of intense pulses in solid state media which can have either saturated absorption or reverse absorption. We model subluminal propagation in ruby and superluminal propagation in alexandrite as three and four level systems, respectively, coupled to Maxwell's equations. We present results well beyond the traditional pump-probe approach and explain the experiments of Bigelow et al. [Phys. Rev. Lett. 90, 113903 (2003)]Science 301, 200 (2003)

  13. H∞ Loop Shaping Control of Input Saturated Systems with Norm-Bounded Parametric Uncertainty

    Directory of Open Access Journals (Sweden)

    Renan Lima Pereira

    2015-01-01

    Full Text Available This paper proposes a gain-scheduling control design strategy for a class of linear systems with the presence of both input saturation constraints and norm-bounded parametric uncertainty. LMI conditions are derived in order to obtain a gain-scheduled controller that ensures the robust stability and performance of the closed loop system. The main steps to obtain such a controller are given. Differently from other gain-scheduled approaches in the literature, this one focuses on the problem of H∞ loop shaping control design with input saturation nonlinearity and norm-bounded uncertainty to reduce the effect of the disturbance input on the controlled outputs. Here, the design problem has been formulated in the four-block H∞ synthesis framework, in which it is possible to describe the parametric uncertainty and the input saturation nonlinearity as perturbations to normalized coprime factors of the shaped plant. As a result, the shaped plant is represented as a linear parameter-varying (LPV system while the norm-bounded uncertainty and input saturation are incorporated. This procedure yields a linear parameter-varying structure for the controller that ensures the stability of the polytopic LPV shaped plant from the vertex property. Finally, the effectiveness of the method is illustrated through application to a physical system: a VTOL “vertical taking-off landing” helicopter.

  14. Subcritical saturation of the magnetorotational instability through mean magnetic field generation

    Science.gov (United States)

    Xie, Jin-Han; Julien, Keith; Knobloch, Edgar

    2018-03-01

    The magnetorotational instability is widely believed to be responsible for outward angular momentum transport in astrophysical accretion discs. The efficiency of this transport depends on the amplitude of this instability in the saturated state. We employ an asymptotic expansion based on an explicit, astrophysically motivated time-scale separation between the orbital period, Alfvén crossing time and viscous or resistive dissipation time-scales, originally proposed by Knobloch and Julien, to formulate a semi-analytical description of the saturated state in an incompressible disc. In our approach a Keplerian shear flow is maintained by the central mass but the instability saturates via the generation of a mean vertical magnetic field. The theory assumes that the time-averaged angular momentum flux and the radial magnetic flux are constant and determines both self-consistently. The results predict that, depending on parameters, steady saturation may be supercritical or subcritical, and in the latter case that the upper (lower) solution branch is always stable (unstable). The angular momentum flux is always outward, consistent with the presence of accretion, and for fixed wavenumber peaks in the subcritical regime. The limit of infinite Reynolds number at large but finite magnetic Reynolds number is also discussed.

  15. Design for performance enhancement in feedback control systems with multiple saturating nonlinearities. Ph.D. Thesis

    Science.gov (United States)

    Kapasouris, Petros

    1988-01-01

    A systematic control design methodology is introduced for multi-input/multi-output systems with multiple saturations. The methodology can be applied to stable and unstable open loop plants with magnitude and/or rate control saturations and to systems in which state limitations are desired. This new methodology is a substantial improvement over previous heuristic single-input/single-output approaches. The idea is to introduce a supervisor loop so that when the references and/or disturbances are sufficiently small, the control system operates linearly as designed. For signals large enough to cause saturations, the control law is modified in such a way to ensure stability and to preserve, to the extent possible, the behavior of the linear control design. Key benefits of this methodology are: the modified compensator never produces saturating control signals, integrators and/or slow dynamics in the compensator never windup, the directional properties of the controls are maintained, and the closed loop system has certain guaranteed stability properties. The advantages of the new design methodology are illustrated by numerous simulations, including the multivariable longitudinal control of modified models of the F-8 (stable) and F-16 (unstable) aircraft.

  16. Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases.

    Science.gov (United States)

    Xiao, W; Chow, B L; Hanna, M; Doetsch, P W

    2001-12-19

    DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to address whether AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We found that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by methyl methanesulfonate (MMS), a model DNA alkylating agent. Interestingly, this sensitivity can be reduced up to 2500-fold by deleting the MAG1 3-methyladenine DNA glycosylase gene, suggesting that Mag1 not only removes lethal base lesions, but also benign lesions and possibly normal bases, and that the resulting AP sites are highly toxic to the cells. This rescuing effect appears to be specific for DNA alkylation damage, since the mag1 mutation reduces killing effects of two other DNA alkylating agents, but does not alter the sensitivity of apn cells to killing by UV, gamma-ray or H(2)O(2). Our mutagenesis assays indicate that nearly half of spontaneous and almost all MMS-induced mutations in the AP endonuclease-deficient cells are due to Mag1 DNA glycosylase activity. Although the DNA replication apparatus appears to be incapable of replicating past AP sites, Polzeta-mediated translesion synthesis is able to bypass AP sites, and accounts for all spontaneous and MMS-induced mutagenesis in the AP endonuclease-deficient cells. These results allow us to delineate base lesion flow within the BER pathway and link AP sites to other DNA damage repair and tolerance pathways.

  17. Elastic velocities of partially gas-saturated unconsolidated sediments

    Science.gov (United States)

    Lee, M.W.

    2004-01-01

    Fluid in sediments significantly affects elastic properties of sediments and gas in the pore space can be identified by a marked reduction of P-wave velocity or a decrease of Poisson's ratio. The elastic properties of gas-saturated sediments can be predicted by the classical Biot-Gassmann theory (BGT). However, parameters for the BGT such as the Biot coefficient or moduli of dry frame of unconsolidated and high porosity sediments are not readily available. Dependence of velocities on differential pressure or porosity for partially gas-saturated sediments is formulated using properties derived from velocities of water-saturated sediments. Laboratory samples for unconsolidated and consolidated sediments and well log data acquired for unconsolidated marine sediments agree well with the predictions. However, because the P-wave velocity depends highly on how the gas is saturated in the pore space such as uniform or patch, the amounts of gas estimated from the P-wave velocity contains high uncertainty. The modeled Vp/Vs ratio of partially gas-saturated sediment using the patch distribution is usually greater than 1.6, whereas the ratio modeled assuming a uniform distribution is about 1.6. Thus, Poisson's ratio or Vp/Vs ratio may be used to differentiate patch from uniform saturation, but differences between various models of patch saturation cannot be easily identified. ?? 2004 Elsevier Ltd. All rights reserved.

  18. Red cell aspartate aminotransferase saturation with oral pyridoxine intake

    Directory of Open Access Journals (Sweden)

    Marilena Oshiro

    Full Text Available CONTEXT AND OBJECTIVE: The coenzyme of aspartate aminotransferase is pyridoxal phosphate, generated from fresh vegetables containing pyridoxine. Vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronie’s syndrome respond to high pyridoxine doses. The objective was to investigate the oral pyridoxine oral dose that would lead to maximized pyridoxal phosphate saturation of red cell aspartate aminotransferase. DESIGN AND SETTING: Controlled trial, in Hematology Division of Instituto Adolfo Lutz. METHODS: Red cell aspartate aminotransferase activity was assayed (before and after in normal volunteers who were given oral pyridoxine for 15-18 days (30 mg, 100 mg and 200 mg daily. In vitro study of blood from seven normal volunteers was also performed, with before and after assaying of aspartate aminotransferase activity. RESULTS: The in vivo study showed increasing aspartate aminotransferase saturation with increasing pyridoxine doses. 83% saturation was reached with 30 mg daily, 88% with 100 mg, and 93% with 200 mg after 20 days of oral supplementation. The in vitro study did not reach 100% saturation. CONCLUSIONS: Neither in vivo nor in vitro study demonstrated thorough aspartate aminotransferase saturation with its coenzyme pyridoxal phosphate in red cells, from increasing pyridoxine supplementation. However, the 200-mg dose could be employed safely in vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronie’s syndrome treatment. Although maximum saturation in circulating red cells is not achieved, erythroblasts and other nucleated and cytoplasmic organelles containing cells certainly will reach thorough saturation, which possibly explains the results obtained in these diseases.

  19. Saturation Detection-Based Blocking Scheme for Transformer Differential Protection

    Directory of Open Access Journals (Sweden)

    Byung Eun Lee

    2014-07-01

    Full Text Available This paper describes a current differential relay for transformer protection that operates in conjunction with a core saturation detection-based blocking algorithm. The differential current for the magnetic inrush or over-excitation has a point of inflection at the start and end of each saturation period of the transformer core. At these instants, discontinuities arise in the first-difference function of the differential current. The second- and third-difference functions convert the points of inflection into pulses, the magnitudes of which are large enough to detect core saturation. The blocking signal is activated if the third-difference of the differential current is larger than the threshold and is maintained for one cycle. In addition, a method to discriminate between transformer saturation and current transformer (CT saturation is included. The performance of the proposed blocking scheme was compared with that of a conventional harmonic blocking method. The test results indicate that the proposed scheme successfully discriminates internal faults even with CT saturation from the magnetic inrush, over-excitation, and external faults with CT saturation, and can significantly reduce the operating time delay of the relay.

  20. On the propagation of a coupled saturation and pressure front

    Energy Technology Data Exchange (ETDEWEB)

    Vasco, D. W.

    2010-12-01

    Using an asymptotic technique, valid for a medium with smoothly varying heterogeneity, I derive an expression for the velocity of a propagating, coupled saturation and pressure front. Due to the nonlinearity of the governing equations, the velocity of the propagating front depends upon the magnitude of the saturation and pressure changes across the front in addition to the properties of the medium. Thus, the expression must be evaluated in conjunction with numerical reservoir simulation. The propagation of the two-phase front is governed by the background saturation distribution, the saturation-dependent component of the fluid mobility, the porosity, the permeability, the capillary pressure function, the medium compressibility, and the ratio of the slopes of the relative permeability curves. Numerical simulation of water injection into a porous layer saturated with a nonaqueous phase liquid indicates that two modes of propagation are important. The fastest mode of propagation is a pressure-dominated disturbance that travels through the saturated layer. This is followed, much later, by a coupled mode with a large saturation change. These two modes are also observed in a simulation using a heterogeneous porous layer. A comparison between the propagation times estimated from the results of the numerical simulation and predictions from the asymptotic expression indicates overall agreement.

  1. Hydrate morphology: Physical properties of sands with patchy hydrate saturation

    Science.gov (United States)

    Dai, S.; Santamarina, J.C.; Waite, William F.; Kneafsey, T.J.

    2012-01-01

    The physical properties of gas hydrate-bearing sediments depend on the volume fraction and spatial distribution of the hydrate phase. The host sediment grain size and the state of effective stress determine the hydrate morphology in sediments; this information can be used to significantly constrain estimates of the physical properties of hydrate-bearing sediments, including the coarse-grained sands subjected to high effective stress that are of interest as potential energy resources. Reported data and physical analyses suggest hydrate-bearing sands contain a heterogeneous, patchy hydrate distribution, whereby zones with 100% pore-space hydrate saturation are embedded in hydrate-free sand. Accounting for patchy rather than homogeneous hydrate distribution yields more tightly constrained estimates of physical properties in hydrate-bearing sands and captures observed physical-property dependencies on hydrate saturation. For example, numerical modeling results of sands with patchy saturation agree with experimental observation, showing a transition in stiffness starting near the series bound at low hydrate saturations but moving toward the parallel bound at high hydrate saturations. The hydrate-patch size itself impacts the physical properties of hydrate-bearing sediments; for example, at constant hydrate saturation, we find that conductivity (electrical, hydraulic and thermal) increases as the number of hydrate-saturated patches increases. This increase reflects the larger number of conductive flow paths that exist in specimens with many small hydrate-saturated patches in comparison to specimens in which a few large hydrate saturated patches can block flow over a significant cross-section of the specimen.

  2. 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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. 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.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Mechanical behavior of saturated, consolidated, alumina powder compacts

    Science.gov (United States)

    Franks, George Vincent, Jr.

    effect of particle size and morphology on the consolidation behavior and the plastic-to-brittle transition of saturated alumina powder compacts was investigated. Large particles pack to higher relative densities than small particles. Blocky particles pack to lower relative densities than particles with rounded surfaces. Consolidated bodies of large particles become brittle at lower consolidation pressures. The effects of pair potential, particle size and strain rate on the flow stress was also investigated. Bodies consolidated from slurries of particles that reside in attractive minima have flow stresses and relaxed (yield) stresses that increase with the depth of the attractive minima. Bodies consolidated from slurries of larger particles possess lower flow stresses than bodies made of small particles. Flow stress for certain bodies formed from the coagulated slurry had a nearly identical flow stress as measured for a commercial, throwing clay. The strain rate sensitivity is greater for bodies that have relative densities approaching the maximum. (Abstract shortened by UMI.)

  10. Dynamics of Saturated Energy Condensation in Two-Dimensional Turbulence

    CERN Document Server

    Chan, Chi-kwan; Brandenburg, Axel

    2011-01-01

    We derive and numerically confirm that the saturation of energy condensation in two-dimensional turbulence is governed by the balance between forcing and small-scale dissipation. The time scale of saturation is inversely proportional to the viscosity but the saturation energy level is determined by both viscosity and the forcing scale. It is shown that, because the energy dissipation is proportional to the enstrophy, which itself is a conserved quantity in the ideal case, it is necessary to resolve the enstrophy spectrum to achieve numerical consistency. We also find that the movement of the condensate vortices can be described as Brownian motion of an inertial particle.

  11. Femtosecond Yb:YAG laser using semiconductor saturable absorbers

    Energy Technology Data Exchange (ETDEWEB)

    Hoenninger, C.; Zhang, G.; Keller, U. [Ultrafast Laser Physics, Institute of Quantum Electronics, Swiss Federal Institute of Technology, ETH Hoenggerberg--HPT, CH-8093 Zuerich (Switzerland); Giesen, A. [Institut fuer Strahlwerkzeuge, Universitaet Stuttgart, Pfaffenwaldring 43, D-70569 Stuttgart (Germany)

    1995-12-01

    We demonstrate a passively mode-locked femtosecond Yb:YAG laser using different semiconductor saturable absorber devices, a high-finesse and a low-finesse antiresonant Fabry{endash}Perot saturable absorber. We achieved pulses as short as 540 fs with dispersion compensation and 1.7-ps pulses without dispersion compensation. We also mode locked the laser at either 1.03 or 1.05 {mu}m by adjusting the band gap and antiresonance wavelength design of the antiresonant Fabry{endash}Perot saturable absorber. {copyright} {ital 1995 Optical Society of America.}

  12. Investigation of saturation effects in ceramic phosphors for laser lighting

    DEFF Research Database (Denmark)

    Krasnoshchoka, Anastasiia; Thorseth, Anders; Dam-Hansen, Carsten

    2017-01-01

    We report observation of saturation effects in a Ce:LuAG and Eu-doped nitride ceramic phosphor for conversion of blue laser light for white light generation. The luminous flux from the phosphors material increases linearly with the input power until saturation effects limit the conversion....... It is shown, that the temperature of the phosphor layer influences the saturation power level and the conversion efficiency. It is also shown that the correlated color temperature (CCT), phosphor conversion efficiency and color rendering index (CRI) are dependent both on incident power and spot size diameter...

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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)

  1. 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.

  2. 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…

  3. 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

  4. 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,

  5. 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...

  6. 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.

  7. 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.

  8. Predicting colloid transport through saturated porous media: A critical review

    Science.gov (United States)

    Molnar, Ian L.; Johnson, William P.; Gerhard, Jason I.; Willson, Clinton S.; O'Carroll, Denis M.

    2015-09-01

    Understanding and predicting colloid transport and retention in water-saturated porous media is important for the protection of human and ecological health. Early applications of colloid transport research before the 1990s included the removal of pathogens in granular drinking water filters. Since then, interest has expanded significantly to include such areas as source zone protection of drinking water systems and injection of nanometals for contaminated site remediation. This review summarizes predictive tools for colloid transport from the pore to field scales. First, we review experimental breakthrough and retention of colloids under favorable and unfavorable colloid/collector interactions (i.e., no significant and significant colloid-surface repulsion, respectively). Second, we review the continuum-scale modeling strategies used to describe observed transport behavior. Third, we review the following two components of colloid filtration theory: (i) mechanistic force/torque balance models of pore-scale colloid trajectories and (ii) approximating correlation equations used to predict colloid retention. The successes and limitations of these approaches for favorable conditions are summarized, as are recent developments to predict colloid retention under the unfavorable conditions particularly relevant to environmental applications. Fourth, we summarize the influences of physical and chemical heterogeneities on colloid transport and avenues for their prediction. Fifth, we review the upscaling of mechanistic model results to rate constants for use in continuum models of colloid behavior at the column and field scales. Overall, this paper clarifies the foundation for existing knowledge of colloid transport and retention, features recent advances in the field, critically assesses where existing approaches are successful and the limits of their application, and highlights outstanding challenges and future research opportunities. These challenges and opportunities

  9. Magnetic resonance sounding measurements for modeling of water flow transport in variably saturated porous media

    Science.gov (United States)

    Legchenko, Anatoly; Legout, Cédric; Descloitres, Marc

    2017-04-01

    Numerical modeling of water flow in partly saturated porous media requires knowledge of hydraulic properties of the media. The straightforward approach consists of directly measuring K(teta) and h(teta), which is challenging in many practically important applications. In-situ non-invasive measurements of K(teta) and h(teta) are even more difficult and probably impossible. Additionally, K(teta) and h(teta) are both scale dependent parameters. Under favorable conditions, surface geophysical methods may allow non-invasive identification of different geological formations and estimate of the porosity. A few papers report hydrogeological modeling considering water-saturated formations with integrated geophysical data (aquifer geometry, K and teta at saturation). However, modeling of water transport in partly saturated subsurface is more difficult task because it requires more extensive knowledge of soil hydraulic properties. We use Magnetic Resonance Sounding (MRS) method for non-invasive time-lapse measurements of the water content as an input into numerical modeling tool for hydrogeological modeling. However, MRS is not able to provide h(teta), which rest inaccessible. We propose an approach, which consists of performing infiltration tests (or observation of natural infiltration and monitoring rain water) and measuring corresponding variation of the water content in the subsurface. Then, we use a data base of soils with accurately known hydraulic properties. We try different soils for modeling water transport under our conditions (reproducing our experiment) and select one, which allows fitting experimentally observed variations in the water content. When such a soil is found we obtain K(teta) and h(teta). Thus, instead of looking for true hydraulic characteristics of the subsurface we obtain some equivalent media that allows reproducing our observations. We demonstrate the feasibility of our approach using simple 1-D models and commercially available software

  10. Using historical biogeography to test for community saturation.

    Science.gov (United States)

    Pinto-Sánchez, Nelsy Rocío; Crawford, Andrew J; Wiens, John J

    2014-09-01

    Saturation is the idea that a community is effectively filled with species, such that no more can be added without extinctions. This concept has important implications for many areas of ecology, such as species richness, community assembly, invasive species and climate change. Here, we illustrate how biogeography can be used to test for community saturation, when combined with data on local species richness, phylogeny and climate. We focus on a clade of frogs (Terrarana) and the impact of the Great American Biotic Interchange on patterns of local richness in Lower Middle America and adjacent regions. We analyse data on species richness at 83 sites and a time-calibrated phylogeny for 363 species. We find no evidence for saturation, and show instead that biotic interchange dramatically increased local richness in the region. We suggest that historical biogeography offers thousands of similar long-term natural experiments that can be used to test for saturation. © 2014 John Wiley & Sons Ltd/CNRS.

  11. Pulse Distortion in Saturated Fiber Optical Parametric Chirped Pulse Amplification

    DEFF Research Database (Denmark)

    Lali-Dastjerdi, Zohreh; Da Ros, Francesco; Rottwitt, Karsten

    2012-01-01

    Fiber optical parametric chirped pulse amplification is experimentally compared for different chirped pulses in the picosecond regime. The amplified chirped pulses show distortion appearing as pedestals after recompression when the amplifier is operated in saturation....

  12. Graph-analytical method for determining saturation in oil formations

    Energy Technology Data Exchange (ETDEWEB)

    Ramazanova, E.E.; Fedortsov, V.K.; Ismaylov, K.K.

    1980-01-01

    Factual material is generalized for a large number of oil fields of the Soviet Union for which probability-statistical models have been selected. A graph-analytical method is developed for determining the saturation pressure of oil by gas.

  13. Delayed system control in presence of actuator saturation

    Directory of Open Access Journals (Sweden)

    A. Mahjoub

    2014-09-01

    Full Text Available The paper is introducing a new design method for systems’ controllers with input delay and actuator saturations and focuses on how to force the system output to track a reference input not necessarily saturation-compatible. We propose a new norm based on the way we quantify tracking performance as a function of saturation errors found using the same norm. The newly defined norm is related to signal average power making possible to account for most common reference signals e.g. step, periodic. It is formally shown that, whatever the reference shape and amplitude, the achievable tracking quality is determined by a well defined reference tracking mismatch error. This latter depends on the reference rate and its compatibility with the actuator saturation constraint. In fact, asymptotic output-reference tracking is achieved in the presence of constraint-compatible step-like references.

  14. Double storey three phase saturated cores fault current limiter

    Science.gov (United States)

    Wolfus, Y.; Nikulshin, Y.; Friedman, A.; Yeshurun, Y.

    2014-05-01

    A novel saturated-cores Fault-Current-Limiter (FCL) configuration is described. This FCL is based on two parallel planes of iron rectangular cores, on which three-phase coils are mounted and connected in series to the grid. Two DC coils are mounted in between the planes on perpendicular core limbs connecting the two AC planes. The DC coils are set to magnetically saturate the AC cores. The transition to three-dimensional, double-storey design enables handling three-phase symmetrical faults while offering better decoupling between the AC and DC circuits. At the same time, it shortens the AC limb lengths and enables deeper magnetic saturation levels in comparison to other saturated cores FCL designs. Hence, this FCL configuration exhibits lower insertion impedance and higher ratio of fault to nominal state impedance in comparison with other designs.

  15. Soil hydraulic properties near saturation, an improved conductivity model

    DEFF Research Database (Denmark)

    Børgesen, Christen Duus; Jacobsen, Ole Hørbye; Hansen, Søren

    2006-01-01

    The hydraulic properties near saturation can change dramatically due to the presence of macropores that are usually difficult to handle in traditional pore size models. The purpose of this study is to establish a data set on hydraulic conductivity near saturation, test the predictive capability...... of commonly used hydraulic conductivity models and give suggestions for improved models. Water retention and near saturated and saturated hydraulic conductivity were measured for a variety of 81 top and subsoils. The hydraulic conductivity models by van Genuchten [van Genuchten, 1980. A closed-form equation...... for predicting the hydraulic conductivity of unsaturated soils. Soil Sci. Soc. Am. J. 44, 892–898.] (vGM) and Brooks and Corey, modified by Jarvis [Jarvis, 1991. MACRO—A Model of Water Movement and Solute Transport in Macroporous Soils. Swedish University of Agricultural Sciences. Department of Soil Sciences...

  16. Saturation of entropy production in quantum many-body systems

    Science.gov (United States)

    Kaneko, Kazuya; Iyoda, Eiki; Sagawa, Takahiro

    2017-12-01

    Bridging the second law of thermodynamics and microscopic reversible dynamics has been a longstanding problem in statistical physics. Here, we address this problem on the basis of quantum many-body physics, and discuss how the entropy production saturates in isolated quantum systems under unitary dynamics. First, we rigorously prove that the entropy production does indeed saturate in the long time regime, even when the total system is in a pure state. Second, we discuss the non-negativity of the entropy production at saturation, implying the second law of thermodynamics. This is based on the eigenstate thermalization hypothesis, which states that even a single energy eigenstate is thermal. We also numerically demonstrate that the entropy production saturates at a non-negative value even when the initial state of a heat bath is a single energy eigenstate. Our results reveal fundamental properties of the entropy production in isolated quantum systems at late times.

  17. Retinal oxygen saturation before and after glaucoma surgery.

    Science.gov (United States)

    Nitta, Eri; Hirooka, Kazuyuki; Shimazaki, Takeru; Sato, Shino; Ukegawa, Kaori; Nakano, Yuki; Tsujikawa, Akitaka

    2017-08-01

    This study compared retinal vessel oxygen saturation before and after glaucoma surgery. Retinal oxygen saturation in glaucoma patients was measured using a non-invasive spectrophotometric retinal oximeter. Adequate image quality was found in 49 of the 108 consecutive glaucoma patients recruited, with 30 undergoing trabeculectomy, 11 EX-PRESS and eight trabeculotomy. Retinal oxygen saturation measurements in the retinal arterioles and venules were performed at 1 day prior to and at approximately 10 days after surgery. Statistical analysis was performed using a Student's t-test. After glaucoma surgery, intraocular pressure (IOP) decreased from 19.8 ± 7.7 mmHg to 9.0 ± 5.7 mmHg (p glaucoma surgery had an effect on the retinal venous oxygen saturation. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  18. On the Stability Criterion in a Saturated Atmosphere

    National Research Council Canada - National Science Library

    Richiardone, R; Giusti, F

    2001-01-01

      The expression of the moist buoyancy frequency indicated that it is not completely correct ot use the moist adiabatic lapse rate as a static stability parameter of a saturated atmosphere, because...

  19. Nonlinear Gain Saturation in Active Slow Light Photonic Crystal Waveguides

    DEFF Research Database (Denmark)

    Chen, Yaohui; Mørk, Jesper

    2013-01-01

    We present a quantitative three-dimensional analysis of slow-light enhanced traveling wave amplification in an active semiconductor photonic crystal waveguides. The impact of slow-light propagation on the nonlinear gain saturation of the device is investigated.......We present a quantitative three-dimensional analysis of slow-light enhanced traveling wave amplification in an active semiconductor photonic crystal waveguides. The impact of slow-light propagation on the nonlinear gain saturation of the device is investigated....

  20. Simulation of Nonlinear Gain Saturation in Active Photonic Crystal Waveguides

    DEFF Research Database (Denmark)

    Chen, Yaohui; Mørk, Jesper

    2012-01-01

    In this paper we present a theoretical analysis of slowlight enhanced traveling wave amplification in an active semiconductor Photonic crystal waveguides. The impact of group index on nonlinear modal gain saturation is investigated.......In this paper we present a theoretical analysis of slowlight enhanced traveling wave amplification in an active semiconductor Photonic crystal waveguides. The impact of group index on nonlinear modal gain saturation is investigated....

  1. Noise and saturation properties of semiconductor quantum dot optical amplifiers

    DEFF Research Database (Denmark)

    Berg, Tommy Winther; Mørk, Jesper

    2002-01-01

    We present a detailed theoretical analysis of quantum dot optical amplifiers. Due to the presence of a reservoir of wetting layer states, the saturation and noise properties differ markedly from bulk or QW amplifiers and may be significantly improved.......We present a detailed theoretical analysis of quantum dot optical amplifiers. Due to the presence of a reservoir of wetting layer states, the saturation and noise properties differ markedly from bulk or QW amplifiers and may be significantly improved....

  2. Succinct synthesis of saturated hydroxy fatty acids and

    DEFF Research Database (Denmark)

    Kaspersen, Mads Holmgaard; Jenkins, Laura; Dunlop, Julia

    2017-01-01

    Saturated hydroxy fatty acids make up a class of underexplored lipids with potentially interesting biological activities. We report a succinct and general synthetic route to saturated hydroxy fatty acids hydroxylated at position 6 or higher, and exemplify this with the synthesis of hydroxylauric...... acids. All regioisomers of hydroxylauric acids were tested on free fatty acid receptors FFA1, FFA4 and GPR84. The results show that the introduction of a hydroxy group and its position have a high impact on receptor activity....

  3. Facile and Green Synthesis of Saturated Cyclic Amines.

    Science.gov (United States)

    Hameed, Arruje; Javed, Sadia; Noreen, Razia; Huma, Tayyaba; Iqbal, Sarosh; Umbreen, Huma; Gulzar, Tahsin; Farooq, Tahir

    2017-10-12

    Single-nitrogen containing saturated cyclic amines are an important part of both natural and synthetic bioactive compounds. A number of methodologies have been developed for the synthesis of aziridines, azetidines, pyrrolidines, piperidines, azepanes and azocanes. This review highlights some facile and green synthetic routes for the synthesis of unsubstituted, multisubstituted and highly functionalized saturated cyclic amines including one-pot, microwave assisted, metal-free, solvent-free and in aqueous media.

  4. Facile and Green Synthesis of Saturated Cyclic Amines

    Directory of Open Access Journals (Sweden)

    Arruje Hameed

    2017-10-01

    Full Text Available Single-nitrogen containing saturated cyclic amines are an important part of both natural and synthetic bioactive compounds. A number of methodologies have been developed for the synthesis of aziridines, azetidines, pyrrolidines, piperidines, azepanes and azocanes. This review highlights some facile and green synthetic routes for the synthesis of unsubstituted, multisubstituted and highly functionalized saturated cyclic amines including one-pot, microwave assisted, metal-free, solvent-free and in aqueous media.

  5. Occupy the Financial Niche: Saturation and Crisis

    Science.gov (United States)

    Purica, Ionut

    The model presented is one theoretical approach within a broader research program that could verify the nonlinear conjectures made, such that to quantify and predict potential discontinuous behaviour. In this case, the crisis behaviour associated with financial funds reallocation among various credit instruments, described as memes with the sense of Dawkins, is shown to be of discontinuous nature stemming from a logistic penetration in the behaviour niche. Actually the logistic penetration is typical in creating cyclic behaviour of economic structures as shown by Marchetti and others from IIASA. A Fokker-Planck equation description results in a stationary solution having a bifurcation like solution with evolution trajectories on a `cusp' type catastrophe that may describe discontinuous decision behaviour.

  6. Generalized paired-agent kinetic model for in vivo quantification of cancer cell-surface receptors under receptor saturation conditions

    Science.gov (United States)

    Sadeghipour, N.; Davis, S. C.; Tichauer, K. M.

    2017-01-01

    New precision medicine drugs oftentimes act through binding to specific cell-surface cancer receptors, and thus their efficacy is highly dependent on the availability of those receptors and the receptor concentration per cell. Paired-agent molecular imaging can provide quantitative information on receptor status in vivo, especially in tumor tissue; however, to date, published approaches to paired-agent quantitative imaging require that only ‘trace’ levels of imaging agent exist compared to receptor concentration. This strict requirement may limit applicability, particularly in drug binding studies, which seek to report on a biological effect in response to saturating receptors with a drug moiety. To extend the regime over which paired-agent imaging may be used, this work presents a generalized simplified reference tissue model (GSRTM) for paired-agent imaging developed to approximate receptor concentration in both non-receptor-saturated and receptor-saturated conditions. Extensive simulation studies show that tumor receptor concentration estimates recovered using the GSRTM are more accurate in receptor-saturation conditions than the standard simple reference tissue model (SRTM) (% error (mean  ±  sd): GSRTM 0  ±  1 and SRTM 50  ±  1) and match the SRTM accuracy in non-saturated conditions (% error (mean  ±  sd): GSRTM 5  ±  5 and SRTM 0  ±  5). To further test the approach, GSRTM-estimated receptor concentration was compared to SRTM-estimated values extracted from tumor xenograft in vivo mouse model data. The GSRTM estimates were observed to deviate from the SRTM in tumors with low receptor saturation (which are likely in a saturated regime). Finally, a general ‘rule-of-thumb’ algorithm is presented to estimate the expected level of receptor saturation that would be achieved in a given tissue provided dose and pharmacokinetic information about the drug or imaging agent being used, and physiological

  7. Biodegradation of crude oil saturated fraction supported on clays.

    Science.gov (United States)

    Ugochukwu, Uzochukwu C; Jones, Martin D; Head, Ian M; Manning, David A C; Fialips, Claire I

    2014-02-01

    The role of clay minerals in crude oil saturated hydrocarbon removal during biodegradation was investigated in aqueous clay/saturated hydrocarbon microcosm experiments with a hydrocarbon degrading microorganism community. The clay minerals used for this study were montmorillonite, palygorskite, saponite and kaolinite. The clay mineral samples were treated with hydrochloric acid and didecyldimethylammonium bromide to produce acid activated- and organoclays respectively which were used in this study. The production of organoclay was restricted to only montmorillonite and saponite because of their relative high CEC. The study indicated that acid activated clays, organoclays and unmodified kaolinite, were inhibitory to biodegradation of the hydrocarbon saturates. Unmodified saponite was neutral to biodegradation of the hydrocarbon saturates. However, unmodified palygorskite and montmorillonite were stimulatory to biodegradation of the hydrocarbon saturated fraction and appears to do so as a result of the clays' ability to provide high surface area for the accumulation of microbes and nutrients such that the nutrients were within the 'vicinity' of the microbes. Adsorption of the saturated hydrocarbons was not significant during biodegradation.

  8. The relation between oxygen saturation level and retionopathy of prematurity

    Directory of Open Access Journals (Sweden)

    Mohammad Gharavi Fard

    2016-03-01

    Full Text Available Introduction: Oxygen therapy used for preterm infant disease might be associated with oxygen toxicity or oxidative stress. The exact oxygen concentration to control and maintain the arterial oxygen saturation balance is not certainly clear. We aimed to compare the efficacy of higher or lower oxygen saturations on the development of severe retinopathy of prematurity which is a major cause of blindness in preterm neonates. Methods: PubMed was searched for obtaining the relevant articles. A total of seven articles were included after studying the titles, abstracts, and the full text of retrieved articles at initial search. Inclusion criteria were all the English language human clinical randomized controlled trials with no time limitation, which studied the efficacy of low versus high oxygen saturation measured by pulse oximetry in preterm infants.Result: It can be suggested that lower limits of oxygen saturations have higher efficacy at postmesetural age of ≤28 weeks in preterm neonates. This relation has been demonstrated in five large clinical trials including three Boost trials, COT, and Support.Discussion: Applying higher concentrations of oxygen supplementations at mesentural age ≥32 weeks reduced the development of retinopathy of prematurity. Lower concentrations of oxygen saturation decreased the incidence and the development of retinopathy of prematurity in preterm neonates while applied soon after the birth.Conclusions: Targeting levels of oxygen saturation in the low or high range should be performed cautiously with attention to the postmesentural age in preterm infants at the time of starting the procedures.

  9. Dynamic Output Feedback Robust MPC with Input Saturation Based on Zonotopic Set-Membership Estimation

    Directory of Open Access Journals (Sweden)

    Xubin Ping

    2016-01-01

    Full Text Available For quasi-linear parameter varying (quasi-LPV systems with bounded disturbance, a synthesis approach of dynamic output feedback robust model predictive control (OFRMPC with the consideration of input saturation is investigated. The saturated dynamic output feedback controller is represented by a convex hull involving the actual dynamic output controller and an introduced auxiliary controller. By taking both the actual output feedback controller and the auxiliary controller with a parameter-dependent form, the main optimization problem can be formulated as convex optimization. The consideration of input saturation in the main optimization problem reduces the conservatism of dynamic output feedback controller design. The estimation error set and bounded disturbance are represented by zonotopes and refreshed by zonotopic set-membership estimation. Compared with the previous results, the proposed algorithm can not only guarantee the recursive feasibility of the optimization problem, but also improve the control performance at the cost of higher computational burden. A nonlinear continuous stirred tank reactor (CSTR example is given to illustrate the effectiveness of the approach.

  10. Micro-poromechanics model of fluid-saturated chemically active fibrous media.

    Science.gov (United States)

    Misra, Anil; Parthasarathy, Ranganathan; Singh, Viraj; Spencer, Paulette

    2015-02-01

    We have developed a micromechanics based model for chemically active saturated fibrous media that incorporates fiber network microstructure, chemical potential driven fluid flow, and micro-poromechanics. The stress-strain relationship of the dry fibrous media is first obtained by considering the fiber behavior. The constitutive relationships applicable to saturated media are then derived in the poromechanics framework using Hill's volume averaging. The advantage of this approach is that the resultant continuum model accounts for the discrete nature of the individual fibers while retaining a form suitable for porous materials. As a result, the model is able to predict the influence of micro-scale phenomena, such as the fiber pre-strain caused by osmotic effects and evolution of fiber network structure with loading, on the overall behavior and in particular, on the poromechanics parameters. Additionally, the model can describe fluid-flow related rate-dependent behavior under confined and unconfined conditions and varying chemical environments. The significance of the approach is demonstrated by simulating unconfined drained monotonic uniaxial compression under different surrounding fluid bath molarity, and fluid-flow related creep and relaxation at different loading-levels and different surrounding fluid bath molarity. The model predictions conform to the experimental observations for saturated soft fibrous materials. The method can potentially be extended to other porous materials such as bone, clays, foams and concrete.

  11. Settlement Analysis of Saturated Tailings Dam Treated by CFG Pile Composite Foundation

    Directory of Open Access Journals (Sweden)

    Jinxing Lai

    2016-01-01

    Full Text Available Cement fly ash gravel (CFG pile composite foundation is an effective and economic foundation treatment approach, which is significant to building foundation, subgrade construction, and so forth. The present paper aims at investigating the settlement behaviors of saturated tailings dam soft ground under CFG pile composite foundation treatment, in which FEM and laboratory model test were utilized. The proposed findings demonstrate that CFG pile treatment is effective in reinforcing saturated tailings dam and loading has little influence on settlement of soil between piles. The variation of soil between piles settlement in FEM has a good agreement with the laboratory model test. Additionally, the cushion deformation modulus has a small effect on the composite foundation settlement, although the cushion thickness will generate certain influence on the settlement distribution of the composite foundation.

  12. A technique for measuring oxygen saturation in biological tissues based on diffuse optical spectroscopy

    Science.gov (United States)

    Kleshnin, Mikhail; Orlova, Anna; Kirillin, Mikhail; Golubiatnikov, German; Turchin, Ilya

    2017-07-01

    A new approach to optical measuring blood oxygen saturation was developed and implemented. This technique is based on an original three-stage algorithm for reconstructing the relative concentration of biological chromophores (hemoglobin, water, lipids) from the measured spectra of diffusely scattered light at different distances from the probing radiation source. The numerical experiments and approbation of the proposed technique on a biological phantom have shown the high reconstruction accuracy and the possibility of correct calculation of hemoglobin oxygenation in the presence of additive noise and calibration errors. The obtained results of animal studies have agreed with the previously published results of other research groups and demonstrated the possibility to apply the developed technique to monitor oxygen saturation in tumor tissue.

  13. Central venous oxygen saturation does not correlate with the venous oxygen saturation at the surgical site during abdominal surgery.

    Science.gov (United States)

    Weinrich, Malte; Scheingraber, Stefan; Stephan, Bernhard; Weiss, Christel; Kayser, Anna; Kopp, Berit; Schilling, Martin K

    2008-01-01

    Measurement of central venous oxygen saturation has become a surrogate parameter for fluid administration, blood transfusions and treatment with catecholamines in (early) goal directed therapy in the treatment of acute septic patients. These strategies are not easily transferred to the postoperative management of abdominal surgery due to the different conditions in surgical patients. A study population of 15 patients (8 females/7 males) underwent elective major abdominal surgery: 6 gastrectomies, 5 major liver resections and 4 lower anterior rectum resections. Surgery was performed for primary or secondary malignancy. The patients' age was 65.4+/-12.7 (mean+/-standard deviation, range 44-84, median 62) years. Blood samples were taken intraoperatively from indwelling central venous lines as well as from draining veins at the surgical site. Blood gas analyses to determine the oxygen saturations were performed immediately. All patients were operated in standardized general anesthesia including epidural analgesia and in a balanced volume status. Central venous oxygen saturations and oxygen saturations in blood from the draining veins of the surgical site showed a wide range with high intra- and interindividual differences intraoperatively. Overall, at most time points no correlation between the two oxygen saturations could be detected in three operation types. A significant correlation was only observed at one time point during liver resections. Our results show a lack of correlation between central venous oxygen saturations and oxygen saturations in the draining veins of the surgical site during major abdominal surgery. Measurement of central venous oxygen saturations does not seem to be a good surrogate for the local oxygen supply in the field of interest in major abdominal surgery even under standardized conditions.

  14. On the water saturation calculation in hydrocarbon sandstone reservoirs

    Energy Technology Data Exchange (ETDEWEB)

    Stalheim, Stein Ottar

    2002-07-01

    The main goal of this work was to identify the most important uncertainty sources in water saturation calculation and examine the possibility for developing new S{sub w} - equations or possibility to develop methods to remove weaknesses and uncertainties in existing S{sub w} - equations. Due to the need for industrial applicability of the equations we aimed for results with the following properties: The accuracy in S{sub w} should increase compared with existing S{sub w} - equations. The equations should be simple to use in petrophysical evaluations. The equations should be based on conventional logs and use as few as possible input parameters. The equations should be numerical stable. This thesis includes an uncertainty and sensitivity analysis of the most common S{sub w} equations. The results are addressed in chapter 3 and were intended to find the most important uncertainty sources in water saturation calculation. To increase the knowledge of the relationship between R{sub t} and S{sub w} in hydrocarbon sandstone reservoirs and to understand how the pore geometry affects the conductivity (n and m) of the rock a theoretical study was done. It was also an aim to examine the possibility for developing new S{sub w} - equations (or investigation an effective medium model) valid inhydrocarbon sandstone reservoirs. The results are presented in paper 1. A new equation for water saturation calculation in clean sandstone oil reservoirs is addressed in paper 2. A recommendation for best practice of water saturation calculation in non water wet formation is addressed in paper 3. Finally a new equation for water saturation calculation in thinly interbedded sandstone/mudstone reservoirs is presented in paper 4. The papers are titled: 1) Is the saturation exponent n a constant. 2) A New Model for Calculating Water Saturation In 3) Influence of wettability on water saturation modeling. 4) Water Saturation Calculations in Thinly Interbedded Sandstone/mudstone Reservoirs. A

  15. The human biology--saturated with experience.

    Science.gov (United States)

    Getz, Linn; Kirkengen, Anna Luise; Ulvestad, Elling

    2011-04-08

    The human being is a self-reflecting, relationship-oriented, goal-directed organism in search of meaning. The process of coordinating and developing knowledge about how experience associated with self-conscience, relationships and values can contribute to development of health and disease is a great challenge for the medical profession. We present a theory-guided synthesis of new scientific knowledge from fields such as epigenetics, psycho-neuro-endocrino-immunology, stress research and systems biology. The sources are articles in acknowledged journals and books, chosen to provide insight into associations between life history (biography) and the human body (biology) in a wide sense. Research shows that information about biography, i.e. experienced meaning and relationships, is literally incorporated into the human organism. Epigenetics illustrates the fundamental biological potential for context-dependent adaptation. Further, studies have shown that different types of existential strain may disturb systems for human physiological adaptation, affect structures in the brain and subsequently render the organism vulnerable for disease. However, a sense of belonging and a perception of being supported and acknowledged can contribute to strengthening or restoring health. The traditional approach to increasing biomedical knowledge has prevented insight into the medical significance of experience. The new knowledge necessitates a reorientation of theory and practice within the medical profession both with respect to individuals and society.

  16. Menopausal estrogen therapy predicts better nocturnal oxyhemoglobin saturation.

    Science.gov (United States)

    Saaresranta, Tarja; Polo-Kantola, Päivi; Virtanen, Irina; Vahlberg, Tero; Irjala, Kerttu; Polo, Olli

    2006-10-20

    The respiratory responses in the few previous studies evaluating the effects of short-term unopposed estrogen therapy on breathing in postmenopausal women have been inconsistent. We performed a study to investigate whether long-term estrogen therapy would prevent age-related decline in nocturnal arterial oxyhemoglobin saturation and whether higher serum estradiol concentration is associated with better arterial oxyhemoglobin saturation. Sixty-four healthy postmenopausal women were followed-up for 5 years in a 5-year prospective open follow-up study. The women were users or non-users of estrogen therapy according to their personal preference. Mean overnight arterial oxyhemoglobin saturation was similar at baseline (94.3 +/- 1.1%) and after follow-up (94.5 +/- 1.6%). Present estrogen users had higher mean arterial oxyhemoglobin saturation (95.2 +/- 1.4%) than present non-users (94.0 +/- 1.5%), when adjusted for age and body mass index (p = 0.042). The change in mean arterial oxyhemoglobin saturation during follow-up was not associated with serum estradiol concentration at baseline but associated with estradiol at follow-up (p = 0.042), when adjusted for age and body mass index. At follow-up, women with higher serum estradiol concentration had also higher mean nocturnal arterial oxyhemoglobin saturation (Pearson r = 0.29, p = 0.019) and lower apnea-hypopnea index (Spearman r = -0.28, p = 0.031). The pooled current estrogen users spent proportionally less time with SaO(2) below 90% than non-users (ANCOVA adjusted for age and BMI, p = 0.017). Estrogen use and especially high serum estradiol concentration predict higher mean overnight arterial oxyhemoglobin saturation. The present data suggest that estrogen therapy has favorable respiratory effects.

  17. Why doesn't the ring current injection rate saturate?

    Science.gov (United States)

    Lopez, R. E.; Lyon, J. G.; Mitchell, E.; Bruntz, R.; Merkin, V. G.; Brogl, S.; Toffoletto, F.; Wiltberger, M.

    2009-02-01

    For low values of the solar wind electric field, the response of the polar cap potential is essentially linear, but at high values of VB s, the polar cap potential saturates and does not increase further with increasing VB s. On the other hand, the ring current injection rate does increase linearly with VB s and shows no evidence of saturating. If enhanced convection is the origin of the ring current, this poses a paradox. How can the polar cap potential, and thus convection, saturate when the ring current does not? We examine a possible explanation based on the reexamination of the Burton equation by Vasyliunas (2006). We show that this explanation is not a viable solution to the paradox since it would require a changing polar cap flux, and we demonstrate that the polar cap flux saturates (at around 1 GWb) as the polar cap potential saturates. Instead, we argue that during storms a quasi-steady reconnection region forms in the tail near the Earth. This reconnection region moves closer to the Earth for higher values of solar wind B s, although the polar cap potential, the dayside merging and nightside reconnection rates, and the amount of open flux do not change much as a function of B s once the polar cap potential has become saturated. As the neutral line moves closer, the volume per unit magnetic flux in the closed field line region is less. Flux tubes leaving the reconnection region in general have lower PV γ as B s increases, and lower PV γ flux tubes can penetrate deeper into the inner magnetosphere, leading to a corresponding greater injection of particles into the inner magnetosphere. Thus a reconnection region that is closer to Earth is more effective in creating a strong ring current. This leads to a continued dependence of the ring current injection rate on VB s, although the polar cap potential has saturated.

  18. 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

  19. 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

  20. Optimal sampling schedule for chemical exchange saturation transfer.

    Science.gov (United States)

    Tee, Y K; Khrapitchev, A A; Sibson, N R; Payne, S J; Chappell, M A

    2013-11-01

    The sampling schedule for chemical exchange saturation transfer imaging is normally uniformly distributed across the saturation frequency offsets. When this kind of evenly distributed sampling schedule is used to quantify the chemical exchange saturation transfer effect using model-based analysis, some of the collected data are minimally informative to the parameters of interest. For example, changes in labile proton exchange rate and concentration mainly affect the magnetization near the resonance frequency of the labile pool. In this study, an optimal sampling schedule was designed for a more accurate quantification of amine proton exchange rate and concentration, and water center frequency shift based on an algorithm previously applied to magnetization transfer and arterial spin labeling. The resulting optimal sampling schedule samples repeatedly around the resonance frequency of the amine pool and also near to the water resonance to maximize the information present within the data for quantitative model-based analysis. Simulation and experimental results on tissue-like phantoms showed that greater accuracy and precision (>30% and >46%, respectively, for some cases) were achieved in the parameters of interest when using optimal sampling schedule compared with evenly distributed sampling schedule. Hence, the proposed optimal sampling schedule could replace evenly distributed sampling schedule in chemical exchange saturation transfer imaging to improve the quantification of the chemical exchange saturation transfer effect and parameter estimation. Copyright © 2013 Wiley Periodicals, Inc.

  1. 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

  2. Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti.

    Science.gov (United States)

    Pobigaylo, Nataliya; Wetter, Danijel; Szymczak, Silke; Schiller, Ulf; Kurtz, Stefan; Meyer, Folker; Nattkemper, Tim W; Becker, Anke

    2006-06-01

    Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis

    Directory of Open Access Journals (Sweden)

    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.

  11. In vitro mutagenesis in embryogenic cell suspensions of banana cv. Grande naine (Musa AAA

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.