WorldWideScience

Sample records for generating conditional mutations

  1. Mutation of Auslander generators

    CERN Document Server

    Lada, Magdalini

    2009-01-01

    Let $\\Lambda$ be an artin algebra with representation dimension equal to three and $M$ an Auslander generator of $\\Lambda$. We show how, under certain assumptions, we can mutate $M$ to get a new Auslander generator whose endomorphism ring is derived equivalent to the endomorphism ring of $M$. We apply our results to selfinjective algebras with radical cube zero of infinite representation type, where we construct an infinite set of Auslander generators.

  2. Rapid generation of hypomorphic mutations

    Science.gov (United States)

    Arthur, Laura L.; Chung, Joyce J.; Jankirama, Preetam; Keefer, Kathryn M.; Kolotilin, Igor; Pavlovic-Djuranovic, Slavica; Chalker, Douglas L.; Grbic, Vojislava; Green, Rachel; Menassa, Rima; True, Heather L.; Skeath, James B.; Djuranovic, Sergej

    2017-01-01

    Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels. PMID:28106166

  3. Biological evolution model with conditional mutation rates

    Science.gov (United States)

    Saakian, David B.; Ghazaryan, Makar; Bratus, Alexander; Hu, Chin-Kun

    2017-05-01

    We consider an evolution model, in which the mutation rates depend on the structure of population: the mutation rates from lower populated sequences to higher populated sequences are reduced. We have applied the Hamilton-Jacobi equation method to solve the model and calculate the mean fitness. We have found that the modulated mutation rates, directed to increase the mean fitness.

  4. Generation of mutation hotspots in ageing bacterial colonies

    DEFF Research Database (Denmark)

    Sekowska, Agnieszka; Wendel, Sofie; Christian Fischer, Emil

    2016-01-01

    How do ageing bacterial colonies generate adaptive mutants? Over a period of two months, we isolated on ageing colonies outgrowing mutants able to use a new carbon source, and sequenced their genomes. This allowed us to uncover exquisite details on the molecular mechanism behind their adaptation......: most mutations were located in just a few hotspots in the genome, and over time, mutations increasingly were consistent with the involvement of 8-oxo-guanosine, formed exclusively on the transcribed strand. This work provides strong support for retromutagenesis as a general process creating adaptive...... mutations during ageing....

  5. Mutation Sampling Technique for the Generation of Structural Test Data

    CERN Document Server

    Scholive, M; Robach, C; Flottes, M L; Rouzeyre, B

    2011-01-01

    Our goal is to produce validation data that can be used as an efficient (pre) test set for structural stuck-at faults. In this paper, we detail an original test-oriented mutation sampling technique used for generating such data and we present a first evaluation on these validation data with regard to a structural test.

  6. Generation of conditional knockout alleles for PRL-3.

    Science.gov (United States)

    Yan, Hong; Kong, Dong; Ge, Xiaomei; Gao, Xiang; Han, Xiao

    2011-11-01

    Phosphatase of regenerating liver-3 (PRL-3) is a member of the protein tyrosine phosphatase (PTP) superfamily and is highly expressed in cancer metastases. For better understanding of the role of PRL-3 in tumor metastasis, we applied a rapid and efficient method for generating PRL-3 floxed mice and investigated its phenotypes. A BAC retrieval strategy was applied to construct the PRL-3 conditional gene-targeting vector. Exon 4 was selected for deletion to generate a nonfunctional prematurely terminated short peptide as it will cause a frame-shift mutation. Conditional knockout PRL-3 mice were generated by using the Cre-loxP system and were validated by Southern blot and RT-PCR analysis. Further analysis revealed the phenotype characteristics of PRL-3 knockout mice and wildtype mice. In this study, we successfully constructed the PRL-3 conditional knockout mice, which will be helpful to clarify the roles of PRL-3 and the mechanisms in tumor metastasis.

  7. Structural Learning of Attack Vectors for Generating Mutated XSS Attacks

    Directory of Open Access Journals (Sweden)

    Yi-Hsun Wang

    2010-09-01

    Full Text Available Web applications suffer from cross-site scripting (XSS attacks that resulting from incomplete or incorrect input sanitization. Learning the structure of attack vectors could enrich the variety of manifestations in generated XSS attacks. In this study, we focus on generating more threatening XSS attacks for the state-of-the-art detection approaches that can find potential XSS vulnerabilities in Web applications, and propose a mechanism for structural learning of attack vectors with the aim of generating mutated XSS attacks in a fully automatic way. Mutated XSS attack generation depends on the analysis of attack vectors and the structural learning mechanism. For the kernel of the learning mechanism, we use a Hidden Markov model (HMM as the structure of the attack vector model to capture the implicit manner of the attack vector, and this manner is benefited from the syntax meanings that are labeled by the proposed tokenizing mechanism. Bayes theorem is used to determine the number of hidden states in the model for generalizing the structure model. The paper has the contributions as following: (1 automatically learn the structure of attack vectors from practical data analysis to modeling a structure model of attack vectors, (2 mimic the manners and the elements of attack vectors to extend the ability of testing tool for identifying XSS vulnerabilities, (3 be helpful to verify the flaws of blacklist sanitization procedures of Web applications. We evaluated the proposed mechanism by Burp Intruder with a dataset collected from public XSS archives. The results show that mutated XSS attack generation can identify potential vulnerabilities.

  8. Structural Learning of Attack Vectors for Generating Mutated XSS Attacks

    CERN Document Server

    Wang, Yi-Hsun; Lee, Hahn-Ming; 10.4204/EPTCS.35.2

    2010-01-01

    Web applications suffer from cross-site scripting (XSS) attacks that resulting from incomplete or incorrect input sanitization. Learning the structure of attack vectors could enrich the variety of manifestations in generated XSS attacks. In this study, we focus on generating more threatening XSS attacks for the state-of-the-art detection approaches that can find potential XSS vulnerabilities in Web applications, and propose a mechanism for structural learning of attack vectors with the aim of generating mutated XSS attacks in a fully automatic way. Mutated XSS attack generation depends on the analysis of attack vectors and the structural learning mechanism. For the kernel of the learning mechanism, we use a Hidden Markov model (HMM) as the structure of the attack vector model to capture the implicit manner of the attack vector, and this manner is benefited from the syntax meanings that are labeled by the proposed tokenizing mechanism. Bayes theorem is used to determine the number of hidden states in the model...

  9. Conditional prediction intervals of wind power generation

    DEFF Research Database (Denmark)

    Pinson, Pierre; Kariniotakis, Georges

    2010-01-01

    A generic method for the providing of prediction intervals of wind power generation is described. Prediction intervals complement the more common wind power point forecasts, by giving a range of potential outcomes for a given probability, their so-called nominal coverage rate. Ideally they inform...... on the characteristics of prediction errors for providing conditional interval forecasts. By simultaneously generating prediction intervals with various nominal coverage rates, one obtains full predictive distributions of wind generation. Adapted resampling is applied here to the case of an onshore Danish wind farm......, for which three point forecasting methods are considered as input. The probabilistic forecasts generated are evaluated based on their reliability and sharpness, while compared to forecasts based on quantile regression and the climatology benchmark. The operational application of adapted resampling...

  10. Test Case Generation using Mutation Operators and Fault Classification

    CERN Document Server

    Jeevarathinam, Mrs R

    2010-01-01

    Software testing is the important phase of software development process. But, this phase can be easily missed by software developers because of their limited time to complete the project. Since, software developers finish their software nearer to the delivery time; they dont get enough time to test their program by creating effective test cases. . One of the major difficulties in software testing is the generation of test cases that satisfy the given adequacy criterion Moreover, creating manual test cases is a tedious work for software developers in the final rush hours. A new approach which generates test cases can help the software developers to create test cases from software specifications in early stage of software development (before coding) and as well as from program execution traces from after software development (after coding). Heuristic techniques can be applied for creating quality test cases. Mutation testing is a technique for testing software units that has great potential for improving the qu...

  11. Generation of RNA in abiotic conditions.

    Science.gov (United States)

    di Mauro, Ernesto

    Generation of RNA in abiotic conditions. Ernesto Di Mauro Dipartimento di Genetica Bi-ologia Molecolare, Universit` "Sapienza" Roma, Italy. a At least four conditions must be satisfied for the spontaneous generation of (pre)-genetic poly-mers: 1) availability of precursors that are activated enough to spontaneously polymerize. Preliminary studies showed that (a) nucleic bases and acyclonucleosides can be synthesized from formamide H2NCOH by simply heating with prebiotically available mineral catalysts [last reviewed in (1)], and that b) nucleic bases can be phosphorylated in every possible posi-tion [2'; 3'; 5'; cyclic 2',3'; cyclic 3',5' (2)]. The higher stability of the cyclic forms allows their accumulation. 2) A polymerization mechanism. A reaction showing the formation of RNA polymers starting from prebiotically plausible precursors (3',5' cyclic GMP and 3', 5'cyclic AMP) was recently reported (3). Polymerization in these conditions is thermodynamically up-hill and an equilibrium is attained that limits the maximum length of the polymer produced to about 40 nucleotides for polyG and 100 nucleotides for polyA. 3) Ligation of the synthesized oligomers. If this type of reaction could occur according to a terminal-joining mechanism and could generate canonical 3',5' phosphodiester bonds, exponential growth would be obtained of the generated oligomers. This type of reaction has been reported (4) , limited to homogeneous polyA sequences and leading to the production of polyA dimers and tetramers. What is still missing are: 4) mechanisms that provide the proof of principle for the generation of sequence complexity. We will show evidence for two mechanisms providing this proof of principle for simple complementary sequences. Namely: abiotic sequence complementary-driven terminal ligation and sequence-complementary terminal growth. In conclusion: all the steps leading to the generation of RNA in abiotic conditions are satisfied. (1) R Saladino, C Crestini, F

  12. Generating new prions by targeted mutation or segment duplication.

    Science.gov (United States)

    Paul, Kacy R; Hendrich, Connor G; Waechter, Aubrey; Harman, Madison R; Ross, Eric D

    2015-07-14

    Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity.

  13. Targeted Next Generation Sequencing reveals previously unidentified and mutations

    NARCIS (Netherlands)

    M.D. Nellist (Mark); R.W.W. Brouwer (Rutger); C. Kockx (Christel); M. van Veghel-Plandsoen (Monique); C.J. Withagen-Hermans (C.); L. Prins-Bakker (Lida); M. Hoogeveen-Westerveld (Marianne); A. Mrsic (Alan); M.M.P. van den Berg (Mike M P); A.E. Koopmans (Anna); M.C.Y. de Wit (Marie Claire); F.H. Jansen (Flip); A.A. Maat-Kievit (Anneke); A.M.W. van den Ouweland (Ans); D.J.J. Halley (Dicky); A. de Klein (Annelies); W.F.J. van IJcken (Wilfred)

    2015-01-01

    textabstractBackground: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in and . Conventional DNA diagnostic screens identify a or mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undete

  14. Operational conditions for random-number generation

    Science.gov (United States)

    Compagner, A.

    1995-11-01

    Ensemble theory is used to describe arbitrary sequences of integers, whether formed by the decimals of π or produced by a roulette or by any other means. Correlation coefficients of any range and order are defined as Fourier transforms of the ensemble weights. Competing definitions of random sequences are considered. Special attention is given to sequences of random numbers needed for Monte Carlo calculations. Different recipes for those sequences lead to correlations that vary in range and order, but the total amount of correlation is the same for all sequences of a given length (without internal periodicities). For maximum-length sequences produced by linear algorithms, most correlation coefficients are zero, but the remaining ones are of absolute value 1. In well-tempered sequences, these complete correlations are of high order or of very long range. General conditions to be obeyed by random-number generators are discussed and a qualitative method for comparing different recipes is given.

  15. Hungarian surveillance of germinal mutations. Lack of detectable increase in indicator conditions caused by germinal mutations following the Chernobyl accident

    Energy Technology Data Exchange (ETDEWEB)

    Czeizel, A. (National Inst. of Hygiene, Budapest (Hungary). Dept. of Human Genetics and Teratology)

    1989-07-01

    The Hungarian surveillance of germinal mutations is based on three indicator conditions seen in offspring, i.e., 15 sentinel anomalies, Down syndrome and component anomaly pairs of unidentified multiple congenital anomalies. It is an 'opportunistic program', because the necessary data are available from the Hungarian Congenital Malformation Registry. This system is described and the criteria of a good registry are summarized. The analysis of indicator conditions caused by germinal mutations did not reveal any measurable mutagenic effects in Hungary following the accident at the Chernobyl nuclear power plant. The pros and cons of germinal mutation surveillance are discussed. (orig.).

  16. Beyond BRAFV600: clinical mutation panel testing by next-generation sequencing in advanced melanoma

    Science.gov (United States)

    Siroy, Alan E.; Boland, Genevieve M.; Milton, Denái R.; Roszik, Jason; Frankian, Silva; Malke, Jared; Haydu, Lauren; Prieto, Victor G.; Tetzlaff, Michael; Ivan, Doina; Wang, Wei-Lien; Torres-Cabala, Carlos; Curry, Jonathan; Roy-Chowdhuri, Sinchita; Broaddus, Russell; Rashid, Asif; Stewart, John; Gershenwald, Jeffrey E.; Amaria, Rodabe N.; Patel, Sapna P.; Papadopoulos, Nicholas E.; Bedikian, Agop; Hwu, Wen-Jen; Hwu, Patrick; Diab, Adi; Woodman, Scott E.; Aldape, Kenneth D.; Luthra, Rajyalakshmi; Patel, Keyur P.; Shaw, Kenna R.; Mills, Gordon B.; Mendelsohn, John; Meric-Bernstam, Funda; Kim, Kevin B.; Routbort, Mark J.; Lazar, Alexander J.; Davies, Michael A.

    2014-01-01

    The management of melanoma has evolved due to improved understanding of its molecular drivers. To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed. Mutations were identified in 43 of the 46 genes on the panel. The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%). Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype. Concurrent TP53 mutations were the most frequent event in tumors with BRAFV600 and NRAS mutations. Melanomas with BRAFNon-V600 mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%). The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location. Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600 and NRAS mutations. These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma. PMID:25148578

  17. The effect of induced mutations on quantitative traits in Arabidopsis thaliana: Natural versus artificial conditions.

    Science.gov (United States)

    Stearns, Frank W; Fenster, Charles B

    2016-12-01

    Mutations are the ultimate source of all genetic variations. New mutations are expected to affect quantitative traits differently depending on the extent to which traits contribute to fitness and the environment in which they are tested. The dogma is that the preponderance of mutations affecting fitness will be skewed toward deleterious while their effects on nonfitness traits will be bidirectionally distributed. There are mixed views on the role of stress in modulating these effects. We quantify mutation effects by inducing mutations in Arabidopsis thaliana (Columbia accession) using the chemical ethylmethane sulfonate. We measured the effects of new mutations relative to a premutation founder for fitness components under both natural (field) and artificial (growth room) conditions. Additionally, we measured three other quantitative traits, not expected to contribute directly to fitness, under artificial conditions. We found that induced mutations were equally as likely to increase as decrease a trait when that trait was not closely related to fitness (traits that were neither survivorship nor reproduction). We also found that new mutations were more likely to decrease fitness or fitness-related traits under more stressful field conditions than under relatively benign artificial conditions. In the benign condition, the effect of new mutations on fitness components was similar to traits not as closely related to fitness. These results highlight the importance of measuring the effects of new mutations on fitness and other traits under a range of conditions.

  18. Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype

    DEFF Research Database (Denmark)

    Hove, Hanne Buciek; Dunø, Morten; Daugaard-Jensen, Jette;

    Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype.......Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype....

  19. Familial mild hyperglycemia associated with a novel ABCC8-V84I mutation within three generations

    DEFF Research Database (Denmark)

    Gonsorcikova, Lucie; Vaxillaire, Martine; Pruhova, Stepanka;

    2011-01-01

    of the young genes (MODY1-4 and 6), we identified a novel ABCC8 V84I mutation, which segregated with autosomal dominant transmission of mild hyperglycemia within three generations. This mutation that is located in a conserved area of transmembrane domain TMD0 seems to be a rare cause of clinical phenotype...

  20. Conditional prediction intervals of wind power generation

    OpenAIRE

    Pinson, Pierre; Kariniotakis, Georges

    2010-01-01

    A generic method for the providing of prediction intervals of wind power generation is described. Prediction intervals complement the more common wind power point forecasts, by giving a range of potential outcomes for a given probability, their so-called nominal coverage rate. Ideally they inform of the situation-specific uncertainty of point forecasts. In order to avoid a restrictive assumption on the shape of forecast error distributions, focus is given to an empirical and nonparametric app...

  1. Profiling of Somatic Mutations in Phaeochromocytoma and Paraganglioma by Targeted Next Generation Sequencing Analysis

    Directory of Open Access Journals (Sweden)

    Andrea Luchetti

    2015-01-01

    Full Text Available At least 12 genes (FH, HIF2A, MAX, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL have been implicated in inherited predisposition to phaeochromocytoma (PCC, paraganglioma (PGL, or head and neck paraganglioma (HNPGL and a germline mutation may be detected in more than 30% of cases. Knowledge of somatic mutations contributing to PCC/PGL/HNPGL pathogenesis has received less attention though mutations in HRAS, HIF2A, NF1, RET, and VHL have been reported. To further elucidate the role of somatic mutation in PCC/PGL/HNPGL tumourigenesis, we employed a next generation sequencing strategy to analyse “mutation hotspots” in 50 human cancer genes. Mutations were identified for HRAS (c.37G>C; p.G13R and c.182A>G; p.Q61R in 7.1% (6/85; for BRAF (c.1799T>A; p.V600E in 1.2% (1/85 of tumours; and for TP53 (c.1010G>A; p.R337H in 2.35% (2/85 of cases. Twenty-one tumours harboured mutations in inherited PCC/PGL/HNPGL genes and no HRAS, BRAF, or TP53 mutations occurred in this group. Combining our data with previous reports of HRAS mutations in PCC/PGL we find that the mean frequency of HRAS/BRAF mutations in sporadic PCC/PGL is 8.9% (24/269 and in PCC/PGL with an inherited gene mutation 0% (0/148 suggesting that HRAS/BRAF mutations and inherited PCC/PGL genes mutations might be mutually exclusive. We report the first evidence for BRAF mutations in the pathogenesis of PCC/PGL/HNPGL.

  2. Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.

    Science.gov (United States)

    Narumi, Satoshi; Matsuo, Kumihiro; Ishii, Tomohiro; Tanahashi, Yusuke; Hasegawa, Tomonobu

    2013-01-01

    Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

  3. Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Satoshi Narumi

    Full Text Available Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS. Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000 and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS] were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

  4. Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations.

    Science.gov (United States)

    Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk; Griewank, Klaus G; Roesch, Alexander

    2017-06-20

    Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

  5. Next-generation sequencing identifies novel CACNA1A gene mutations in episodic ataxia type 2.

    Science.gov (United States)

    Maksemous, Neven; Roy, Bishakha; Smith, Robert A; Griffiths, Lyn R

    2016-03-01

    Episodic Ataxia type 2 (EA2) is a rare autosomal dominantly inherited neurological disorder characterized by recurrent disabling imbalance, vertigo, and episodes of ataxia lasting minutes to hours. EA2 is caused most often by loss of function mutations of the calcium channel gene CACNA1A. In addition to EA2, mutations in CACNA1A are responsible for two other allelic disorders: familial hemiplegic migraine type 1 (FHM1) and spinocerebellar ataxia type 6 (SCA6). Herein, we have utilized next-generation sequencing (NGS) to screen the coding sequence, exon-intron boundaries, and Untranslated Regions (UTRs) of five genes where mutation is known to produce symptoms related to EA2, including CACNA1A. We performed this screening in a group of 31 unrelated patients with EA2 symptoms. Both novel and known mutations were detected through NGS technology, and confirmed through Sanger sequencing. Genetic testing showed in total 15 mutation bearing patients (48%), of which nine were novel mutations (6 missense and 3 small frameshift deletion mutations) and six known mutations (4 missense and 2 nonsense).These results demonstrate the efficiency of our NGS-panel for detecting known and novel mutations for EA2 in the CACNA1A gene, also identifying a novel missense mutation in ATP1A2 which is not a normal target for EA2 screening.

  6. Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Kazuko Sakai

    Full Text Available Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5. The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100. KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100. DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

  7. Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.

    Science.gov (United States)

    Sakai, Kazuko; Tsurutani, Junji; Yamanaka, Takeharu; Yoneshige, Azusa; Ito, Akihiko; Togashi, Yosuke; De Velasco, Marco A; Terashima, Masato; Fujita, Yoshihiko; Tomida, Shuta; Tamura, Takao; Nakagawa, Kazuhiko; Nishio, Kazuto

    2015-01-01

    Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

  8. Adhesion of Escherichia coli under flow conditions reveals potential novel effects of FimH mutations

    DEFF Research Database (Denmark)

    Feenstra, T.; Schmidt Thøgersen, Mariane; Wieser, E.

    2017-01-01

    H mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial...... mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel...

  9. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations.

    Science.gov (United States)

    Soldner, Frank; Laganière, Josée; Cheng, Albert W; Hockemeyer, Dirk; Gao, Qing; Alagappan, Raaji; Khurana, Vikram; Golbe, Lawrence I; Myers, Richard H; Lindquist, Susan; Zhang, Lei; Guschin, Dmitry; Fong, Lauren K; Vu, B Joseph; Meng, Xiangdong; Urnov, Fyodor D; Rebar, Edward J; Gregory, Philip D; Zhang, H Steve; Jaenisch, Rudolf

    2011-07-22

    Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Considerations for clinical read alignment and mutational profiling using next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Gavin R Oliver

    2012-07-01

    Full Text Available Next-generation sequencing technologies are increasingly being applied in clinical settings, however the data are characterized by a range of platform-specific artifacts making downstream analysis problematic and error prone. One major application of NGS is in the profiling of clinically relevant mutations whereby sequences are aligned to a reference genome and potential mutations assessed and scored. Accurate sequence alignment is pivotal in reliable assessment of potential mutations however selection of appropriate alignment tools is a non-trivial task complicated by the availability of multiple solutions each with its own performance characteristics. Using BRCA1 as an example, we have simulated and mutated a test dataset based on Illumina sequencing technology. Our findings reveal key differences in the performances of a range of common commercial and open source tools and will be of importance to anyone using NGS to profile mutations in clinical or basic research.

  11. Clinical relevance of sensitive and quantitative STAT3 mutation analysis using next-generation sequencing in T-cell large granular lymphocytic leukemia

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Larsen, Martin; Rewes, Annika;

    2014-01-01

    Diagnosis of T-cell large granular lymphocytic leukemia (T-LGL) is often challenging because clinical and laboratory characteristics are overlapping with nonneoplastic conditions. Recently, mutation in the STAT3 gene has been identified as a recurrent genetic abnormality in T-LGL. STAT3 mutation......, therefore, represents a promising marker in T-LGL diagnostics. We developed a new quantitative next-generation sequencing assay that allows sensitive analysis of the STAT3 gene. The assay was used to study the utility of STAT3 mutation analysis as a diagnostic tool in T-LGL. The study included 16 T...... donors all tested negative, thus demonstrating the specificity of the assay. The results also indicated that mutation levels in blood and bone marrow are not systematically different, and next-generation sequencing-based STAT3 mutation analysis represents a sensitive method for monitoring residual...

  12. Next Generation Sequencing to Determine the Cystic Fibrosis Mutation Spectrum in Palestinian Population

    Directory of Open Access Journals (Sweden)

    O. Essawi

    2015-01-01

    Full Text Available An extensive molecular analysis of the CF transmembrane regulator (CFTR gene was performed to establish the CFTR mutation spectrum and frequencies in the Palestinian population, which can be considered as an understudied population. We used a targeted Next Generation Sequencing approach to sequence the entire coding region and the adjacent sequences of the CFTR gene combined with MLPA analysis of 60 unrelated CF patients. Eighteen different CF-causing mutations, including one previously undescribed mutation p.(Gly1265Arg, were identified. The overall detection rate is up to 67%, and when we consider only CF patients with sweat chloride concentrations >70 mEq/L, we even have a pickup rate of 92%. Whereas p.(Phe508del is the most frequent allele (35% of the positive cases, 3 other mutations c.2988+1Kbdel8.6Kb, c.1393-1G>A, and p.(Gly85Glu showed frequencies higher than 5% and a total of 9 mutations account for 84% of the mutations. This limited spectrum of CF mutations is in agreement with the homozygous ethnic origin of the Palestinian population. The relative large portion of patients without a mutation is most likely due to clinical misdiagnosis. Our results will be important in the development of an adequate molecular diagnostic test for CF in Palestine.

  13. CDH1 mutations in gastric cancer patients from northern Brazil identified by Next- Generation Sequencing (NGS).

    Science.gov (United States)

    El-Husny, Antonette; Raiol-Moraes, Milene; Amador, Marcos; Ribeiro-Dos-Santos, André M; Montagnini, André; Barbosa, Silvanira; Silva, Artur; Assumpção, Paulo; Ishak, Geraldo; Santos, Sidney; Pinto, Pablo; Cruz, Aline; Ribeiro-Dos-Santos, Ândrea

    2016-05-13

    Gastric cancer is considered to be the fifth highest incident tumor worldwide and the third leading cause of cancer deaths. Developing regions report a higher number of sporadic cases, but there are only a few local studies related to hereditary cases of gastric cancer in Brazil to confirm this fact. CDH1 germline mutations have been described both in familial and sporadic cases, but there is only one recent molecular description of individuals from Brazil. In this study we performed Next Generation Sequencing (NGS) to assess CDH1 germline mutations in individuals who match the clinical criteria for Hereditary Diffuse Gastric Cancer (HDGC), or who exhibit very early diagnosis of gastric cancer. Among five probands we detected CDH1 germline mutations in two cases (40%). The mutation c.1023T > G was found in a HDGC family and the mutation c.1849G > A, which is nearly exclusive to African populations, was found in an early-onset case of gastric adenocarcinoma. The mutations described highlight the existence of gastric cancer cases caused by CDH1 germline mutations in northern Brazil, although such information is frequently ignored due to the existence of a large number of environmental factors locally. Our report represent the first CDH1 mutations in HDGC described from Brazil by an NGS platform.

  14. Within-generation mutation variance for litter size in inbred mice.

    Science.gov (United States)

    Casellas, Joaquim; Medrano, Juan F

    2008-08-01

    The mutational input of genetic variance per generation (sigma(m)(2)) is the lower limit of the genetic variability in inbred strains of mice, although greater values could be expected due to the accumulation of new mutations in successive generations. A mixed-model analysis using Bayesian methods was applied to estimate sigma(m)(2) and the across-generation accumulated genetic variability on litter size in 46 generations of a C57BL/6J inbred strain. This allowed for a separate inference on sigma(m)(2) and on the additive genetic variance in the base population (sigma(a)(2)). The additive genetic variance in the base generation was 0.151 and quickly decreased to almost null estimates in generation 10. On the other hand, sigma(m)(2) was moderate (0.035) and the within-generation mutational variance increased up to generation 14, then oscillating between 0.102 and 0.234 in remaining generations. This pattern suggested the existence of a continuous uploading of genetic variability for litter size (h(2)=0.045). Relevant genetic drift was not detected in this population. In conclusion, our approach allowed for separate estimation of sigma(a)(2) and sigma(m)(2) within the mixed-model framework, and the heritability obtained highlighted the significant and continuous influence of new genetic variability affecting the genetic stability of inbred strains.

  15. Detecting novel genetic mutations in Chinese Usher syndrome families using next-generation sequencing technology.

    Science.gov (United States)

    Qu, Ling-Hui; Jin, Xin; Xu, Hai-Wei; Li, Shi-Ying; Yin, Zheng-Qin

    2015-02-01

    Usher syndrome (USH) is the most common cause of combined blindness and deafness inherited in an autosomal recessive mode. Molecular diagnosis is of great significance in revealing the molecular pathogenesis and aiding the clinical diagnosis of this disease. However, molecular diagnosis remains a challenge due to high phenotypic and genetic heterogeneity in USH. This study explored an approach for detecting disease-causing genetic mutations in candidate genes in five index cases from unrelated USH families based on targeted next-generation sequencing (NGS) technology. Through systematic data analysis using an established bioinformatics pipeline and segregation analysis, 10 pathogenic mutations in the USH disease genes were identified in the five USH families. Six of these mutations were novel: c.4398G > A and EX38-49del in MYO7A, c.988_989delAT in USH1C, c.15104_15105delCA and c.6875_6876insG in USH2A. All novel variations segregated with the disease phenotypes in their respective families and were absent from ethnically matched control individuals. This study expanded the mutation spectrum of USH and revealed the genotype-phenotype relationships of the novel USH mutations in Chinese patients. Moreover, this study proved that targeted NGS is an accurate and effective method for detecting genetic mutations related to USH. The identification of pathogenic mutations is of great significance for elucidating the underlying pathophysiology of USH.

  16. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing

    Science.gov (United States)

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T.; Scarpa, Aldo

    2016-01-01

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

  17. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing.

    Science.gov (United States)

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T; Scarpa, Aldo

    2016-01-12

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue.

  18. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Jordan Eboreime

    Full Text Available We used targeted next generation deep-sequencing (Safe Sequencing System to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11 were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10-8 suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

  19. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

    Science.gov (United States)

    Eboreime, Jordan; Choi, Soo-Kung; Yoon, Song-Ro; Arnheim, Norman; Calabrese, Peter

    2016-01-01

    We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10-8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

  20. Overcoming mTOR resistance mutations with a new-generation mTOR inhibitor.

    Science.gov (United States)

    Rodrik-Outmezguine, Vanessa S; Okaniwa, Masanori; Yao, Zhan; Novotny, Chris J; McWhirter, Claire; Banaji, Arpitha; Won, Helen; Wong, Wai; Berger, Mike; de Stanchina, Elisa; Barratt, Derek G; Cosulich, Sabina; Klinowska, Teresa; Rosen, Neal; Shokat, Kevan M

    2016-06-09

    Precision medicines exert selective pressure on tumour cells that leads to the preferential growth of resistant subpopulations, necessitating the development of next-generation therapies to treat the evolving cancer. The PIK3CA-AKT-mTOR pathway is one of the most commonly activated pathways in human cancers, which has led to the development of small-molecule inhibitors that target various nodes in the pathway. Among these agents, first-generation mTOR inhibitors (rapalogs) have caused responses in 'N-of-1' cases, and second-generation mTOR kinase inhibitors (TORKi) are currently in clinical trials. Here we sought to delineate the likely resistance mechanisms to existing mTOR inhibitors in human cell lines, as a guide for next-generation therapies. The mechanism of resistance to the TORKi was unusual in that intrinsic kinase activity of mTOR was increased, rather than a direct active-site mutation interfering with drug binding. Indeed, identical drug-resistant mutations have been also identified in drug-naive patients, suggesting that tumours with activating MTOR mutations will be intrinsically resistant to second-generation mTOR inhibitors. We report the development of a new class of mTOR inhibitors that overcomes resistance to existing first- and second-generation inhibitors. The third-generation mTOR inhibitor exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.

  1. Automated generation of formal safety conditions from railway interlocking tables

    DEFF Research Database (Denmark)

    Haxthausen, Anne Elisabeth

    2014-01-01

    This paper describes a tool for extracting formal safety conditions from interlocking tables for railway interlocking systems. The tool has been applied to generate safety conditions for the interlocking system at Stenstrup station in Denmark, and the SAL model checker tool has been used to check...

  2. Wide spetcrum mutational analysis of metastatic renal cell cancer: a retrospective next generation sequencing approach

    Science.gov (United States)

    Fiorentino, Michelangelo; Gruppioni, Elisa; Massari, Francesco; Giunchi, Francesca; Altimari, Annalisa; Ciccarese, Chiara; Bimbatti, Davide; Scarpa, Aldo; Iacovelli, Roberto; Porta, Camillo; Virinder, Sarhadi; Tortora, Giampaolo; Artibani, Walter; Schiavina, Riccardo; Ardizzoni, Andrea; Brunelli, Matteo; Knuutila, Sakari; Martignoni, Guido

    2017-01-01

    Renal cell cancer (RCC) is characterized by histological and molecular heterogeneity that may account for variable response to targeted therapies. We evaluated retrospectively with a next generation sequencing (NGS) approach using a pre-designed cancer panel the mutation burden of 32 lesions from 22 metastatic RCC patients treated with at least one tyrosine kinase or mTOR inhibitor. We identified mutations in the VHL, PTEN, JAK3, MET, ERBB4, APC, CDKN2A, FGFR3, EGFR, RB1, TP53 genes. Somatic alterations were correlated with response to therapy. Most mutations hit VHL1 (31,8%) followed by PTEN (13,6%), JAK3, FGFR and TP53 (9% each). Eight (36%) patients were wild-type at least for the genes included in the panel. A genotype concordance between primary RCC and its secondary lesion was found in 3/6 cases. Patients were treated with Sorafenib, Sunitinib and Temsirolimus with partial responses in 4 (18,2%) and disease stabilization in 7 (31,8%). Among the 4 partial responders, 1 (25%) was wild-type and 3 (75%) harbored different VHL1 variants. Among the 7 patients with disease stabilization 2 (29%) were wild-type, 2 (29%) PTEN mutated, and single patients (14% each) displayed mutations in VHL1, JAK3 and APC/CDKN2A. Among the 11 non-responders 7 (64%) were wild-type, 2 (18%) were p53 mutated and 2 (18%) VHL1 mutated. No significant associations were found among RCC histotype, mutation variants and response to therapies. In the absence of predictive biomarkers for metastatic RCC treatment, a NGS approach may address single patients to basket clinical trials according to actionable molecular specific alterations. PMID:27741505

  3. Wide spetcrum mutational analysis of metastatic renal cell cancer: a retrospective next generation sequencing approach.

    Science.gov (United States)

    Fiorentino, Michelangelo; Gruppioni, Elisa; Massari, Francesco; Giunchi, Francesca; Altimari, Annalisa; Ciccarese, Chiara; Bimbatti, Davide; Scarpa, Aldo; Iacovelli, Roberto; Porta, Camillo; Virinder, Sarhadi; Tortora, Giampaolo; Artibani, Walter; Schiavina, Riccardo; Ardizzoni, Andrea; Brunelli, Matteo; Knuutila, Sakari; Martignoni, Guido

    2017-01-31

    Renal cell cancer (RCC) is characterized by histological and molecular heterogeneity that may account for variable response to targeted therapies. We evaluated retrospectively with a next generation sequencing (NGS) approach using a pre-designed cancer panel the mutation burden of 32 lesions from 22 metastatic RCC patients treated with at least one tyrosine kinase or mTOR inhibitor. We identified mutations in the VHL, PTEN, JAK3, MET, ERBB4, APC, CDKN2A, FGFR3, EGFR, RB1, TP53 genes. Somatic alterations were correlated with response to therapy. Most mutations hit VHL1 (31,8%) followed by PTEN (13,6%), JAK3, FGFR and TP53 (9% each). Eight (36%) patients were wild-type at least for the genes included in the panel.A genotype concordance between primary RCC and its secondary lesion was found in 3/6 cases. Patients were treated with Sorafenib, Sunitinib and Temsirolimus with partial responses in 4 (18,2%) and disease stabilization in 7 (31,8%). Among the 4 partial responders, 1 (25%) was wild-type and 3 (75%) harbored different VHL1 variants. Among the 7 patients with disease stabilization 2 (29%) were wild-type, 2 (29%) PTEN mutated, and single patients (14% each) displayed mutations in VHL1, JAK3 and APC/CDKN2A. Among the 11 non-responders 7 (64%) were wild-type, 2 (18%) were p53 mutated and 2 (18%) VHL1 mutated.No significant associations were found among RCC histotype, mutation variants and response to therapies. In the absence of predictive biomarkers for metastatic RCC treatment, a NGS approach may address single patients to basket clinical trials according to actionable molecular specific alterations.

  4. Gauge-fixing Condition on Generating Superfield of Chiral Multiplet

    CERN Document Server

    Kimura, Tetsuji

    2015-01-01

    We study a supergauge transformation of a complex superfield which generates a chiral superfield in two-dimensional ${\\cal N}=(2,2)$ theory. We refer to this complex superfield as the generating superfield. The chiral superfield is not sensitive of the supergauge transformation. Since there exist redundant component fields in the generating superfield, we remove some of them by a gauge-fixing condition. This situation is parallel to that of a vector superfield. In order to obtain a suitable configuration of the GLSM for the exotic five-brane which gives rise to a nongeometric background, we impose a relatively relaxed gauge-fixing condition. It turns out that the gauge-fixed generating superfield is different from a semichiral superfield whose scalar field represents a coordinate of generalized K\\"{a}hler geometry.

  5. Beyond BRAF(V600): clinical mutation panel testing by next-generation sequencing in advanced melanoma.

    Science.gov (United States)

    Siroy, Alan E; Boland, Genevieve M; Milton, Denái R; Roszik, Jason; Frankian, Silva; Malke, Jared; Haydu, Lauren; Prieto, Victor G; Tetzlaff, Michael; Ivan, Doina; Wang, Wei-Lien; Torres-Cabala, Carlos; Curry, Jonathan; Roy-Chowdhuri, Sinchita; Broaddus, Russell; Rashid, Asif; Stewart, John; Gershenwald, Jeffrey E; Amaria, Rodabe N; Patel, Sapna P; Papadopoulos, Nicholas E; Bedikian, Agop; Hwu, Wen-Jen; Hwu, Patrick; Diab, Adi; Woodman, Scott E; Aldape, Kenneth D; Luthra, Rajyalakshmi; Patel, Keyur P; Shaw, Kenna R; Mills, Gordon B; Mendelsohn, John; Meric-Bernstam, Funda; Kim, Kevin B; Routbort, Mark J; Lazar, Alexander J; Davies, Michael A

    2015-02-01

    The management of melanoma has evolved owing to improved understanding of its molecular drivers. To augment the current understanding of the prevalence, patterns, and associations of mutations in this disease, the results of clinical testing of 699 advanced melanoma patients using a pan-cancer next-generation sequencing (NGS) panel of hotspot regions in 46 genes were reviewed. Mutations were identified in 43 of the 46 genes on the panel. The most common mutations were BRAFV600 (36%), NRAS (21%), TP53 (16%), BRAFNon-V600 (6%), and KIT (4%). Approximately one-third of melanomas had >1 mutation detected, and the number of mutations per tumor was associated with melanoma subtype. Concurrent TP53 mutations were the most frequent events in tumors with BRAFV600 and NRAS mutations. Melanomas with BRAFNon-V600mutations frequently harbored concurrent NRAS mutations (18%), which were rare in tumors with BRAFV600 mutations (1.6%). The prevalence of BRAFV600 and KIT mutations were significantly associated with melanoma subtypes, and BRAFV600 and TP53 mutations were significantly associated with cutaneous primary tumor location. Multiple potential therapeutic targets were identified in metastatic unknown primary and cutaneous melanomas that lacked BRAFV600 and NRAS mutations. These results enrich our understanding of the patterns and clinical associations of oncogenic mutations in melanoma.

  6. Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer

    Science.gov (United States)

    Galardi, Francesca; Pestrin, Marta; Gabellini, Stefano; Simi, Lisa; Mancini, Irene; Vannucchi, Alessandro Maria; Pazzagli, Mario; Di Leo, Angelo; Pinzani, Pamela

    2016-01-01

    Circulating Tumor Cells (CTCs) represent a “liquid biopsy” of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients. The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach. CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray. Upon whole genome amplification 3–5 single CTCs per patient were analyzed by NGS for 50 cancer-related genes. We found 51 sequence variants in 25 genes. We observed inter- and intra-patient heterogeneity in the mutational status of CTCs. The highest number of somatic deleterious mutations was found in the gene TP53, whose mutation is associated with adverse prognosis in breast cancer. The discordance between the mutational status of the primary tumor and CTCs observed in 3 patients suggests that, in advanced stages of cancer, CTC characteristics are more closely linked to the dynamic modifications of the disease status. In one patient the mutational profiles of CTCs before and during treatment shared only few sequence variants. This study supports the applicability of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients which, in perspective, should allow investigating the clonal evolution of the tumor for the development of new therapeutic strategies in precision medicine. PMID:27034166

  7. Generation of prion transmission barriers by mutational control of amyloid conformations.

    Science.gov (United States)

    Chien, Peter; DePace, Angela H; Collins, Sean R; Weissman, Jonathan S

    2003-08-21

    Self-propagating beta-sheet-rich protein aggregates are implicated in a wide range of protein-misfolding phenomena, including amyloid diseases and prion-based inheritance. Two properties have emerged as common features of amyloids. Amyloid formation is ubiquitous: many unrelated proteins form such aggregates and even a single polypeptide can misfold into multiple forms--a process that is thought to underlie prion strain variation. Despite this promiscuity, amyloid propagation can be highly sequence specific: amyloid fibres often fail to catalyse the aggregation of other amyloidogenic proteins. In prions, this specificity leads to barriers that limit transmission between species. Using the yeast prion [PSI+], we show in vitro that point mutations in Sup35p, the protein determinant of [PSI+], alter the range of 'infectious' conformations, which in turn changes amyloid seeding specificity. We generate a new transmission barrier in vivo by using these mutations to specifically disfavour subsets of prion strains. The ability of mutations to alter the conformations of amyloid states without preventing amyloid formation altogether provides a general mechanism for the generation of prion transmission barriers and may help to explain how mutations alter toxicity in conformational diseases.

  8. Current Control Method for Distributed Generation Power Generation Plants under Grid Fault Conditions

    DEFF Research Database (Denmark)

    Rodriguez, Pedro; Luna, Alvaro; Hermoso, Juan Ramon

    2011-01-01

    The operation of distributed power generation systems under grid fault conditions is a key issue for the massive integration of renewable energy systems. Several studies have been conducted to improve the response of such distributed generation systems under voltage dips. In spite of being less s...

  9. Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9.

    Science.gov (United States)

    Lv, Qingyan; Yuan, Lin; Deng, Jichao; Chen, Mao; Wang, Yong; Zeng, Jian; Li, Zhanjun; Lai, Liangxue

    2016-04-26

    CRISPR/Cas9 has been widely used in generating site-specific genetically modified animal models. Myostatin (MSTN) is a negative regulator of muscle mass, related to muscle growth and differentiation. The knockout of MSTN with the desired phenotype of double muscle has been successfully generated in mice, goats, pigs and cattle, but not in rabbits. In this study, the MSTN knockout (KO) rabbits were generated by co-injection of Cas9 mRNA and sgRNA into zygotes. The typical phenotype of double muscle with hyperplasia or hypertrophy of muscle fiber was observed in MSTN KO rabbits. Furthermore, a similar phenotype was found in the F1 generation, suggesting that the mutation of MSTN could be stably inherited in the MSTN KO rabbits. In summary, we have successfully generated MSTN KO rabbits using CRISPR/Cas9 system with high efficiency, which is a reliable and effective animal model for the study of muscle development and related diseases.

  10. Next generation sequencing on patients with LGMD and nonspecific myopathies: findings associated with ANO5 mutations

    OpenAIRE

    2015-01-01

    We studied 786 undiagnosed patients with LGMD or nonspecific myopathic features to investigate the role of ANO5 mutations in limb-girdle muscular dystrophies (LGMDs) and in nonspecific myopathies using the next generation sequencing (NGS) approach. In 160 LGMD patients, we first sequenced hotspot exons 5 and 20 and then sequenced the remaining part of the coding region. Another 626 patients, recruited using broader inclusion criteria, were directly analyzed by targeted NGS. By combining NGS a...

  11. Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

    Science.gov (United States)

    Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

    2014-06-01

    Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

  12. Pitfalls of improperly procured adjacent non-neoplastic tissue for somatic mutation analysis using next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Lei Wei

    2016-10-01

    Full Text Available Abstract Background The rapid adoption of next-generation sequencing provides an efficient system for detecting somatic alterations in neoplasms. The detection of such alterations requires a matched non-neoplastic sample for adequate filtering of non-somatic events such as germline polymorphisms. Non-neoplastic tissue adjacent to the excised neoplasm is often used for this purpose as it is simultaneously collected and generally contains the same tissue type as the neoplasm. Following NGS analysis, we and others have frequently observed low-level somatic mutations in these non-neoplastic tissues, which may impose additional challenges to somatic mutation detection as it complicates germline variant filtering. Methods We hypothesized that the low-level somatic mutation observed in non-neoplastic tissues may be entirely or partially caused by inadvertent contamination by neoplastic cells during the surgical pathology gross assessment or tissue procurement process. To test this hypothesis, we applied a systematic protocol designed to collect multiple grossly non-neoplastic tissues using different methods surrounding each single neoplasm. The procedure was applied in two breast cancer lumpectomy specimens. In each case, all samples were first sequenced by whole-exome sequencing to identify somatic mutations in the neoplasm and determine their presence in the adjacent non-neoplastic tissues. We then generated ultra-deep coverage using targeted sequencing to assess the levels of contamination in non-neoplastic tissue samples collected under different conditions. Results Contamination levels in non-neoplastic tissues ranged up to 3.5 and 20.9 % respectively in the two cases tested, with consistent pattern correlated with the manner of grossing and procurement. By carefully controlling the conditions of various steps during this process, we were able to eliminate any detectable contamination in both patients. Conclusion The results demonstrated that the

  13. ERP generator anomalies in presymptomatic carriers of the Alzheimer's disease E280A PS-1 mutation.

    Science.gov (United States)

    Bobes, María A; García, Yuriem Fernández; Lopera, Francisco; Quiroz, Yakeel T; Galán, Lídice; Vega, Mayrim; Trujillo, Nelson; Valdes-Sosa, Mitchell; Valdes-Sosa, Pedro

    2010-02-01

    Although subtle anatomical anomalies long precede the onset of clinical symptoms in Alzheimer's disease, their impact on the reorganization of brain networks underlying cognitive functions has not been fully explored. A unique window into this reorganization is provided by presymptomatic cases of familial Alzheimer's disease (FAD). Here we studied neural circuitry related to semantic processing in presymptomatic FAD cases by estimating the intracranial sources of the N400 event-related potential (ERP). ERPs were obtained during a semantic-matching task from 24 presymptomatic carriers and 25 symptomatic carriers of the E280A presenilin-1 (PS-1) mutation, as well as 27 noncarriers (from the same families). As expected, the symptomatic-carrier group performed worse in the matching task and had lower N400 amplitudes than both asymptomatic groups, which did not differ from each other on these variables. However, N400 topography differed in mutation carrier groups with respect to the noncarriers. Intracranial source analysis evinced that the presymptomatic-carriers presented a decrease of N400 generator strength in right inferior-temporal and medial cingulate areas and increased generator strength in the left hippocampus and parahippocampus compared to the controls. This represents alterations in neural function without translation into behavioral impairments. Compared to controls, the symptomatic-carriers presented a similar anatomical shift in the distribution of N400 generators to that found in presymptomatic-carriers, albeit with a larger reduction in generator strength. The redistribution of N400 generators in presymptomatic-carriers indicates that early focal degeneration associated with the mutation induces neural reorganization, possibly contributing to a functional compensation that enables normal performance in the semantic task.

  14. A sea spray generation function for fetch-limited conditions

    Directory of Open Access Journals (Sweden)

    J. Piazzola

    Full Text Available This paper presents a sea spray generation function for aerosols produced by bubbles bursting that accounts for the effects of fetch. This is achieved by introducing a fetch-dependent model for the whitecap fraction, which is valid for fetch-limited conditions, i.e. steady-state conditions of the wave field in the whitecap droplet flux derived by Monahan et al. (1986. The aerosol generation fluxes calculated by this method show an enhancement of the aerosol production with increasing fetch. However, the predicted generation fluxes are lower than those calculated by using the classical model for the whitecap fraction proposed by Monahan and O’Muircheartaigh (1980. The results are then compared to aerosol size distributions measured in a Mediterranean coastal site at various fetch lengths. The data confirm the role of fetch, through its influence on marine aerosol generation and subsequent particle dispersion. The aerosol size distributions are divided into "fine" and "coarse" fractions. Submicrometer particles decrease in concentration at larger fetch, while the concentrations of super micron aerosols increase with increasing fetch.

    Key words. Atmospheric composition and structure (aerosols and particles Meteorology and atmospheric dynamics (waves and ties Oceanography: physical (air-sea interactions

  15. Cosegregation of the ND4 G11696A mutation with the LHON-associated ND4 G11778A mutation in a four generation Chinese family.

    Science.gov (United States)

    Qu, Jia; Li, Ronghua; Zhou, Xiangtian; Tong, Yi; Yang, Li; Chen, Jie; Zhao, Fuxing; Lu, Chunjie; Qian, Yaping; Lu, Fan; Guan, Min-Xin

    2007-01-01

    We report here the characterization of a four-generation Han Chinese family with Leber's hereditary optic neuropathy (LHON). This Chinese family exhibited a variable severity and age-at-onset of visual loss. Notably, the average age-at-onset of vision impairment changed from 26 years (generation III) to 14 years (generation IV), with the average of 18 years in this family. In addition, 30% and 50% of matrilineal relatives in generation III and IV of this family developed visual loss with a variability of severity, ranging from blindness to normal vision. Sequence analysis of the complete mitochondrial DNA in this pedigree revealed the presence of the homoplasmic ND4 G11778A mutation and 33 other variants, belonging to the Asian haplogroup D4. Of other variants, the homoplasmic G11696A mutation in the ND4 gene is of special interest as it was implicated to be associated with LHON in a large Dutch family and five Chinese pedigrees with extremely penetrance of visual loss. In fact, the G11696A mutation caused the substitution of an isoleucine for valine at amino acid position 313, located in a predicted transmembrane region of ND4. These imply that the G11696A mutation may act in synergy with the primary LHON-associated G11778A mutation in this Chinese pedigree.

  16. Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Robert King

    Full Text Available Targeted Induced Local Lesions in Genomes (TILLING is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.

  17. Current Control Method for Distributed Generation Power Generation Plants under Grid Fault Conditions

    DEFF Research Database (Denmark)

    Rodriguez, Pedro; Luna, Alvaro; Hermoso, Juan Ramon

    2011-01-01

    The operation of distributed power generation systems under grid fault conditions is a key issue for the massive integration of renewable energy systems. Several studies have been conducted to improve the response of such distributed generation systems under voltage dips. In spite of being less s...... the current ratings of the converter is introduced. Moreover, a novel flexible algorithm has been proposed in order to regulate easily the injection of positive and negative currents for general purpose applications....

  18. Optimal working conditions for thermoelectric generators with realistic thermal coupling

    CERN Document Server

    Apertet, Y; Glavatskaya, O; Goupil, C; Lecoeur, P

    2011-01-01

    We study how maximum output power can be obtained from a thermoelectric generator(TEG) with nonideal heat exchangers. We demonstrate with an analytic approach based on a force-flux formalism that the sole improvement of the intrinsic characteristics of thermoelectric modules including the enhancement of the figure of merit is of limited interest: the constraints imposed by the working conditions of the TEG must be considered on the same footing. Introducing an effective thermal conductance we derive the conditions which permit maximization of both efficiency and power production of the TEG dissipatively coupled to heat reservoirs. Thermal impedance matching must be accounted for as well as electrical impedance matching in order to maximize the output power. Our calculations also show that the thermal impedance does not only depend on the thermal conductivity at zero electrical current: it also depends on the TEG figure of merit. Our analysis thus yields both electrical and thermal conditions permitting optima...

  19. Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

    Science.gov (United States)

    Birket, Matthew J; Ribeiro, Marcelo C; Kosmidis, Georgios; Ward, Dorien; Leitoguinho, Ana Rita; van de Pol, Vera; Dambrot, Cheryl; Devalla, Harsha D; Davis, Richard P; Mastroberardino, Pier G; Atsma, Douwe E; Passier, Robert; Mummery, Christine L

    2015-10-27

    Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

  20. Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function

    Directory of Open Access Journals (Sweden)

    Matthew J. Birket

    2015-10-01

    Full Text Available Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC model of hypertrophic cardiomyopathy (HCM. A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

  1. Infra-red parametric generation: Phase mismatch condition

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, S.; Dubey, Swati; Jain, Kamal, E-mail: Jainrupam1987@gmail.com [School of Studies in Physics, Vikram University, Ujjain (M.P.) India-456010 (India)

    2015-07-31

    An analytical investigation is made for the Infrared parametric generation in doped semiconductor plasma under phase mismatch condition. Theoretical formulations are undertaken to determine induced polarization and threshold pump field for the onset of parametric generation in semiconductor plasma medium. The origin of this nonlinear interaction lies in the second order optical susceptibility arising due to the induced nonlinear current density in piezoelectric medium. Numerical estimations are made for n- type InSb at 77 K duly irradiated by a pulsed 10.6µm CO{sub 2} laser. It is very difficult to attain exact phase matching in experimental frame so we have considered a tolerable small phase mismatch in order to attain a new result. Its effect on the Infrared parametric generation in compound semiconductor is examined through induced polarization. Transmitted intensity is determined to have an idea about conversion efficiency of the said process. Phase mismatch tends to raise the required pump field to stimulate the parametric generation. Transmitted intensity is found to decrease with coherence length lc and increase carrier concentration n{sub 0}, which is favorable for improved conversion efficiency.

  2. Infra-red parametric generation: Phase mismatch condition

    Science.gov (United States)

    Ghosh, S.; Dubey, Swati; Jain, Kamal

    2015-07-01

    An analytical investigation is made for the Infrared parametric generation in doped semiconductor plasma under phase mismatch condition. Theoretical formulations are undertaken to determine induced polarization and threshold pump field for the onset of parametric generation in semiconductor plasma medium. The origin of this nonlinear interaction lies in the second order optical susceptibility arising due to the induced nonlinear current density in piezoelectric medium. Numerical estimations are made for n- type InSb at 77 K duly irradiated by a pulsed 10.6µm CO2 laser. It is very difficult to attain exact phase matching in experimental frame so we have considered a tolerable small phase mismatch in order to attain a new result. Its effect on the Infrared parametric generation in compound semiconductor is examined through induced polarization. Transmitted intensity is determined to have an idea about conversion efficiency of the said process. Phase mismatch tends to raise the required pump field to stimulate the parametric generation. Transmitted intensity is found to decrease with coherence length lc and increase carrier concentration n0, which is favorable for improved conversion efficiency.

  3. Mutation Detection with Next-Generation Resequencing through a Mediator Genome

    Energy Technology Data Exchange (ETDEWEB)

    Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel; Jurkevitch, Edouard; Sorek, Rotem; Ben-Jacob, Eshel

    2010-12-31

    The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

  4. Mutation detection with next-generation resequencing through a mediator genome.

    Directory of Open Access Journals (Sweden)

    Omri Wurtzel

    Full Text Available The affordability of next generation sequencing (NGS is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

  5. Mutation Detection with Next-Generation Resequencing through a Mediator Genome

    Energy Technology Data Exchange (ETDEWEB)

    Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel; Jurkevitch, Edouard; Sorek, Rotem

    2010-12-20

    The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WT and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.

  6. Novel compact sorption generators for car air conditioning

    Energy Technology Data Exchange (ETDEWEB)

    Tamainot-Telto, Z.; Metcalf, S.J.; Critoph, R.E. [School of Engineering, University of Warwick, Gibet Hill Road, Coventry CV4 7AL (United Kingdom)

    2009-06-15

    A prototype compact generator using the activated carbon-ammonia pair based on the plate heat exchanger concept has been designed and built at Warwick University. The novel generator has low thermal mass and good heat transfer. The heat exchanger uses nickel-brazed shims and spacers to create adsorbent layers only 4 mm thick between pairs of liquid flow channels of very low thermal mass. The prototype sorption generator manufactured was evaluated under EU car air conditioning test conditions. The prototype sorption generator is described and its experimental performance reported. While driven with waste heat from the engine coolant water (at 90 C), a pair of the current prototype generators (loaded with about 1 kg of activated carbon) operating out of phase has produced an average cooling power 1.6 kW with about 2 kW peaks. The typical average COP obtained is 0.22. (author) [French] Un prototype du generateur compact, base sur le concept des echangeurs de chaleur a plaques et utilisant la paire charbon actif-ammoniac, a ete concu et construit a l'Universite de Warwick. Le nouveau generateur a une faible inertie thermique et un excellent transfert de chaleur. L' echangeur utilise des plaques ayant des micro-canaux et des intercalaires brases au Nickel pour creer des couches d'adsorbant de 4 mm d'epaisseur entre les paires de plaques a l'interieur desquelles circule le fluide liquide. Le prototype du generateur a sorption ainsi fabrique a ete teste suivant des conditions prescrites par la Norme Europeenne de la Climatisation Automobile. Le prototype du generateur a sorption est decrit et ses performances experimentales presentees. Une paire dudit prototype (contenant chacun 1 kg the charbon actif), operant avec dephasage et ulisant des pertes thermiques en provenance de l'eau de refroidissement de moteur (a 90 C), a produit une puissance frigorifique moyenne de 1.6 kW avec une valeur maximum de 2 kW. La valeur typique du COP moyen est de

  7. Rapid Conditioning for the Next Generation Melting System

    Energy Technology Data Exchange (ETDEWEB)

    Rue, David M. [Gas Technology Institute, Des Plaines, IL (United States)

    2015-06-17

    This report describes work on Rapid Conditioning for the Next Generation Melting System under US Department of Energy Contract DE-FC36-06GO16010. The project lead was the Gas Technology Institute (GTI). Partners included Owens Corning and Johns Manville. Cost share for this project was provided by NYSERDA (the New York State Energy Research and Development Authority), Owens Corning, Johns Manville, Owens Illinois, and the US natural gas industry through GTI’s SMP and UTD programs. The overreaching focus of this project was to study and develop rapid refining approaches for segmented glass manufacturing processes using high-intensity melters such as the submerged combustion melter. The objectives of this project were to 1) test and evaluate the most promising approaches to rapidly condition the homogeneous glass produced from the submerged combustion melter, and 2) to design a pilot-scale NGMS system for fiberglass recycle.

  8. Hydrologic conditions controlling runoff generation immediately after wildfire

    Science.gov (United States)

    Ebel, Brian A.; Moody, John A.; Martin, Deborah A.

    2012-01-01

    We investigated the control of postwildfire runoff by physical and hydraulic properties of soil, hydrologic states, and an ash layer immediately following wildfire. The field site is within the area burned by the 2010 Fourmile Canyon Fire in Colorado, USA. Physical and hydraulic property characterization included ash thickness, particle size distribution, hydraulic conductivity, and soil water retention curves. Soil water content and matric potential were measured indirectly at several depths below the soil surface to document hydrologic states underneath the ash layer in the unsaturated zone, whereas precipitation and surface runoff were measured directly. Measurements of soil water content showed that almost no water infiltrated below the ash layer into the near-surface soil in the burned site at the storm time scale (i.e., minutes to hours). Runoff generation processes were controlled by and highly sensitive to ash thickness and ash hydraulic properties. The ash layer stored from 97% to 99% of rainfall, which was critical for reducing runoff amounts. The hydrologic response to two rain storms with different rainfall amounts, rainfall intensity, and durations, only ten days apart, indicated that runoff generation was predominantly by the saturation-excess mechanism perched at the ash-soil interface during the first storm and predominantly by the infiltration-excess mechanism at the ash surface during the second storm. Contributing area was not static for the two storms and was 4% (saturation excess) to 68% (infiltration excess) of the catchment area. Our results showed the importance of including hydrologic conditions and hydraulic properties of the ash layer in postwildfire runoff generation models.

  9. Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

    Science.gov (United States)

    Russo, Giancarlo; Patrignani, Andrea; Poveda, Lucy; Hoehn, Frederic; Scholtka, Bettina; Schlapbach, Ralph; Garvin, Alex M

    2015-12-01

    Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS. We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

  10. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

    Directory of Open Access Journals (Sweden)

    Wu Bai-Lin

    2009-10-01

    Full Text Available Abstract Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other

  11. Identifying the lost generation of adults with autism spectrum conditions.

    Science.gov (United States)

    Lai, Meng-Chuan; Baron-Cohen, Simon

    2015-11-01

    Autism spectrum conditions comprise a set of early-onset neurodevelopmental syndromes with a prevalence of 1% across all ages. First diagnosis in adulthood has finally become recognised as an important clinical issue due to the increasing awareness of autism, broadening of diagnostic criteria, and the introduction of the spectrum concept. Thus, the idea of a lost generation of people who were previously excluded from a diagnosis of classic autism has arisen. Making a first diagnosis of autism spectrum conditions in adults can be challenging for practical reasons (eg, no person to provide a developmental history), developmental reasons (eg, the acquisition of learnt or camouflaging strategies), and clinical reasons (eg, high frequency of co-occurring disorders). The diagnostic process includes referral, screening, interviews with informants and patients, and functional assessments. In delineating differential diagnoses, true comorbidities, and overlapping behaviour with other psychiatric diagnoses, particular attention should be paid to anxiety, depression, obsessive-compulsive disorder, psychosis, personality disorders, and other neurodevelopmental disorders. Possible misdiagnosis, especially in women, should be explored. The creation of supportive, accepting, and autism-friendly social and physical environments is important and requires a coordinated effort across agencies and needs support from government policies.

  12. Mutations in the BLOC-1 Subunits Dysbindin and Muted Generate Divergent and Dosage-dependent Phenotypes*

    Science.gov (United States)

    Larimore, Jennifer; Zlatic, Stephanie A.; Gokhale, Avanti; Tornieri, Karine; Singleton, Kaela S.; Mullin, Ariana P.; Tang, Junxia; Talbot, Konrad; Faundez, Victor

    2014-01-01

    Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5mu/mu) and dysbindin (Bloc1s8sdy/sdy). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5mu/mu and Bloc1s8sdy/sdy mice. Unlike Bloc1s8sdy/sdy, elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders. PMID:24713699

  13. Targeted next-generation sequencing of cancer genes identified frequent TP53 and ATRX mutations in leiomyosarcoma.

    Science.gov (United States)

    Yang, Ching-Yao; Liau, Jau-Yu; Huang, Wei-Ju; Chang, Yu-Ting; Chang, Ming-Chu; Lee, Jen-Chieh; Tsai, Jia-Huei; Su, Yi-Ning; Hung, Chia-Cheng; Jeng, Yung-Ming

    2015-01-01

    Leiomyosarcoma is an aggressive soft tissue sarcoma with poor patient survival. The genetic changes of leiomyosarcoma remain to be discovered. In this study, we analyzed the genetic changes of 44 cancer-related genes by using next-generation sequencing in 54 leiomyosarcomas. We identified TP53 mutations in 19 of the 54 tumors (35%) and ATRX mutations in 9 of the 54 tumors (17%). The TP53-mutated leiomyosarcomas were limited to female patients (P = 0.006). All but 2 of the TP53-mutated leiomyosarcomas were located in the uterus (n = 11) or retroperitoneum (n = 6). The ATRX mutations were associated with poorly differentiated leiomyosarcomas (P = 0.028) and the presence of tumor necrosis (P = 0.015). Kaplan-Meier survival analysis showed that patients with ATRX-mutated leiomyosarcomas had worse overall survival than did patients with ATRX-wild-type leiomyosarcomas. All of the ATRX-mutated leiomyosarcomas showed the alternative lengthening of telomere phenotype. The ATRX mutations did not correlate with ATRX protein expression, as detected using immunohistochemistry. In conclusion, we identified loss of function of the p53 and ATRX pathways being the main mechanisms for leiomyosarcomas. The molecular mechanisms may provide new opportunities to treat these aggressive neoplasms.

  14. A System To Generate Chromosomal Mutations in Lactococcus lactis Which Allows Fast Analysis of Targeted Genes

    NARCIS (Netherlands)

    Law, Jean; Buist, Girbe; Haandrikman, Alfred; Kok, Jan; Venema, Gerhardus; Leenhouts, Kees

    1995-01-01

    A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori+ RepA- vector pORI19, containing lacZα and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19

  15. Inner Magnetosphere Simulations: Exploring Magnetosonic Wave Generation Conditions

    Science.gov (United States)

    Zaharia, S. G.; Jordanova, V. K.; MacDonald, E.; Thomsen, M. F.

    2012-12-01

    We investigate the conditions for magnetosonic wave generation in the near-Earth magnetosphere by performing numerical simulations with our newly improved self-consistent model, RAM-SCB. The magnetosonic (ion Bernstein) instability, a potential electron acceleration mechanism in the outer radiation belt, is driven by a positive slope in the ion distribution function perpendicular to the magnetic field, a so-called "velocity ring" distribution at energies above 1 keV. The formation of such distributions is dependent on the interplay of magnetic and electric drifts, as well as ring current losses, and therefore its study requires a realistic treatment of both plasma and field dynamics. The RAM-SCB model represents a 2-way coupling of the kinetic ring current-atmosphere interactions model (RAM) with a 3D plasma equilibrium code. In RAM-SCB the magnetic field is computed in force balance with the RAM anisotropic pressures and then returned to RAM to guide the particle dynamics. RAM-SCB thus properly treats both the kinetic drift physics crucial in the inner magnetosphere and the self-consistent interaction between plasma and magnetic field (required due to the strong field depressions during storms, depressions that strongly affect particle drifts). In order to provide output at geosynchronous locations, recently the RAM-SCB boundary has been expanded to 9 RE from Earth, with plasma pressure and magnetic field boundary conditions prescribed there from empirical models. This presentation will analyze, using event simulations with the improved model and comparisons with LANL MPA geosynchronous observations, the occurrence and location of magnetosonic unstable regions in the inner magnetosphere and their dependence on the following factors: 1). geomagnetic activity level (including quiet time, storm main phase and recovery); 2). magnetic field self-consistency (stretched vs. dipole fields). We will also discuss the physical mechanism for the occurrence of the velocity

  16. Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.

    Directory of Open Access Journals (Sweden)

    Timothy J Dahlem

    Full Text Available The zebrafish is a powerful experimental system for uncovering gene function in vertebrate organisms. Nevertheless, studies in the zebrafish have been limited by the approaches available for eliminating gene function. Here we present simple and efficient methods for inducing, detecting, and recovering mutations at virtually any locus in the zebrafish. Briefly, double-strand DNA breaks are induced at a locus of interest by synthetic nucleases, called TALENs. Subsequent host repair of the DNA lesions leads to the generation of insertion and deletion mutations at the targeted locus. To detect the induced DNA sequence alterations at targeted loci, genomes are examined using High Resolution Melt Analysis, an efficient and sensitive method for detecting the presence of newly arising sequence polymorphisms. As the DNA binding specificity of a TALEN is determined by a custom designed array of DNA recognition modules, each of which interacts with a single target nucleotide, TALENs with very high target sequence specificities can be easily generated. Using freely accessible reagents and Web-based software, and a very simple cloning strategy, a TALEN that uniquely recognizes a specific pre-determined locus in the zebrafish genome can be generated within days. Here we develop and test the activity of four TALENs directed at different target genes. Using the experimental approach described here, every embryo injected with RNA encoding a TALEN will acquire targeted mutations. Multiple independently arising mutations are produced in each growing embryo, and up to 50% of the host genomes may acquire a targeted mutation. Upon reaching adulthood, approximately 90% of these animals transmit targeted mutations to their progeny. Results presented here indicate the TALENs are highly sequence-specific and produce minimal off-target effects. In all, it takes about two weeks to create a target-specific TALEN and generate growing embryos that harbor an array of germ line

  17. Targeted 'Next-Generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations

    Directory of Open Access Journals (Sweden)

    Lopez Jimenez Nelson

    2011-12-01

    Full Text Available Abstract Background Anophthalmia/microphthalmia (A/M is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M. Methods We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP calling software. We verified predicted sequence alterations using Sanger sequencing. Results We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15 that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp deletion and one 3 bp duplication in SOX2. Conclusions Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However, next-generation sequencing was less useful for small, intragenic deletions and duplications. We did not find mutations in 10/15 patients and conclude that there is a need for further gene discovery in A/M.

  18. Conditional linear-optical measurement schemes generate effective photon nonlinearities

    CERN Document Server

    Lapaire, G G; Dowling, J P; Sipe, J E; Dowling, Jonathan P.

    2003-01-01

    We provide a general approach for the analysis of optical state evolution under conditional measurement schemes, and identify the necessary and sufficient conditions for such schemes to simulate unitary evolution on the freely propagating modes. If such unitary evolution holds, an effective photon nonlinearity can be identified. Our analysis extends to conditional measurement schemes more general than those based solely on linear optics.

  19. Conjugation between quinolone-susceptible bacteria can generate mutations in the quinolone resistance-determining region, inducing quinolone resistance.

    Science.gov (United States)

    Pitondo-Silva, André; Martins, Vinicius Vicente; Silva, Carolina Fávero da; Stehling, Eliana Guedes

    2015-02-01

    Quinolones are an important group of antibacterial agents that can inhibit DNA gyrase and topoisomerase IV activity. DNA gyrase is responsible for maintaining bacteria in a negatively supercoiled state, being composed of subunits A and B. Topoisomerase IV is a homologue of DNA gyrase and consists of two subunits codified by the parC and parE genes. Mutations in gyrA and gyrB of DNA gyrase may confer resistance to quinolones, and the majority of resistant strains show mutations between positions 67 and 106 of gyrA, a region denoted the quinolone resistance-determining region (QRDR). The most frequent substitutions occur at positions 83 and 87, but little is known about the mechanisms promoting appearance of mutations in the QRDR. The present study proposes that some mutations in the QRDR could be generated as a result of the natural mechanism of conjugation between bacteria in their natural habitat. This event was observed following conjugation in vitro of two different isolates of quinolone-susceptible Pseudomonas aeruginosa, which transferred plasmids of different molecular weights to a recipient strain of Escherichia coli (HB101), also quinolone-susceptible, generating two different transconjugants that presented mutations in DNA gyrase and acquisition of resistance to all quinolones tested.

  20. Somatic mutation profiling of follicular thyroid cancer by next generation sequencing.

    Science.gov (United States)

    Swierniak, Michal; Pfeifer, Aleksandra; Stokowy, Tomasz; Rusinek, Dagmara; Chekan, Mykola; Lange, Dariusz; Krajewska, Jolanta; Oczko-Wojciechowska, Małgorzata; Czarniecka, Agnieszka; Jarzab, Michal; Jarzab, Barbara; Wojtas, Bartosz

    2016-09-15

    The molecular etiology of follicular thyroid tumors is largely unknown, rendering the diagnostics of these tumors challenging. The somatic alterations present in these tumors apart from RAS gene mutations and PAX8/PPARG translocations are not well described. To evaluate the profile of somatic alteration in follicular thyroid tumors, a total of 82 thyroid tissue samples derived from 48 patients were subjected to targeted Illumina HiSeq next generation sequencing of 372 cancer-related genes. New somatic alterations were identified in oncogenes (MDM2, FLI1), transcription factors and repressors (MITF, FLI1, ZNF331), epigenetic enzymes (KMT2A, NSD1, NCOA1, NCOA2), and protein kinases (JAK3, CHEK2, ALK). Single nucleotide and large structural variants were most and least frequently identified, respectively. A novel translocation in DERL/COX6C was detected. Many somatic alterations in non-coding gene regions with high penetrance were observed. Thus, follicular thyroid tumor somatic alterations exhibit complex patterns. Most tumors contained distinct somatic alterations, suggesting previously unreported heterogeneity.

  1. Next-Generation Sequencing Reveals Deep Intronic Cryptic ABCC8 and HADH Splicing Founder Mutations Causing Hyperinsulinism by Pseudoexon Activation

    Science.gov (United States)

    Flanagan, Sarah E.; Xie, Weijia; Caswell, Richard; Damhuis, Annet; Vianey-Saban, Christine; Akcay, Teoman; Darendeliler, Feyza; Bas, Firdevs; Guven, Ayla; Siklar, Zeynep; Ocal, Gonul; Berberoglu, Merih; Murphy, Nuala; O’Sullivan, Maureen; Green, Andrew; Clayton, Peter E.; Banerjee, Indraneel; Clayton, Peter T.; Hussain, Khalid; Weedon, Michael N.; Ellard, Sian

    2013-01-01

    Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ∼10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals’ fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease. PMID:23273570

  2. Biological conditions for oscillations and chaos generated by multispecies competition

    NARCIS (Netherlands)

    Huisman, J; Weissing, FJ

    2001-01-01

    We investigate biological mechanisms that generate oscillations and chaos in multispecies competition models. For this purpose, we use a competition model concerned with competition for abiotic essential resources. Because phytoplankton and plants consume quite a number of abiotic essential resource

  3. Hydrogen generation at ambient conditions: application in fuel cells.

    Science.gov (United States)

    Boddien, Albert; Loges, Björn; Junge, Henrik; Beller, Matthias

    2008-01-01

    The efficient generation of hydrogen from formic acid/amine adducts at ambient temperature is demonstrated. The highest catalytic activity (TOF up to 3630 h(-1) after 20 min) was observed in the presence of in situ generated ruthenium phosphine catalysts. Compared to the previously known methods to generate hydrogen from liquid feedstocks, the systems presented here can be operated at room temperature without the need for any high-temperature reforming processes, and the hydrogen produced can then be directly used in fuel cells. A variety of Ru precursors and phosphine ligands were investigated for the decomposition of formic acid/amine adducts. These catalytic systems are particularly interesting for the generation of H2 for new applications in portable electric devices.

  4. Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L. Using the CRISPR/Cas System.

    Directory of Open Access Journals (Sweden)

    Nathaniel M Butler

    Full Text Available Genome editing using sequence-specific nucleases (SSNs offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated systems (Cas have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.. In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA targeting the potato ACETOLACTATE SYNTHASE1 (StALS1 gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This

  5. IDH1 R132H Mutation Generates a Distinct Phospholipid Metabolite Profile in Glioma

    NARCIS (Netherlands)

    Esmaeili, M.; Hamans, B.C.; Navis, A.C.; Horssen, R. van; Bathen, T.F.; Gribbestad, I.S.; Leenders, W.P.; Heerschap, A.

    2014-01-01

    Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate. Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized

  6. Mutation of a conserved residue enhances the sensitivity of analogue-sensitised kinases to generate a novel approach to the study of mitosis in fission yeast.

    Science.gov (United States)

    Tay, Ye-Dee; Patel, Avinash; Kaemena, Daniel F; Hagan, Iain M

    2013-11-01

    The chemical genetic strategy in which mutational enlargement of the ATP-binding site sensitises of a protein kinase to bulky ATP analogues has proved to be an elegant tool for the generation of conditional analogue-sensitive kinase alleles in a variety of model organisms. Here, we describe a novel substitution mutation in the kinase domain that can enhance the sensitivity of analogue-sensitive kinases. Substitution of a methionine residue to phenylalanine in the +2 position after HRDLKxxN motif of the subdomain VIb within the kinase domain markedly increased the sensitivities of the analogue-sensitive kinases to ATP analogues in three out of five S. pombe kinases (i.e. Plo1, Orb5 and Wee1) that harbor this conserved methionine residue. Kinome alignment established that a methionine residue is found at this site in 5-9% of kinases in key model organisms, suggesting that a broader application of this structural modification may enhance ATP analogue sensitivity of analogue-sensitive kinases in future studies. We also show that the enhanced sensitivity of the wee1.as8 allele in a cdc25.22 background can be exploited to generate highly synchronised mitotic and S phase progression at 36°C. Proof-of-principle experiments show how this novel synchronisation technique will prove of great use in the interrogation of the mitotic or S-phase functions through temperature sensitivity mutation of molecules of interest in fission yeast.

  7. Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a danish five-generation family with a novel FAM83H nonsense mutation

    DEFF Research Database (Denmark)

    Haubek, Dorte; Gjørup, Hans; Jensen, Lillian Gryesten

    2011-01-01

    Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a danish five-generation family with a novel FAM83H nonsense mutation......Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a danish five-generation family with a novel FAM83H nonsense mutation...

  8. Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a danish five-generation family with a novel FAM83H nonsense mutation

    DEFF Research Database (Denmark)

    Haubek, Dorte; Gjørup, Hans; Jensen, Lillian Gryesten

    2011-01-01

    Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a danish five-generation family with a novel FAM83H nonsense mutation......Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a danish five-generation family with a novel FAM83H nonsense mutation...

  9. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β -Globin Gene with the IVSI-6 Thalassemia Mutation.

    Science.gov (United States)

    Breveglieri, Giulia; Mancini, Irene; Bianchi, Nicoletta; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Rubini, Michele; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto; Finotti, Alessia

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin (mu) α-globin2/(hu) β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia.

  10. Genetic diagnosis of Duchenne/Becker muscular dystrophy using next-generation sequencing: validation analysis of DMD mutations

    Science.gov (United States)

    Okubo, Mariko; Minami, Narihiro; Goto, Kanako; Goto, Yuichi; Noguchi, Satoru; Mitsuhashi, Satomi; Nishino, Ichizo

    2016-01-01

    Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited neuromuscular disease. The genetic diagnosis is not easily made because of the large size of the dystrophin gene, complex mutational spectrum and high number of tests patients undergo for diagnosis. Multiplex ligation-dependent probe amplification (MLPA) has been used as the initial diagnostic test of choice. Although MLPA can diagnose 70% of DMD/BMD patients having deletions/duplications, the remaining 30% of patients with small mutations require further analysis, such as Sanger sequencing. We applied a high-throughput method using Ion Torrent next-generation sequencing technology and diagnosed 92% of patients with DMD/BMD in a single analysis. We designed a multiplex primer pool for DMD and sequenced 67 cases having different mutations: 37 with deletions/duplications and 30 with small mutations or short insertions/deletions in DMD, using an Ion PGM sequencer. The results were compared with those from MLPA or Sanger sequencing. All deletions were detected. In contrast, 50% of duplications were correctly identified compared with the MLPA method. Small insertions in consecutive bases could not be detected. We estimated that Ion Torrent sequencing could diagnose ~92% of DMD/BMD patients according to the mutational spectrum of our cohort. Our results clearly indicate that this method is suitable for routine clinical practice providing novel insights into comprehensive genetic information for future molecular therapy. PMID:26911353

  11. Non-invasive detection of somatic mutations using next-generation sequencing in primary central nervous system lymphoma.

    Science.gov (United States)

    Fontanilles, Maxime; Marguet, Florent; Bohers, Élodie; Viailly, Pierre-Julien; Dubois, Sydney; Bertrand, Philippe; Camus, Vincent; Mareschal, Sylvain; Ruminy, Philippe; Maingonnat, Catherine; Lepretre, Stéphane; Veresezan, Elena-Liana; Derrey, Stéphane; Tilly, Hervé; Picquenot, Jean-Michel; Laquerrière, Annie; Jardin, Fabrice

    2017-07-18

    Primary central nervous system lymphomas (PCNSL) have recurrent genomic alterations. The main objective of our study was to demonstrate that targeted sequencing of circulating cell-free DNA (cfDNA) released by PCNSL at the time of diagnosis could identify somatic mutations by next-generation sequencing (NGS). PlasmacfDNA and matched tumor DNA (tDNA) from 25 PCNSL patients were sequenced using an Ion Torrent Personal Genome Machine (Life Technologies®). First, patient-specific targeted sequencing of identified somatic mutations in tDNA was performed. Then, a second sequencing targeting MYD88 c.T778C was performed and compared to plasma samples from 25 age-matched control patients suffering from other types of cancer. According to the patient-specific targeted sequencing, eight patients (32% [95% CI 15-54%]) had detectable somatic mutations in cfDNA. Considering MYD88 sequencing, six patients had the specific c.T778C alteration detected in plasma. Using a control group, the sensitivity was 24% [9-45%] and the specificity was 100%. Tumor volume or deep brain structure involvement did not influence the detection of somatic mutations in plasma. This pilot study provided evidence that somatic mutations can be detected by NGS in the cfDNA of a subset of patients suffering from PCNSL.

  12. Ethyl methane sulfonate induced mutations in M2 generation and physiological variations in M1 generation of peppers (Capsicum annuum L.

    Directory of Open Access Journals (Sweden)

    Mohamed Hamed Arisha

    2015-06-01

    Full Text Available This study was conducted to enhance genetic variability in peppers (Capsicum annuum, cv B12 using ethyl methansulphonate (EMS. Exposure to an EMS concentration of 0.6%, v/v for 12 hours was used to mutagenize 2000 seeds for the first generation (M1. It was observed that the growth behaviors including plant height, flowering date and number of seeds per first fruit were different in the M1 generation than in wild type plants. In addition one phenotypic mutation (leaf shape and plant architecture was observed during the M1 generation. During the seedling stage in the M2 generation, the observed changes were in the form of slow growth or chlorophyll defect (e.g., albino, pale green and yellow seedlings. At maturity, there were three kinds of phenotypic mutations observed in three different families of the mutant population. The first observed change was a plant with yellow leaf color, and the leaves of this mutant plant contained 62.19% less chlorophyll a and 64.06% less chlorophyll b as compared to the wild-type. The second mutation resulted in one dwarf plant with a very short stature (6 cm, compact internodes and the leaves and stem were rough and thick. The third type of mutation occurred in four plants and resulted in the leaves of these plants being very thick and longer than those of wild type plants. Furthermore, anatomical observations of the leaf blade section of this mutant plant type contained more xylem and collenchyma tissue in the leaf midrib of the mutant plant than wild type. In addition, its leaf blade contained thicker palisade and spongy tissue than the wild type.

  13. Atmospheric wind field conditions generated by active grids

    Science.gov (United States)

    Knebel, Pascal; Kittel, Achim; Peinke, Joachim

    2011-08-01

    An active grid for turbulence generation of several rotatable axes with surmounted vanes that can be driven via stepper or servo motors is presented. We investigate the impact of different excitation protocols for the grid. Using such protocols that already have the intermittent structure of turbulence, higher intermittent flows can be achieved. This concept can also be used to generate turbulent flows of high turbulence intensities (>25%) exhibiting integral length scales beyond the typical size of the test section of the wind tunnel. Similar two-point correlations measured by the intermittent statistics of velocity increments that are characteristic for flows of high Reynolds number, i.e. in the atmospheric boundary layer, can be reproduced.

  14. Less frequently mutated genes in colorectal cancer: evidences from next-generation sequencing of 653 routine cases

    Science.gov (United States)

    Malapelle, Umberto; Pisapia, Pasquale; Sgariglia, Roberta; Vigliar, Elena; Biglietto, Maria; Carlomagno, Chiara; Giuffrè, Giuseppe; Troncone, Giancarlo

    2016-01-01

    Aims The incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers. Methods Following a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes. Results A total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%). Conclusions In a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments. PMID:26797410

  15. Updating Small Generator Interconnection Procedures for New Market Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Coddington, M.; Fox, K.; Stanfield, S.; Varnado, L.; Culley, T.; Sheehan, M.

    2012-12-01

    Federal and state regulators are faced with the challenge of keeping interconnection procedures updated against a backdrop of evolving technology, new codes and standards, and considerably transformed market conditions. This report is intended to educate policymakers and stakeholders on beneficial reforms that will keep interconnection processes efficient and cost-effective while maintaining a safe and reliable power system.

  16. Third-Generation Tyrosine Kinase Inhibitors Targeting Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Tristan A. Barnes

    2017-05-01

    Full Text Available Sensitizing mutations in the epidermal growth factor receptor (EGFR predict response to EGFR tyrosine kinase inhibitors (TKIs and both first- and second-generation TKIs are available as first-line treatment options in patients with advanced EGFR-mutant non-small cell lung cancer. Eventual resistance develops with multiple mechanisms identifiable both upon repeat biopsy and in plasma circulating tumor DNA. The T790M gatekeeper mutation is responsible for almost 60% of cases. A number of third-generation TKIs are in clinical development, and osimertinib has been approved by the US Food and Drug Administration for the treatment of patients with EGFR T790M mutant lung cancer after failure of initial EGFR kinase therapy. Resistance mechanisms are being identified to these novel agents, and the treatment landscape of EGFR-mutant lung cancer continues to evolve. The sequence of EGFR TKIs may change in the future and combination therapies targeting resistance appear highly promising.

  17. Third-Generation Tyrosine Kinase Inhibitors Targeting Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Barnes, Tristan A; O'Kane, Grainne M; Vincent, Mark David; Leighl, Natasha B

    2017-01-01

    Sensitizing mutations in the epidermal growth factor receptor (EGFR) predict response to EGFR tyrosine kinase inhibitors (TKIs) and both first- and second-generation TKIs are available as first-line treatment options in patients with advanced EGFR-mutant non-small cell lung cancer. Eventual resistance develops with multiple mechanisms identifiable both upon repeat biopsy and in plasma circulating tumor DNA. The T790M gatekeeper mutation is responsible for almost 60% of cases. A number of third-generation TKIs are in clinical development, and osimertinib has been approved by the US Food and Drug Administration for the treatment of patients with EGFR T790M mutant lung cancer after failure of initial EGFR kinase therapy. Resistance mechanisms are being identified to these novel agents, and the treatment landscape of EGFR-mutant lung cancer continues to evolve. The sequence of EGFR TKIs may change in the future and combination therapies targeting resistance appear highly promising.

  18. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

    Science.gov (United States)

    Traxler, Elizabeth A; Yao, Yu; Wang, Yong-Dong; Woodard, Kaitly J; Kurita, Ryo; Nakamura, Yukio; Hughes, Jim R; Hardison, Ross C; Blobel, Gerd A; Li, Chunliang; Weiss, Mitchell J

    2016-09-01

    Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes β-globin), mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult β-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or α2γ2) to adult (referred to as HbA or α2β2) form. These disorders are alleviated when postnatal expression of fetal γ-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate γ-globin-to-β-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with β-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of β-hemoglobinopathies.

  19. Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene.

    Science.gov (United States)

    Cunha, Karin Soares; Oliveira, Nathalia Silva; Fausto, Anna Karoline; de Souza, Carolina Cruz; Gros, Audrey; Bandres, Thomas; Idrissi, Yamina; Merlio, Jean-Philippe; de Moura Neto, Rodrigo Soares; Silva, Rosane; Geller, Mauro; Cappellen, David

    2016-12-17

    Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

  20. Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

    Science.gov (United States)

    Cunha, Karin Soares; Oliveira, Nathalia Silva; Fausto, Anna Karoline; de Souza, Carolina Cruz; Gros, Audrey; Bandres, Thomas; Idrissi, Yamina; Merlio, Jean-Philippe; de Moura Neto, Rodrigo Soares; Silva, Rosane; Geller, Mauro; Cappellen, David

    2016-01-01

    Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations. PMID:27999334

  1. Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

    Directory of Open Access Journals (Sweden)

    Karin Soares Cunha

    2016-12-01

    Full Text Available Neurofibromatosis 1 (NF1 is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11. We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G. Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns for different types of pathogenic variations, including the deep intronic splicing mutations.

  2. Generating optimal initial conditions for smooth particle hydrodynamics (SPH) simulations

    Energy Technology Data Exchange (ETDEWEB)

    Diehl, Steven [Los Alamos National Laboratory; Rockefeller, Gabriel M [Los Alamos National Laboratory; Fryer, Christopher L [Los Alamos National Laboratory

    2008-01-01

    We present a new optimal method to set up initial conditions for Smooth Particle Hydrodynamics Simulations, which may also be of interest for N-body simulations. This new method is based on weighted Voronoi tesselations (WVTs) and can meet arbitrarily complex spatial resolution requirements. We conduct a comprehensive review of existing SPH setup methods, and outline their advantages, limitations and drawbacks. A serial version of our WVT setup method is publicly available and we give detailed instruction on how to easily implement the new method on top of an existing parallel SPH code.

  3. Generation of transgenic dogs that conditionally express green fluorescent protein.

    Science.gov (United States)

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  4. [Mutation screening for the causative gene in a four-generation Chinese pedigree with progressive cardiac conduction defect].

    Science.gov (United States)

    Tan, X J; Huang, H; He, F; Zhu, L; Li, H; Jiang, Y S; Li, H; Huang, X H; Sun, Z S; Li, Z H

    2016-05-24

    To define the potential causative gene mutation in a Chinese pedigree with progressive cardiac conduction defect (PCCD). Sanger sequencing was performed to define potential causative gene mutation in a four-generation family with 68 members including seven PCCD patients (5 male) from 2010 to 2015.No causative gene was detected by screening known candidate genes related to PCCD including SCN5A, NKX2.5 and LMNA.High-throughput sequencing technology on exon-enriched DNA was then used to search the causative genes in 2 patients and one normal family member. Eight new non-synonymous single nucleotide variants including AQP7 gene (exon5: c.T343C: p.Y115H), CACNA1B gene (NM_001243812: exon19: c.A2986G: p.T996A), CATSPERB gene (exon27: c.C3254G: p.P1085R), CLCA2 gene (exon11: c.G1725T: p.W575C), CLCA3P gene (ncRNA_intronic), MYLK-AS1 gene (ncRNA_intronic), TTN gene (ncRNA_UTR3), LMNA gene (LMNA: NM_170708: exon5: c.C922T: p.Q308X) were identified by comparing and filtering the results with known public databases.Then, more detailed biological analysis on these 8 genes was conducted.Traditional Sanger sequencing validated the exome sequencing results, and found that the mutation c. 1725G﹥T in gene CLCA2 segregated with the phenotype of this PCCD pedigree.The mutation c. 1725G﹥T in gene CLCA2 was thus be considered as the causative PCCD gene in this pedigree from the perspective of genetics and genomics. The heterozygote mutation c. 1725G﹥T in gene CLCA2 might be causative gene in this PCCD pedigree.This finding adds new gene mutation variant responsible for PCCD.

  5. Sea Slot Cone Generator Overtopping Performance in 3D Conditions

    DEFF Research Database (Denmark)

    Margheritini, Lucia; Vicinanza, Diego; Frigaard, Peter

    2008-01-01

    This note describes the influence of wave spreading, directionality and local bathymetry on the efficiency of the SSG wave energy converter Pilot plant in Kvitsøy, Norway. This is an overtopping device i.e. its efficiency is directly proportional to the overtopping flows in the three reservoirs...... is also described. It has been found that the performance of the SSG Pilot will be negatively affected by spreading and directionality of the incoming waves as direct consequence of reduction on the overtopping flow rates of 10% - 35% compared to 2D conditions....... the device has one on top of each others. The overtopping flow rates have been measured separately for each one of them, together with incoming waves during physical model tests at Aalborg University. The influence of the significant wave height Hs and of the wave length L on the captured overtopping water...

  6. Planar light bullets under conditions of second-harmonic generation

    Science.gov (United States)

    Sazonov, Sergey V.; Mamaikin, Mikhail S.; Komissarova, Maria V.; Zakharova, Irina G.

    2017-08-01

    We study solutions to second-harmonic-generation equations in two-dimensional media with anomalous dispersion. The analytical solution is obtained in an approximate form of the planar spatiotemporal two-component soliton by means of the averaged Lagrangian method. It is shown that a decrease in the amplitudes of both soliton components and an increase in the value of the transverse coordinate are accompanied by an increase in their temporal duration. Within this variational approach, we have managed to find a stability criterion for the light bullet and a period of oscillations of soliton parameters. Then, we use the obtained form as an initial configuration to carry out the direct numerical simulation of soliton dynamics. We demonstrate stable propagation of spatiotemporal solitons undergoing small oscillations predicted analytically for a long distance. The formation of a two-component light bullet is shown when we launch a pulse only at the fundamental frequency. In addition, we investigate the phase and group-velocity mismatch effects on the propagation of pulses.

  7. A miniature condition in Brahman cattle is associated with a single nucleotide mutation within the growth hormone gene.

    Science.gov (United States)

    McCormack, B L; Chase, C C; Olson, T A; Elsasser, T H; Hammond, A C; Welsh, T H; Jiang, H; Randel, R D; Okamura, C A; Lucy, M C

    2009-08-01

    Miniature Brahman cattle at the USDA ARS Subtropical Agriculture Research Station in Brooksville, FL have normal proportioned growth but are approximately 70% of mature height and weight when compared with Brahman cattle in the same herd. Pedigree analyses suggest that the condition is inherited through a recessive allele. The miniature Brahman cattle in the Brooksville herd have been used for studies of growth and reproduction, but the underlying causative mutation is unknown. Presumably, the miniature condition could arise from a mutation in the GH gene. The objective, therefore, was to clone the GH cDNA from Brooksville miniature Brahman cattle, compare its sequence to normal Brahman cattle, and test the biological activity of the native GH protein. Messenger RNA was isolated from the pituitary, and a cDNA for the protein coding region of the GH gene was amplified by reverse-transcription polymerase chain reaction (PCR) from each of 2 miniature Brahman bulls. The cDNA were cloned into plasmid vectors, and top and bottom strands were sequenced by automated DNA sequencing. The sequence of both cDNA clones derived from miniature cattle differed from Bos indicus GH (GenBank AF034386) at base number 641 because there was a cytosine (C) instead of a thymine (T). The C to T change encoded a mutation (threonine to methionine) at amino acid 200 (T200M mutation). The mutation was confirmed by sequencing of an additional 2 miniature cattle and comparing their sequence to 2 normal cattle. The threonine is located in the fourth alpha helix of GH and is 1 of 8 amino acids that participate in binding of GH to the GH receptor. Twelve miniature Brahman and 9 normal Brahman cattle were tested by using a restriction fragment length polymorphism analysis that employed the BsmBI restriction enzyme (specific for the mutated nucleotide). The 12 miniature Brahman cattle were homozygous for the mutation (-/-). Seven of the normal Brahman cattle were homozygous for the wild-type allele

  8. A simple method to generate chromosomal mutations in Lactobacillus plantarum strain TF103 to eliminate undesired fermentation products.

    Science.gov (United States)

    Liu, Siqing

    2006-01-01

    Gram-positive bacteria have been explored to convert lignocellulosic biomass to biofuel and bioproducts. Our long-term goal is to create genetically engineered lactic acid bacteria (LAB) strains that convert agricultural biomass into ethanol and other value-added products. The immediate approaches toward this goal involve genetic manipulations by either introducing ethanol production pathway genes or inactivating pathways genes that lead to production of undesired byproducts. The widely studied species Lactobacillus plantarum is now considered a model for genetic manipulations of LAB. In this study, L. plantarum TF103 strain, in which two of the chromosomal L-ldh and D-ldh genes are inactivated, was used to introduce additional mutations on the chromosome to eliminate undesired fermentation products. We targeted the acetolactate synthase gene (als) that converts pyruvate to acetolactate, to eliminate the production of acetoin and 2,3-butanodial. A pBluescript derivative containing sections of the als coding region and an erythromycin resistance gene was directly introduced into L. plantarum TF103 cells to create mutations under selection pressure. The resulting erythromycin resistant (Emr) TF103 strain appears to have chromosomal mutations of both the als and the adjacent lysP genes as revealed by polymerase chain reaction and Southern blot analyses. Mutations were thus generated via targeted homologous recombination using a Gram-negative cloning vector, eliminating the use of a shuttle vector. This method should facilitate research in targeted inactivation of other genes in LAB.

  9. Identification of novel mutations in hemochromatosis genes by targeted next generation sequencing in Italian patients with unexplained iron overload.

    Science.gov (United States)

    Badar, Sadaf; Busti, Fabiana; Ferrarini, Alberto; Xumerle, Luciano; Bozzini, Paolo; Capelli, Paola; Pozzi-Mucelli, Roberto; Campostrini, Natascia; De Matteis, Giovanna; Marin Vargas, Sergio; Giorgetti, Alejandro; Delledonne, Massimo; Olivieri, Oliviero; Girelli, Domenico

    2016-06-01

    Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available "first level" molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non-diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of "non-HFE" HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)-based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non-diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of "non-HFE" HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS-based "second level" genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations.

  10. New Mutations in NEB Gene Discovered by Targeted Next-Generation Sequencing in Nemaline Myopathy Italian Patients.

    Science.gov (United States)

    Piga, Daniela; Magri, Francesca; Ronchi, Dario; Corti, Stefania; Cassandrini, Denise; Mercuri, Eugenio; Tasca, Giorgio; Bertini, Enrico; Fattori, Fabiana; Toscano, Antonio; Messina, Sonia; Moroni, Isabella; Mora, Marina; Moggio, Maurizio; Colombo, Irene; Giugliano, Teresa; Pane, Marika; Fiorillo, Chiara; D'Amico, Adele; Bruno, Claudio; Nigro, Vincenzo; Bresolin, Nereo; Comi, Giacomo Pietro

    2016-07-01

    Nemaline myopathy represents a group of clinically and genetically heterogeneous neuromuscular disorders. Different clinical-genetic entities have been characterized in the last few years, with implications for diagnostics and genetic counseling. Fifty percent of nemaline myopathy forms are due to NEB mutations, but genetic analysis of this large and complex gene by Sanger sequencing is time consuming and expensive. We selected 10 Italian patients with clinical and biopsy features suggestive for nemaline myopathy and negative for ACTA1, TPM2 and TPM3 mutations. We applied a targeted next-generation sequencing strategy designed to analyse NEB coding regions, the relative full introns and the promoter. We also evaluated copy number variations (by CGH array) and transcriptional changes by RNA Sanger sequencing, whenever possible. This combined strategy revealed 11 likely pathogenic variants in 8 of 10 patients. The molecular diagnosis was fully achieved in 3 of 8 patients, while only one heterozygous mutation was observed in 5 subjects. This approach revealed to be a fast and cost-effective way to analyse the large NEB gene in a small group of patients and might be promising for the detection of pathological variants of other genes featuring large coding regions and lacking mutational hotspots.

  11. Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

    Directory of Open Access Journals (Sweden)

    Giancarlo Russo

    2015-12-01

    We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

  12. Generation and characterization of two novel low phytate mutations in soybean (Glycine max L. Merr.).

    Science.gov (United States)

    Yuan, Feng-Jie; Zhao, Hai-Jun; Ren, Xue-Liang; Zhu, Shen-Long; Fu, Xu-Jun; Shu, Qing-Yao

    2007-11-01

    Phytic acid (PA, myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is important to the nutritional quality of soybean meal. Organic phosphorus (P) in PA is indigestible in humans and non-ruminant animals, which affects nutrition and causes P pollution of ground water from animal wastes. Two novel soybean [(Glycine max L. (Merr.)] low phytic acid (lpa) mutations were isolated and characterized. Gm-lpa-TW-1 had a phytic acid P (PA-P) reduction of 66.6% and a sixfold increase in inorganic P (Pi), and Gm-lpa-ZC-2 had a PA-P reduction of 46.3% and a 1.4-fold increase in Pi, compared with their respective non-mutant progenitor lines. The reduction of PA-P and increase of Pi in Gm-lpa-TW-1 were molar equivalent; the decrease of PA-P in Gm-lpa-ZC-2, however, was accompanied by the increase of both Pi and lower inositol phosphates. In both mutant lines, the total P content remained similar to their wild type parents. The two lpa mutations were both inherited in a single recessive gene model but were non-allelic. Sequence data and progeny analysis indicate that Gm-lpa-TW-1 lpa mutation resulted from a 2 bp deletion in the soybean D: -myo-inositol 3-phosphate synthase (MIPS1 EC 5.5.1.4) gene 1 (MIPS1). The lpa mutation in Gm-lpa-ZC-2 was mapped on LG B2, closely linked with microsatellite loci Satt416 and Satt168, at genetic distances of approximately 4.63 and approximately 9.25 cM, respectively. Thus this mutation probably represents a novel soybean lpa locus. The seed emergence rate of Gm-lpa-ZC-2 was similar to its progenitor line and was not affected by seed source and its lpa mutation. However, Gm-lpa-TW-1 had a significantly reduced field emergence when seeds were produced in a subtropic environment. Field tests of the mutants and their progenies further demonstrated that the lpa mutation in Gm-lpa-ZC-2 does not negatively affect plant yield traits. These results will advance understanding of the genetic, biochemical and molecular control of PA synthesis in soybean. The

  13. Conditioning, Correlation and Entropy Generation in Maxwell’s Demon

    Directory of Open Access Journals (Sweden)

    Neal G. Anderson

    2013-10-01

    Full Text Available Maxwell’s Demon conspires to use information about the state of a confined molecule in a Szilard engine (randomly frozen into a state subspace by his own actions to derive work from a single-temperature heat bath. It is widely accepted that, if the Demon can achieve this at all, he can do so without violating the Second Law only because of a counterbalancing price that must be paid to erase information when the Demon’s memory is reset at the end of his operating cycle. In this paper, Maxwell’s Demon is analyzed within a “referential” approach to physical information that defines and quantifies the Demon’s information via correlations between the joint physical state of the confined molecule and that of the Demon’s memory. On this view, which received early emphasis in Fahn’s 1996 classical analysis of Maxwell’s Demon, information is erased not during the memory reset step of the Demon’s cycle, but rather during the expansion step, when these correlations are destroyed. Dissipation and work extraction are analyzed here for a Demon that operates a generalized quantum mechanical Szilard engine embedded in a globally closed composite, which also includes a work reservoir, a heat bath and the remainder of the Demon’s environment. Memory-engine correlations lost during the expansion step, which enable extraction of work from the Demon via operations conditioned on the memory contents, are shown to be dissipative when this decorrelation is achieved unconditionally so no work can be extracted. Fahn’s essential conclusions are upheld in generalized form, and his quantitative results supported via appropriate specialization to the Demon of his classical analysis, all without external appeal to classical thermodynamics, the Second Law, phase space conservation arguments or Landauer’s Principle.

  14. Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis.

    Directory of Open Access Journals (Sweden)

    Bernd Timmermann

    Full Text Available BACKGROUND: Colorectal cancer (CRC is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS and microsatellite instable (MSI colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. CONCLUSIONS/SIGNIFICANCE: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.

  15. Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing

    Directory of Open Access Journals (Sweden)

    Hyunseok P. Kang

    2016-06-01

    Full Text Available Hereditary breast and ovarian cancer syndrome, caused by a germline pathogenic variant in the BRCA1 or BRCA2 (BRCA1/2 genes, is characterized by an increased risk for breast, ovarian, pancreatic and other cancers. Identification of those who have a BRCA1/2 mutation is important so that they can take advantage of genetic counseling, screening, and potentially life-saving prevention strategies. We describe the design and analytic validation of the Counsyl Inherited Cancer Screen, a next-generation-sequencing-based test to detect pathogenic variation in the BRCA1 and BRCA2 genes. We demonstrate that the test is capable of detecting single-nucleotide variants (SNVs, short insertions and deletions (indels, and copy-number variants (CNVs, also known as large rearrangements with zero errors over a 114-sample validation set consisting of samples from cell lines and deidentified patient samples, including 36 samples with BRCA1/2pathogenic germline mutations.

  16. EFFECT OF GAMMA RADIATION OF MACRO MUTATIONS, EFFECTIVENESS AND EFFICIENCY UNDER M2 GENERATION IN PEA (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    Arvind KUMAR

    2016-06-01

    Full Text Available The present investigation was undertaken to study the spectrum of macro mutants, effectiveness and efficiency of different doses of gamma rays in pea variety (Arkel. The seeds were treated with gamma rays viz., 00kR (dry control, 05kR, 10kR, 15kR, 20kR, 25kR, 30kR, 35kR, 40kR (dry seeds and presoaked seeds of the same was exposed to 00kR (wet control, 05kR, 10kR, 15kR, 20kR (kilo Roentgen biological damage was calculated in M1 and M2 generation based on lethality (L and pollen sterility. The irradiated seeds were sown in the M1 field their respective controls and harvested in bulk to raise the M2 generation in Randomized Block Design (RBD with three replications. The spectrum of macro mutants i.e., plant stature (tall, dwarf, small dwarf, maturity (early, late, pod shape (bold, long, short, seed colour (brown, light white, light green and seed shape (small, bold, wrinkled were observed in M2 generation. The usefulness of any mutagen in plant breeding depends not only on its effectiveness but also upon if efficiency. Mutagenic effectiveness is a measure of the frequency of mutations induced by unit mutagen dose, whereas mutagenic efficiency is measure of proportion of mutations in relation of undesirable changes like lethality and sterility are used for gamma rays. A result of the indicated positive relationship in M2 generation with macro mutation, effectiveness and efficiency was found to be highest at lowest doses.

  17. Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle.

    Science.gov (United States)

    Deng, Maoxian; Boopathi, Ettickan; Hypolite, Joseph A; Raabe, Tobias; Chang, Shaohua; Zderic, Stephen; Wein, Alan J; Chacko, Samuel

    2013-11-15

    Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

  18. NSD1 mutations generate a genome-wide DNA methylation signature.

    LENUS (Irish Health Repository)

    Choufani, S

    2015-12-22

    Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth\\/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1(+\\/-)-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1(+\\/-) signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

  19. Optimizing the Structure of Distribution Smart Grids with Renewable Generation against Abnormal Conditions: A Complex Networks Approach with Evolutionary Algorithms

    Directory of Open Access Journals (Sweden)

    Lucas Cuadra

    2017-07-01

    Full Text Available In this work, we describe an approach that allows for optimizing the structure of a smart grid (SG with renewable energy (RE generation against abnormal conditions (imbalances between generation and consumption, overloads or failures arising from the inherent SG complexity by combining the complex network (CN and evolutionary algorithm (EA concepts. We propose a novel objective function (to be minimized that combines cost elements, related to the number of electric cables, and several metrics that quantify properties that are beneficial for SGs (energy exchange at the local scale and high robustness and resilience. The optimized SG structure is obtained by applying an EA in which the chromosome that encodes each potential network (or individual is the upper triangular matrix of its adjacency matrix. This allows for fully tailoring the crossover and mutation operators. We also propose a domain-specific initial population that includes both small-world and random networks, helping the EA converge quickly. The experimental work points out that the proposed method works well and generates the optimum, synthetic, small-world structure that leads to beneficial properties such as improving both the local energy exchange and the robustness. The optimum structure fulfills a balance between moderate cost and robustness against abnormal conditions. Our approach should be considered as an analysis, planning and decision-making tool to gain insight into smart grid structures so that the low level detailed design is carried out by using electrical engineering techniques.

  20. Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.

    Science.gov (United States)

    Zhou, Xiaoyang; Wang, Lulu; Du, Yinan; Xie, Fei; Li, Liang; Liu, Yu; Liu, Chuanhong; Wang, Shiqiang; Zhang, Shibing; Huang, Xingxu; Wang, Yong; Wei, Hong

    2016-01-01

    Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.

  1. Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing

    DEFF Research Database (Denmark)

    Rockenbauer, Eszter; Hansen, Stine; Mikkelsen, Martin;

    2014-01-01

    We sequenced the D21S11 locus in 77 individuals from Danish paternity cases using 454 FLX next generation sequencing (NGS) technology. All samples were also typed with the AmpFlSTR(®) Profiler Plus(®) or the AmpFlSTR(®) Identifiler(®) PCR Amplification kits as part of paternity investigations....... In 18 of the confirmed trios, a genetic inconsistency was observed between one of the parents and the child at the D21S11 locus. NGS of the D21S11 locus revealed which allele had mutated from which parent to the child in 13 of these trios. All characterized mutations could be explained by single......-step mutations in the longest sub-repeat of D21S11. A total of 53 of the 77 sequenced samples originated from unrelated individuals. Twenty different D21S11 alleles were detected by NGS in these individuals whereas only 13 different alleles were observed with fragment analysis. Several alleles had the same...

  2. An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Lucy F Stead

    Full Text Available Squamous cell carcinoma (SCC of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.

  3. Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

    Science.gov (United States)

    Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

    2015-12-15

    We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis.

  4. Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra

    Science.gov (United States)

    Shewaramani, Sonal; Finn, Thomas J.; Kassen, Rees; Rainey, Paul B.

    2017-01-01

    Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes. PMID:28103245

  5. Simulation of Entropy Generation under Stall Conditions in a Centrifugal Fan

    Directory of Open Access Journals (Sweden)

    Lei Zhang

    2014-06-01

    Full Text Available Rotating stalls are generally the first instability met in turbomachinery, before surges. This 3D phenomenon is characterized by one or more stalled flow cells which rotate at a fraction of the impeller speed. The goal of the present work is to shed some light on the entropy generation in a centrifugal fan under rotating stall conditions. A numerical simulation of entropy generation is carried out with the ANSYS Fluent software which solves the Navier-Stokes equations and user defined function (UDF. The entropy generation characteristics in the centrifugal fan for five typical conditions are presented and discussed, involving the design condition, conditions on occurrence and development of stall inception, the rotating stall conditions with two throttle coefficients. The results show that the entropy generation increases after the occurrence of stall inception. The high entropy generation areas move along the circumferential and axial directions, and finally merge into one stall cell. The entropy generation rate during circumferential propagation of the stall cell is also discussed, showing that the entropy generation history is similar to sine curves in impeller and volute, and the volute tongue has a great influence on entropy generation in the centrifugal fan.

  6. The generation of successive unmarked mutations and chromosomal insertion of heterologous genes in Actinobacillus pleuropneumoniae using natural transformation.

    Directory of Open Access Journals (Sweden)

    Janine T Bossé

    Full Text Available We have developed a simple method of generating scarless, unmarked mutations in Actinobacillus pleuropneumoniae by exploiting the ability of this bacterium to undergo natural transformation, and with no need to introduce plasmids encoding recombinases or resolvases. This method involves two successive rounds of natural transformation using linear DNA: the first introduces a cassette carrying cat (which allows selection by chloramphenicol and sacB (which allows counter-selection using sucrose flanked by sequences to either side of the target gene; the second transformation utilises the flanking sequences ligated directly to each other in order to remove the cat-sacB cassette. In order to ensure efficient uptake of the target DNA during transformation, A. pleuropneumoniae uptake sequences are added into the constructs used in both rounds of transformation. This method can be used to generate multiple successive deletions and can also be used to introduce targeted point mutations or insertions of heterologous genes into the A. pleuropneumoniae chromosome for development of live attenuated vaccine strains. So far, we have applied this method to highly transformable isolates of serovars 8 (MIDG2331, which is the most prevalent in the UK, and 15 (HS143. By screening clinical isolates of other serovars, it should be possible to identify other amenable strains.

  7. An online technique for condition monitoring the induction generators used in wind and marine turbines

    Science.gov (United States)

    Yang, Wenxian; Tavner, P. J.; Court, R.

    2013-07-01

    Induction generators have been successfully applied to a variety of industries. However, their operation and maintenance in renewable wind and marine energy industries still face challenges due to harsh environments, limited access to site and relevant reliability issues. Hence, further enhancing their condition monitoring is regarded as one of the essential measures for improving their availability. To date, much effort has been made to monitor induction motors, which can be equally applied to monitoring induction generators. However, the achieved techniques still have constrains in particular when dealing with the condition monitoring problems in wind and marine turbine generators. For example, physical measurements of partial discharge, noise and temperature have been widely applied to monitoring induction machinery. They are simple and cost-effective, but unable to be used for fault diagnosis. The spectral analysis of vibration and stator current signals is also a mature technique popularly used in motor/generator condition monitoring practice. However, it often requires sufficient expertise for data interpretation, and significant pre-knowledge about the machines and their components. In particular in renewable wind and marine industries, the condition monitoring results are usually coupled with load variations, which further increases the difficulty of obtaining a reliable condition monitoring result. In view of these issues, a new condition monitoring technique is developed in this paper dedicated for wind and marine turbine generators. It is simple, informative and less load-dependent thus more reliable to deal with the online motor/generator condition monitoring problems under varying loading conditions. The technique has been verified through both simulated and practical experiments. It has been shown that with the aid of the proposed technique, not only the electrical faults but also the shaft unbalance occurring in the generator become detectable

  8. Prevalence and coexistence of KRAS, BRAF, PIK3CA, NRAS, TP53, and APC mutations in Indian colorectal cancer patients: Next-generation sequencing-based cohort study.

    Science.gov (United States)

    Jauhri, Mayank; Bhatnagar, Akanksha; Gupta, Satish; Bp, Manasa; Minhas, Sachin; Shokeen, Yogender; Aggarwal, Shyam

    2017-02-01

    Colorectal cancer incidences are on a rise in India. In this study, we have analyzed the mutation frequencies of six potential biomarkers, their coexistence, association with clinicopathological characteristics, and tumor location in Indian colorectal cancer patients. Next-generation sequencing was performed to identify mutations in the six potential biomarker genes using formalin-fixed paraffin-embedded tissue blocks of 112 colorectal cancer patients. The mutation frequency observed in KRAS, BRAF, PIK3CA, NRAS, TP53, and APC was 35.7%, 7.1%, 16.1%, 6.3%, 39.3%, and 29.5%, respectively. The significant associations of mutations were KRAS with age less than 60 years (p = 0.041), PIK3CA with males (p = 0.032), tumor stage I-II (p = 0.013), lack of metastasis in lymph nodes (p = 0.040), NRAS with rectum (p = 0.002), and APC with T2 stage of tumor growth (p = 0.013). No single patient harbored mutations in these six genes or any five genes simultaneously. Significance was noted in coexistence of KRAS with APC (p = 0.024) and mutual exclusion of KRAS with BRAF (p = 0.029). PIK3CA exon 9 was observed to be more frequently associated with KRAS mutations than PIK3CA exon 20 (p = 0.072). NRAS mutations were mutually exclusive with BRAF and PIK3CA mutations. As per our knowledge, this is the first next-generation sequencing-based biomarker study in Indian colorectal cancer patients. Frequent coexistence of gene mutations in pairs and triplets suggests that synergistic effect of overlapping mutations might further trigger the disease. In addition, infrequent coexistence of multiple gene mutations hints toward different signaling pathways for colorectal cancer tumorigenesis.

  9. Fuel Savings and Emission Reductions from Next-Generation Mobile Air Conditioning Technology in India: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Chaney, L.; Thundiyil, K.; Chidambaram, S.; Abbi, Y. P.; Anderson, S.

    2007-05-01

    This paper quantifies the mobile air-conditioning fuel consumption of the typical Indian vehicle, exploring potential fuel savings and emissions reductions these systems for the next generation of vehicles.

  10. Temporal dynamics of 'HoBi'-like pestivirus quasispecies in persistently infected calves generated under experimental conditions.

    Science.gov (United States)

    Weber, Matheus N; Bauermann, Fernando V; Canal, Cláudio W; Bayles, Darrell O; Neill, John D; Ridpath, Julia F

    2017-01-02

    'HoBi'-like virus is an atypical group within the Pestivirus genus that is implicated in economic losses for cattle producers due to both acute and persistent infections. Pestivirus strains exist as quasispecies (swarms of individual viruses) in infected animals and the viral populations making up the quasispecies differ widely in size and diversity in each animal. In the present study the viral quasispecies circulating in persistently infected (PI) calves, generated and maintained under experimental conditions using two different 'HoBi'-like strains, was observed over time. An increase in genetic variability and the development of certain mutations was observed over time. Mutations observed included the loss of a putative N-linked glycosylation site in the E2 region and the change of specific residues in E1/E2. It is hypothesized that these changes may be the results on continued adaption of the pestivirus to individual hosts. This is the first study characterizing variation in the viral swarms of animals persistently infected with HoBi-like viruses over time. Studies of the shifts in PI viral swarms will contribute to our understanding of the host and viral mechanisms that function in the maintenance of pestivirus persistent infections.

  11. Single amino acid mutation in alpha-helical peptide affect second harmonic generation hyperpolarizability

    Science.gov (United States)

    Wei, Jing; Wang, Jin-Yun; Zhang, Min-Yi; Chai, Guo-Liang; Lin, Chen-Sheng; Cheng, Wen-Dan

    2013-01-01

    We investigate the effect of side chain on the first-order hyperpolarizability in α-helical polyalanine peptide with the 10th alanine mutation (Acetyl(ala)9X(ala)7NH2). Structures of various substituted peptides are optimized by ONIOM (DFT: AM1) scheme, and then linear and nonlinear optical properties are calculated by SOS//CIS/6-31G∗ method. The polarizability and first-order hyperpolarizability increase obviously only when 'X' represents phenylalanine, tyrosine and tryptophan. We also discuss the origin of nonlinear optical response and determine what caused the increase of first-order hyperpolarizability. Our results strongly suggest that side chains containing benzene, phenol and indole have important contributions to first-order hyperpolarizability.

  12. Muscle 3243A -> G mutation load and capacity of the mitochondrial energy-generating system

    NARCIS (Netherlands)

    Janssen, Antoon J. M.; Schuelke, Markus; Smeitink, Jan A. M.; Trijbels, Frans J. M.; Sengers, Rob C. A.; Lucke, Barbara; Wintjes, Liesbeth T. M.; Morava, Eva; van Engelen, Baziel G. M.; Struts, Bart W.; Hol, Frans A.; Siers, Marloes H.; ter Laak, Henk; van der Knaap, Marjo S.; van Spronsen, Francjan J.; Rodenburg, Richard J. T.; van den Heuvel, Lambert P.

    2008-01-01

    Objective: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate productio

  13. Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system.

    NARCIS (Netherlands)

    Janssen, A.J.; Schuelke, M.; Smeitink, J.A.M.; Trijbels, F.J.; Sengers, R.C.; Lucke, B.; Wintjes, L.T.; Morava, E.; Engelen, B.G.M. van; Smits, B.W.; Hol, F.A.; Siers, M.H.; Laak, H. ter; Knaap, M.S. van der; Spronsen, F.J. van; Rodenburg, R.J.; Heuvel, L.P.v.d.

    2008-01-01

    OBJECTIVE: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate productio

  14. Muscle 3243A -> G mutation load and capacity of the mitochondrial energy-generating system

    NARCIS (Netherlands)

    Janssen, Antoon J. M.; Schuelke, Markus; Smeitink, Jan A. M.; Trijbels, Frans J. M.; Sengers, Rob C. A.; Lucke, Barbara; Wintjes, Liesbeth T. M.; Morava, Eva; van Engelen, Baziel G. M.; Struts, Bart W.; Hol, Frans A.; Siers, Marloes H.; ter Laak, Henk; van der Knaap, Marjo S.; van Spronsen, Francjan J.; Rodenburg, Richard J. T.; van den Heuvel, Lambert P.

    Objective: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate

  15. Spontaneous mutation parameters for Arabidopsis thaliana measured in the wild.

    Science.gov (United States)

    Rutter, Matthew T; Shaw, Frank H; Fenster, Charles B

    2010-06-01

    Mutations are the ultimate source of genetic diversity and their contributions to evolutionary process depend critically on their rate and their effects on traits, notably fitness. Mutation rate and mutation effect can be measured simultaneously through the use of mutation accumulation lines, and previous mutation accumulation studies measuring these parameters have been performed in laboratory conditions. However, estimation of mutation parameters for fitness in wild populations requires assays in environments where mutations are exposed to natural selection and natural environmental variation. Here we quantify mutation parameters in both the wild and greenhouse environments using 100 25th generation Arabidopsis thaliana mutation accumulation lines. We found significantly greater mutational variance and a higher mutation rate for fitness under field conditions relative to greenhouse conditions. However, our field estimates were low when scaled to natural environmental variation. Many of the mutation accumulation lines have increased fitness, counter to the expectation that nearly all mutations decrease fitness. A high mutation rate and a low mutational contribution to phenotypic variation may explain observed levels of natural genetic variation. Our findings indicate that mutation parameters are not fixed, but are variables whose values may reflect the specific environment in which mutations are tested.

  16. Control of power converters in distributed generation applications under grid fault conditions

    DEFF Research Database (Denmark)

    Rodriguez, Pedro; Luna, Alvaro; Munoz-Aguilar, Raul;

    2011-01-01

    The operation of distributed power generation systems under grid fault conditions is a key issue for the massive integration of renewable energy systems. Several studies have been conducted to improve the response of such distributed generation systems under voltage dips. In spite of being less s...

  17. Condition Monitoring and Faults Diagnosis for Synchronous Generator Using Artificial Neural Networks

    Directory of Open Access Journals (Sweden)

    Omer Elfaki Elbashir

    2013-09-01

    Full Text Available Early detection and diagnosis of incipient fault is desirable for on line condition assessment production quality assurance and improved operational efficiency of synchronous generator running of power supply. Artificial Intelligent techniques are increasly used for condition monitoring and fault diagnosis of machines. In this paper, Artificial Neural Network (ANN approach employed for fault diagnosis in the generator, based on monitoring generator currents to give indication of the winding faults. Feed-forward Network, error back propagation training algorithm are used to perform the generator faults diagnosis and their values. NN which has been trained for all possible operating condition of the machine used to classify the incoming data. The inputs of the NN are the stator and rotor currents, and the output represents the running condition of the generator. The training of the NN achieved by the data through a mathematical model based approach to simulate the generator faults at various degree of severity.This paper evaluates through simulation line currents magnitude of the generator .The final results have been represented on a monitoring unit, built using matlab program, to give early warning of the generator failure.

  18. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples.

    Science.gov (United States)

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D'Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.

  19. A simple two-step, 'hit and fix' method to generate subtle mutations in BACs using short denatured PCR fragments.

    Science.gov (United States)

    Yang, Yongping; Sharan, Shyam K

    2003-08-01

    The bacteriophage lambda recombination system has proven to be a valuable tool for engineering bacterial artificial chromosomes (BAC). Due to its high efficiency, subtle alterations in the BACs can be generated using oligonucleotides as targeting vectors. Since no selection marker is used, recombinant clones are identified utilizing a selective PCR screening method. However, occasionally the selective PCR screening is not feasible. We describe here a two-step 'hit and fix' method that can be reliably used for generating any subtle alteration in BACs using short denatured PCR fragments as targeting vectors. In the first step of this method, 6-20 nucleotides are changed around the base where the mutation has to be generated. In the second step, these altered nucleotides are reverted to the original sequence and simultaneously a subtle alteration is introduced. Since in each step several nucleotides are changed, PCR primers specific for such alterations can be designed. This two-step method provides a simple and efficient tool for generating subtle alterations in BACs that can be very valuable for functional analysis of genes.

  20. Performance Analysis of Doubly Fed Induction Generator Based Wind turbine under Faulty and RLC Load Conditions

    Directory of Open Access Journals (Sweden)

    Rekha Parashar

    2015-02-01

    Full Text Available This paper presents the performance of Doubly Fed Induction Generator based wind turbine system during different types of grid fault. The doubly fed induction generator (DFIG based wind turbine (WT system provides better power delivery towards the demand. The design and response of the DFIG based wind turbine system during different fault conditions, various load conditions and integrated system consisting of DFIG based WT system have been verified using MATLAB/ Simulink. The simulation results are shown with the model of DFIG based wind energy generation system.

  1. Test Feasibility of Next-Generation Sequencing Assays in Clinical Mutation Detection of Small Biopsy and Fine Needle Aspiration Specimens.

    Science.gov (United States)

    Zheng, Gang; Tsai, Harrison; Tseng, Li-Hui; Illei, Peter; Gocke, Christopher D; Eshleman, James R; Netto, George; Lin, Ming-Tseh

    2016-05-01

    To evaluate preanalytic factors contributing to failure of next-generation sequencing (NGS) assays. AmpliSeq Cancer Hotspot Panel was conducted in 1,121 of 1,152 formalin-fixed paraffin-embedded tissues submitted to a clinical laboratory, including 493 small biopsy or fine needle aspiration (FNA) specimens (44%) and 25 metastatic bone specimens (2.2%). Single nucleotide mutations and/or insertion/deletion mutations were detected in 702 specimens. Thirty-eight specimens (3.4%) were reported as "no results" due to NGS assay failure. Higher failure rates were observed in specimens submitted for lung cancer panel and melanoma panel (3.1% and 3.7% vs 1.0% colorectal cancer panel), metastatic bone specimens (36% vs 2.6% nonbone specimens), referred specimens (5.0% vs 1.8% in-house specimens), and small biopsy and FNA specimens (5.8% and 3.1% vs 0.7% resection/excision specimens). Test feasibility was higher in in-house specimens than referred specimens (99.1% vs 96.9% in resection specimens, 94.4% vs 87.3% in small biopsy specimens, and 94.3% vs 58.8% in FNA specimens). NGS assays demonstrated clinical utility in solid tumor specimens, including those taken by biopsy or FNA. Preanalytic factors identified by this study that may contribute to NGS assay failure highlight the need for pathologists to revisit tissue processing protocols in order to better optimize cancer mutational profiling. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Operational-Condition-Independent Criteria Dedicated to Monitoring Wind Turbine Generators

    Directory of Open Access Journals (Sweden)

    Richard Court

    2013-01-01

    Full Text Available Condition monitoring is beneficial to the wind industry for both onshore and offshore plants. However, due to the variations in operational conditions, its potential has not been fully explored. There is a need to develop an operational-condition-independent condition monitoring technique, which has motivated the research presented here. In this paper, three operational-condition-independent criteria are developed. The criteria accomplish the condition monitoring by analyzing the wind turbine electrical signals in the time domain. Therefore, they are simple to calculate and ideal for online use. All proposed criteria were tested through both simulated and practical experiments. The experiments have shown that these criteria not only provide a solution for detecting both mechanical and electrical faults that occur in wind turbine generators, but provide a potential tool for diagnosing generator winding faults.

  3. Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes.

    Science.gov (United States)

    Skora, Andrew D; Douglass, Jacqueline; Hwang, Michael S; Tam, Ada J; Blosser, Richard L; Gabelli, Sandra B; Cao, Jianhong; Diaz, Luis A; Papadopoulos, Nickolas; Kinzler, Kenneth W; Vogelstein, Bert; Zhou, Shibin

    2015-08-11

    Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.

  4. Generation of an induced pluripotent stem cell line from a patient with hereditary multiple endocrine neoplasia 2A (MEN2A syndrome with RET mutation

    Directory of Open Access Journals (Sweden)

    J. Hadoux

    2016-07-01

    Currently, there is no satisfactory animal model recapitulating all the features of the disease especially at the level of stem cells. We generated induced pluripotent stem cells (iPSCs from a patient with RET mutation at codon 634 who developed pheochromocytoma and MTC. RETC634Y-mutated cells were reprogrammed by non-integrative viral transduction. These iPSCs had normal karyotype, harboured the RETC634Y mutation and expressed pluripotency hallmarks as well as RET. A comprehensive pathological assessment of teratoma was performed after injection in immunodeficient mice.

  5. Targeted Next-Generation Sequencing Revealed Novel Mutations in Chinese Ataxia Telangiectasia Patients: A Precision Medicine Perspective.

    Science.gov (United States)

    Chen, Zhao; Ye, Wei; Long, Zhe; Ding, Dongxue; Peng, Huirong; Hou, Xuan; Qiu, Rong; Xia, Kun; Tang, Beisha; Jiang, Hong

    2015-01-01

    Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia and immunodeficiency due to mutations in the ATM gene. We performed targeted next-generation sequencing (NGS) on three unrelated patients and identified five disease-causing variants in three probands, including two pairs of heterozygous variants (FAT-1:c.4396C>T/p.R1466X, c.1608-2A>G; FAT-2:c.4412_4413insT/p.L1472Ffs*19, c.8824C>T/p.Q2942X) and one pair of homozygous variants (FAT-3: c.8110T>G/p.C2704G, Hom). With regard to precision medicine for rare genetic diseases, targeted NGS currently enables the rapid and cost-effective identification of causative mutations and is an updated molecular diagnostic tool that merits further optimization. This high-throughput data-based strategy would propel the development of precision diagnostic methods and establish a foundation for precision medicine.

  6. Efficient CRISPR-Cas9-Mediated Generation of Knockin Human Pluripotent Stem Cells Lacking Undesired Mutations at the Targeted Locus

    Directory of Open Access Journals (Sweden)

    Florian T. Merkle

    2015-05-01

    Full Text Available The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs, but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.

  7. Targeted Next-Generation Sequencing Revealed Novel Mutations in Chinese Ataxia Telangiectasia Patients: A Precision Medicine Perspective.

    Directory of Open Access Journals (Sweden)

    Zhao Chen

    Full Text Available Ataxia telangiectasia (AT is an autosomal recessive disease characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia and immunodeficiency due to mutations in the ATM gene. We performed targeted next-generation sequencing (NGS on three unrelated patients and identified five disease-causing variants in three probands, including two pairs of heterozygous variants (FAT-1:c.4396C>T/p.R1466X, c.1608-2A>G; FAT-2:c.4412_4413insT/p.L1472Ffs*19, c.8824C>T/p.Q2942X and one pair of homozygous variants (FAT-3: c.8110T>G/p.C2704G, Hom. With regard to precision medicine for rare genetic diseases, targeted NGS currently enables the rapid and cost-effective identification of causative mutations and is an updated molecular diagnostic tool that merits further optimization. This high-throughput data-based strategy would propel the development of precision diagnostic methods and establish a foundation for precision medicine.

  8. Panel-based next generation sequencing as a reliable and efficient technique to detect mutations in unselected patients with retinal dystrophies

    Science.gov (United States)

    Glöckle, Nicola; Kohl, Susanne; Mohr, Julia; Scheurenbrand, Tim; Sprecher, Andrea; Weisschuh, Nicole; Bernd, Antje; Rudolph, Günther; Schubach, Max; Poloschek, Charlotte; Zrenner, Eberhart; Biskup, Saskia; Berger, Wolfgang; Wissinger, Bernd; Neidhardt, John

    2014-01-01

    Hereditary retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different forms of RD can be caused by mutations in >100 genes, including >1600 exons. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. So far, NGS is not routinely used in gene diagnostics. We developed a diagnostic NGS pipeline to identify mutations in 170 genetically and clinically unselected RD patients. NGS was applied to 105 RD-associated genes. Underrepresented regions were examined by Sanger sequencing. The NGS approach was successfully established using cases with known sequence alterations. Depending on the initial clinical diagnosis, we identified likely causative mutations in 55% of retinitis pigmentosa and 80% of Bardet–Biedl or Usher syndrome cases. Seventy-one novel mutations in 40 genes were newly associated with RD. The genes USH2A, EYS, ABCA4, and RHO were more frequently affected than others. Occasionally, cases carried mutations in more than one RD-associated gene. In addition, we found possible dominant de-novo mutations in cases with sporadic RD, which implies consequences for counseling of patients and families. NGS-based mutation analyses are reliable and cost-efficient approaches in gene diagnostics of genetically heterogeneous diseases like RD. PMID:23591405

  9. Generation of Evc2/Limbin global and conditional KO mice and its roles during mineralized tissue formation.

    Science.gov (United States)

    Zhang, Honghao; Takeda, Haruko; Tsuji, Takehito; Kamiya, Nobuhiro; Rajderkar, Sudha; Louie, Ke'Ale; Collier, Crystal; Scott, Greg; Ray, Manas; Mochida, Yoshiyuki; Kaartinen, Vesa; Kunieda, Tetsuo; Mishina, Yuji

    2015-07-28

    Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis, 2015. © 2015 Wiley Periodicals, Inc.

  10. New murine Niemann-Pick type C models bearing a pseudoexon-generating mutation recapitulate the main neurobehavioural and molecular features of the disease

    Science.gov (United States)

    Gómez-Grau, Marta; Albaigès, Júlia; Casas, Josefina; Auladell, Carme; Dierssen, Mara; Vilageliu, Lluïsa; Grinberg, Daniel

    2017-01-01

    Niemann-Pick disease type C (NPC) is a rare neurovisceral disease caused mainly by mutations in the NPC1 gene. This autosomal recessive lysosomal disorder is characterised by the defective lysosomal secretion of cholesterol and sphingolipids. No effective therapy exists for the disease. We previously described a deep intronic point mutation (c.1554-1009 G > A) in NPC1 that generated a pseudoexon, which could be corrected at the cellular level using antisense oligonucleotides. Here, we describe the generation of two mouse models bearing this mutation, one in homozygosity and the other in compound heterozygosity with the c.1920delG mutation. Both the homozygotes for the c.1554-1009 G > A mutation and the compound heterozygotes recapitulated the hallmarks of NPC. Lipid analysis revealed accumulation of cholesterol in the liver and sphingolipids in the brain, with both types of transgenic mice displaying tremor and ataxia at 7–8 weeks of age. Behavioural tests showed motor impairment, hyperactivity, reduced anxiety-like behaviour and impaired learning and memory performances, features consistent with those reported previously in NPC animal models and human patients. These mutant mice, the first NPC models bearing a pseudoexon-generating mutation, could be suitable for assessing the efficacy of specific splicing-targeted therapeutic strategies against NPC. PMID:28167839

  11. Mutation Profiling of Clinically Advanced Cancers Using Next-Generation Sequencing for Targeted Therapy: A Lifespan Experience.

    Science.gov (United States)

    Friedman, Kenneth; Resnick, Murray B; Safran, Howard

    2015-10-01

    The application of modern molecular tests such as next-generation sequencing (NGS) to human malignancies has led to better understanding of tumor biology and the design of targeted molecular therapies. In the research setting, important genomic alterations in tumors have been discovered with potential therapeutic implications but data regarding the impact of this technology in a real world oncology practice is limited. As a result, we decided to review the results of NGS in 144 advanced-stage cancer patients referred to the oncology practices of Lifespan-affiliated centers in Rhode Island. Most cancers revealed genomic alterations in genes commonly mutated in cancer. However, several unexpected genomic alterations were discovered in certain cancers with potential therapeutic intervention. Most cancers contained "actionable" genomic alterations despite being of advanced stage. Our experience demonstrates that application of NGS in the clinical setting contributes both to increasing the therapeutic armamentarium as well as our understanding of tumor biology.

  12. Recent developments in the third generation inhibitors of Bcr-Abl for overriding T315I mutation.

    Science.gov (United States)

    Lu, X Y; Cai, Q; Ding, K

    2011-01-01

    In the treatment of chronic myeloid leukemia (CML) with Bcr-Abl kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents, such as imatinib, nilotinib and dasatinib, by discrupting important contact interactions between the inhibitors and the enzyme. To overcome this particular resistance, several different strategies have been explored and many molecules have been investigated as capable of potently inhibiting Bcr-Abl T315I. Herein, this review reports on some predominant examples of third generation inhibitors of Bcr-Abl active against the T315I mutation, and special attentions are paid to the "hybrid-design" strategy for creating type-II class ATP-competitive inhibitors.

  13. A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma.

    Science.gov (United States)

    Liu, Chunqiao; Widen, Sonya A; Williamson, Kathleen A; Ratnapriya, Rinki; Gerth-Kahlert, Christina; Rainger, Joe; Alur, Ramakrishna P; Strachan, Erin; Manjunath, Souparnika H; Balakrishnan, Archana; Floyd, James A; Li, Tiansen; Waskiewicz, Andrew; Brooks, Brian P; Lehmann, Ordan J; FitzPatrick, David R; Swaroop, Anand

    2016-04-01

    Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt-Frizzled (FZD) signaling in ocular development and directly implicate WNT-FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma.

  14. Program Code Generator for Cardiac Electrophysiology Simulation with Automatic PDE Boundary Condition Handling.

    Science.gov (United States)

    Punzalan, Florencio Rusty; Kunieda, Yoshitoshi; Amano, Akira

    2015-01-01

    Clinical and experimental studies involving human hearts can have certain limitations. Methods such as computer simulations can be an important alternative or supplemental tool. Physiological simulation at the tissue or organ level typically involves the handling of partial differential equations (PDEs). Boundary conditions and distributed parameters, such as those used in pharmacokinetics simulation, add to the complexity of the PDE solution. These factors can tailor PDE solutions and their corresponding program code to specific problems. Boundary condition and parameter changes in the customized code are usually prone to errors and time-consuming. We propose a general approach for handling PDEs and boundary conditions in computational models using a replacement scheme for discretization. This study is an extension of a program generator that we introduced in a previous publication. The program generator can generate code for multi-cell simulations of cardiac electrophysiology. Improvements to the system allow it to handle simultaneous equations in the biological function model as well as implicit PDE numerical schemes. The replacement scheme involves substituting all partial differential terms with numerical solution equations. Once the model and boundary equations are discretized with the numerical solution scheme, instances of the equations are generated to undergo dependency analysis. The result of the dependency analysis is then used to generate the program code. The resulting program code are in Java or C programming language. To validate the automatic handling of boundary conditions in the program code generator, we generated simulation code using the FHN, Luo-Rudy 1, and Hund-Rudy cell models and run cell-to-cell coupling and action potential propagation simulations. One of the simulations is based on a published experiment and simulation results are compared with the experimental data. We conclude that the proposed program code generator can be used to

  15. Program Code Generator for Cardiac Electrophysiology Simulation with Automatic PDE Boundary Condition Handling.

    Directory of Open Access Journals (Sweden)

    Florencio Rusty Punzalan

    Full Text Available Clinical and experimental studies involving human hearts can have certain limitations. Methods such as computer simulations can be an important alternative or supplemental tool. Physiological simulation at the tissue or organ level typically involves the handling of partial differential equations (PDEs. Boundary conditions and distributed parameters, such as those used in pharmacokinetics simulation, add to the complexity of the PDE solution. These factors can tailor PDE solutions and their corresponding program code to specific problems. Boundary condition and parameter changes in the customized code are usually prone to errors and time-consuming. We propose a general approach for handling PDEs and boundary conditions in computational models using a replacement scheme for discretization. This study is an extension of a program generator that we introduced in a previous publication. The program generator can generate code for multi-cell simulations of cardiac electrophysiology. Improvements to the system allow it to handle simultaneous equations in the biological function model as well as implicit PDE numerical schemes. The replacement scheme involves substituting all partial differential terms with numerical solution equations. Once the model and boundary equations are discretized with the numerical solution scheme, instances of the equations are generated to undergo dependency analysis. The result of the dependency analysis is then used to generate the program code. The resulting program code are in Java or C programming language. To validate the automatic handling of boundary conditions in the program code generator, we generated simulation code using the FHN, Luo-Rudy 1, and Hund-Rudy cell models and run cell-to-cell coupling and action potential propagation simulations. One of the simulations is based on a published experiment and simulation results are compared with the experimental data. We conclude that the proposed program code

  16. Comparison of three generations of ActiGraph activity monitors under free-living conditions

    DEFF Research Database (Denmark)

    Grydeland, May; Hansen, Bjørge Herman; Ried-Larsen, M.

    2014-01-01

    + in children in free-living conditions. The generations GT1M and GT3X+ provided comparable outputs. The differences between the old and the newer monitors were more complex when investigating time spent at different intensities. Comparisons of data assessed by the AM7164 with data assessed by newer generations......BACKGROUND: A recent review concludes that the agreement of data across ActiGraph accelerometer models for children and youth still is uncertain. The aim of this study was to evaluate the agreement of three generations of ActiGraph accelerometers in children in a free-living condition. METHODS......: Sixteen 9-year-olds wore the ActiGraph AM7164, GT1M and GT3X+ simultaneously for three consecutive days. We compared mean counts per minute (mcpm) and time spent at different intensities from the three generations of monitors, and the agreement of outputs were evaluated by intra-class correlation...

  17. Optimal trajectory generation for generalization of discrete movements with boundary condition

    DEFF Research Database (Denmark)

    Herzog, Sebastian; Wörgötter, Florentin; Kulvicius, Tomas

    2016-01-01

    with zero error. Moreover, it has most of the properties of the state-of-the-art trajectory generation methods such as robustness to perturbations and generalisation to new boundary position and velocity conditions. We believe that, due to these features, our method has great potential for various robotic......Trajectory generation methods play an important role in robotics since they are essential for the execution of actions. In this paper we present a novel trajectory generation method for generalization of accurate movements with boundary conditions. Our approach originates from optimal control...... theory and is based on a second order dynamic system. We evaluate our method and compare it to state-of-the-art movement generation methods in both simulations and a real robot experiment. We show that the new method is very compact in its representation and can reproduce demonstrated trajectories...

  18. Factor XII mutations, estrogen-dependent inherited angioedema, and related conditions

    Directory of Open Access Journals (Sweden)

    Binkley Karen E

    2010-07-01

    Full Text Available Abstract The clinical, biochemical and genetic features of the conditions known as estrogen-dependent inherited angioedema, estrogen-associated angioedema, hereditary angioedema with normal C-1 inhibitor, type III angioedema, or factor XII angioedema are reviewed. Discussion emphasizes pathogenesis, diagnosis, and management.

  19. Factor XII mutations, estrogen-dependent inherited angioedema, and related conditions

    OpenAIRE

    Binkley Karen E

    2010-01-01

    Abstract The clinical, biochemical and genetic features of the conditions known as estrogen-dependent inherited angioedema, estrogen-associated angioedema, hereditary angioedema with normal C-1 inhibitor, type III angioedema, or factor XII angioedema are reviewed. Discussion emphasizes pathogenesis, diagnosis, and management.

  20. A mitochondrial mutator plasmid that causes senescence under dietary restricted conditions

    NARCIS (Netherlands)

    Maas, M.F.P.M.; Hoekstra, R.F.; Debets, A.J.M.

    2007-01-01

    Background Calorie or dietary restriction extends life span in a wide range of organisms including the filamentous fungus Podospora anserina. Under dietary restricted conditions, P. anserina isolates are several-fold longer lived. This is however not the case in isolates that carry one of the pAL2-1

  1. The Spectrum of BRCA Mutations and Characteristics of BRCA-associated Breast Cancers in China: Screening of 2991 Patients and 1043 Controls by Next-Generation Sequencing.

    Science.gov (United States)

    Lang, Guan-Tian; Shi, Jin-Xiu; Hu, Xin; Zhang, Chen-Hui; Shan, Ling; Song, Chuan-Gui; Zhuang, Zhi-Gang; Cao, A-Yong; Ling, Hong; Yu, Ke-Da; Shan, Li; Sun, Meng-Hong; Zhou, Xiao-Yan; Huang, Wei; Shao, Zhi-Ming

    2017-03-14

    To characterize the prevalence of BRCA mutations and characteristics of BRCA carriers in China and to update the clinical recommendations for BRCA testing, we conducted a wide screen for BRCA mutations using next-generation sequencing (NGS). A total of 4,034 Chinese subjects were screened for germline BRCA1/2 mutations, including 2,991 breast cancer patients and 1,043 healthy individuals from the community enrolled as controls. We developed an NGS-based approach to perform BRCA1/2 screening. BRCA mutations were identified in 9.1% (232/2,560) of cases with at least one risk factor, in 3.5% (15/431) of sporadic patients and in 0.38% (4/1,043) of healthy controls. The mutation frequency ranged from 8.9-15.2% in cohorts with a single risk factor to 16.6-100% in groups with multiple risk factors. We identified 70 novel BRCA mutations. A high frequency of BRCA1 c.5470_5477del was detected, accounting for 13.9% (16/115) of the BRCA1 mutations detected in our study. Clinical characteristics such as family history, invasive carcinoma, negative human epidermal growth factor receptor 2 (HER2), high Ki67 index, lymph node status, and high tumour grade were closely related to BRCA mutations. BRCA2 carriers had poorer disease-free survival among HER2- or hormone receptor-positive patients (hazard ratio=1.892; 95% confidence interval: 1.132-3.161; P=0.013). This study shows that BRCA mutation carriers could be frequently identified among breast cancer patients with multiple risk factors. Importantly, we established an NGS-based pipeline for BRCA1/2 testing in clinical practice, and strongly suggest that breast cancer patients of premier- and moderate-grade risk receive BRCA1/2 mutations testing in China. This article is protected by copyright. All rights reserved.

  2. Consequences of Femtosecond Laser Filament Generation Conditions in Standoff Laser Induced Breakdown Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Harilal, Sivanandan S.; Yeak, J.; Brumfield, Brian E.; Phillips, Mark C.

    2016-08-08

    We investigate the role of femtosecond laser focusing conditions on ablation properties and its implications on analytical merits and standoff detection applications. Femtosecond laser pulses can be used for ablation either by tightly focusing or by using filaments generated during its propagation. We evaluated the persistence of atomic, and molecular emission features as well as time evolution of the fundamental properties (temperature and density) of ablation plumes generated using different methods.

  3. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1

    DEFF Research Database (Denmark)

    Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co-electroporati......Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co...

  4. Targeted ultradeep next-generation sequencing as a method for KIT D816V mutation analysis in mastocytosis

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Broesby-Olsen, Sigurd; Vestergaard, Hanne;

    2016-01-01

    mutation levels. In this study, we established an NGS-based KIT mutation analysis and analyzed the sensitivity of D816V detection using the Ion Torrent platform. Eighty-two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation...

  5. In vitro correction of a pseudoexon-generating deep intronic mutation in LGMD2A by antisense oligonucleotides and modified small nuclear RNAs.

    Science.gov (United States)

    Blázquez, Lorea; Aiastui, Ana; Goicoechea, Maria; Martins de Araujo, Mafalda; Avril, Aurélie; Beley, Cyriaque; García, Luis; Valcárcel, Juan; Fortes, Puri; López de Munain, Adolfo

    2013-10-01

    Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation. © 2013 WILEY PERIODICALS, INC.

  6. An improved fuzzy synthetic condition assessment of a wind turbine generator system

    DEFF Research Database (Denmark)

    Li, H.; Hu, Y. G.; Yang, Chao;

    2013-01-01

    This paper presents an improved fuzzy synthetic model that is based on a real-time condition assessment method of a grid-connected wind turbine generator system (WTGS) to improve the operational reliability and optimize the maintenance strategy. First, a condition assessment framework is proposed...... by analyzing the monitored physical quantities of an actual WTGS with an electrically excited synchronous generator (EESG) and a full-scale converter. To examine the variable speed operational performances, the dynamic limits and the deterioration degree functions of the characteristic variables are determined...... 850 kW WTGS, real-time condition assessments are performed that utilize the proposed fuzzy synthetic method; the model’s effectiveness is also compared to a traditional fuzzy assessment method in which constant limited values and constant weights are adopted. The results show that the condition...

  7. Mutations of nuclear localization signals in mNANOG generate dominant negative mutants

    Institute of Scientific and Technical Information of China (English)

    ZHANG Juan; ZHANG XiaoFei; PEI DuanQing

    2009-01-01

    Mouse NANOG plays a critical role in maintaining self-renewal and pluripotency of embryonic stem cells.Yet,the precise mechanism of how mNANOG functions is still less known.Here,we report that mouse NANOG has two nuclear localization signals (NLS,RKQKMR and RMKCKR) which are respon-sible for the nuclear localization and transcriptional activity in the conserved homeobox domain.NLS mutants of mouse NANOG generate:3 mutants that are localized throughout the cells and lose the transectivation function.We further prove that all three NLS mutants may interact with the wild-type mouse NANOG like NANOG dimerization itself and inhibit the wild-type mouse NANOG activity,acting as dominant negative mutants.The NLS mutants of mouse NANOG may also inhibit activity of oct4 promoter in pluripotent cells,indicating that the NLS mutants can affect the endogenous mouse NANOG function in vivo.These data suggest that the NLS mutants of mouse NANOG may be used as a tool to regulate NANOG activity in pluripotent cells.

  8. Operational-Condition-Independent Criteria Dedicated to Monitoring Wind Turbine Generators: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Yang, W.; Sheng, S.; Court, R.

    2012-08-01

    To date the existing wind turbine condition monitoring technologies and commercially available systems have not been fully accepted for improving wind turbine availability and reducing their operation and maintenance costs. One of the main reasons is that wind turbines are subject to constantly varying loads and operate at variable rotational speeds. As a consequence, the influences of turbine faults and the effects of varying load and speed are coupled together in wind turbine condition monitoring signals. So, there is an urgent need to either introduce some operational condition de-coupling procedures into the current wind turbine condition monitoring techniques or develop a new operational condition independent wind turbine condition monitoring technique to maintain high turbine availability and achieve the expected economic benefits from wind. The purpose of this paper is to develop such a technique. In the paper, three operational condition independent criteria are developed dedicated for monitoring the operation and health condition of wind turbine generators. All proposed criteria have been tested through both simulated and practical experiments. The experiments have shown that these criteria provide a solution for detecting both mechanical and electrical faults occurring in wind turbine generators.

  9. Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning.

    Science.gov (United States)

    Qiu, X; Wu, L; Huang, H; McDonel, P E; Palumbo, A V; Tiedje, J M; Zhou, J

    2001-02-01

    To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

  10. Enhancing inulinase yield by irradiation mutation associated with optimization of culture conditions

    Directory of Open Access Journals (Sweden)

    Yafeng Gou

    2015-09-01

    Full Text Available A new inulinase-producing strain was isolated from rhizosphere soils of Jerusalem artichoke collected from Shihezi (Xinjiang, China using Jerusalem artichoke power (JAP as sole carbon source. It was identified as an Aspergillus niger strain by analysis of 16S rRNA. To improve inulinase production, this fungus was subjected to mutagenesis induced by 60Co γ-irradiation. A genetically stable mutant (designated E12 was obtained and it showed 2.7-fold higher inulinase activity (128 U/mL than the parental strain in the supernatant of a submerged culture. Sequential methodology was used to optimize the inulinase production of stain E12. A screening trial was first performed using Plackett-Burman design and variables with statistically significant effects on inulinase bio-production were identified. These significant factors were further optimized by central composite design experiments and response surface methodology. Finally, it was found that the maximum inulinase production (185 U/mL could be achieved under the optimized conditions namely pH 7.0, yeast extract concentration of 5.0 g/L, JAP concentration of 66.5 g/L, peptone concentration of 29.1 g/L, solution volume of 49.4 mL in 250-mL shake flasks, agitation speed of 180 rpm, and fermentation time of 60 h. The yield of inulinase under optimized culture conditions was approximately 1.4-fold of that obtained by using basal culture medium. These findings are of significance for the potential industrial application of the mutant E12.

  11. Conditional generation of arbitrary single-mode quantum states of light by repeated photon subtractions

    CERN Document Server

    Fiurasek, J; Cerf, N J; Fiurasek, Jaromir; Garcia-Patron, Raul; Cerf, Nicolas J.

    2005-01-01

    We propose a scheme for the conditional generation of arbitrary finite superpositions of (squeezed) Fock states in a single mode of a traveling optical field. The suggested setup requires only a source of squeezed states, beam splitters, strong coherent beams, and photodetectors with single-photon sensitivity. The method does not require photodetectors with a high efficiency nor with a single-photon resolution.

  12. A study on the gas generation from radioactive waste packages under disposal conditions in Korea

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joo wan; Kim, Chang Lak; Choi, Heui Joo; Yoon, Jeong Hyoun [Korea Electric Power Corporation, Nuclear Environment Institute, Taejon (Korea, Republic of)

    1999-07-01

    In order to confirm the compliance to acceptance criteria , the performance of radioactive waste packages currently used at the nuclear power plants in Korea in aspect of gas generation is investigated. As the principal gas generation mechanisms radiolysis, corrosion of metals, and microbial activity of organic materials are considered. For calculating rates and total volumes of radiolytic hydrogen gas generated in waste packages a computer program that accommodates interactions among adjacent packages is used. Gas production due to metal corrosion and microbial degradation of Dry Active Waste (DAW) packages and the others is estimated over an assessment period of one thousand years under a given set of repository condition, respectively. Flammability hazard caused by radiolytic hydrogen formation inside a sealed waste package, pressure build-up inside the engineered barrier structure under repository condition is also assessed. (author)

  13. Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease patient iPSC line carrying a A79V mutation in PSEN1

    Directory of Open Access Journals (Sweden)

    Carlota Pires

    2016-09-01

    Full Text Available Alzheimer's disease (AD is a progressive and irreversible neurodegenerative disease causing neural cell degeneration and brain atrophy and is considered to be the most common form of dementia. We previously generated an induced pluripotent stem cell (iPSC line from an AD patient carrying an A79V mutation in PSEN1 as an in vitro disease model. Here we generated a gene-corrected version from this hiPSC line by substituting the point mutation with the wild-type sequence. The reported A79V-GC-iPSCs line is a very useful resource in combination with the A79V-iPSC line in order to study pathological cellular phenotypes related to this particular mutation.

  14. MEK inhibitor treatment is effective in a patient with metastatic carcinoma of the ampulla of Vater with BRAF and NRAS mutations shown by next-generation sequencing.

    Science.gov (United States)

    Tahover, Esther; Bar Shalom, Rachel; Bogot, Naama; Kelsen, David; Gabizon, Alberto

    2016-07-01

    Here, we present a case of an 84-year-old woman who developed obstructive jaundice and was diagnosed with nonoperable adenocarcinoma originating from the ampulla of Vater, a lethal disease with a median overall survival of less than a year. Her tumor was examined by next-generation sequencing, which showed BRAF and NRAS mutations. To target these mutations, a MEK inhibitor was chosen for treatment. The patient has been treated with a MEK inhibitor for the last 12 months since diagnosis, with clinical and laboratory improvement and manageable side effects. PET-computed tomography imaging has shown stable disease or improvement in the primary and metastatic lesions. This is the first case report of an ampulla of a Vater cancer patient with NRAS and BRAF mutations, identified in next-generation sequencing, and treated successfully with a MEK inhibitor.

  15. Generation of a human induced pluripotent stem cell (iPSC line from a patient carrying a P33T mutation in the PDX1 gene

    Directory of Open Access Journals (Sweden)

    Xianming Wang

    2016-09-01

    Full Text Available Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4 and Type 2 Diabetes Mellitus (T2DM. To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a P33T heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 P33T iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms.

  16. Inspection of generator end bells in their installed condition; Inspektion von Rotorkappen im eingebauten Zustand

    Energy Technology Data Exchange (ETDEWEB)

    Noteboom, J.W.; Boxma, C.J. [KEMA Nederland B.V., Arnheim (Netherlands)

    1998-08-01

    Interest in evaluating the condition of generator end bells is the result of reports by leading generator manufacturers on the incidence of stress corrosion cracking of generator end bells made of X 55 MnC (N) 18 K and X 40 MnCr 18. KEMA (of the Netherlands) has developed a technique for the measurement of generator end bells, whereby the rotor does not have to be removed first. Fracture mechanisms calculations provide the evaluation of the non-homogeneous spots discovered. (orig.) [Deutsch] Das Interesse an der Zustandsbeurteilung von Generatorkappen ist das Ergebnis von Berichten bedeutender Generatorhersteller ueber die Spannungsrisskorrosionsanfaelligkeit von Generatorkappen aus X 55 MnC (N) 18 K und X 40 MnCr 18. KEMA hat ein Verfahren zur Messung von Generatorkappen entwickelt, bei dem der Rotor nicht zuerst auseinandergebaut werden muss. Bruchmechanikberechnungen dienen der Bewertung von aufgefundenen inhomogenen Stellen. (orig.)

  17. Structural Evaluation of a PGSFR Steam Generator for a Steady State Condition

    Energy Technology Data Exchange (ETDEWEB)

    Park, Chang-Gyu; Kim, Jong-Bum; Kim, Hoe-Woong; Koo, Gyeong-Hoi [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-05-15

    In this study, design loads for design condition and normal operating steady state condition were classified and the structural analyses for each design loads were carried out. And, structural integrities under each service level were evaluated according to ASME design code. The structural analyses of a steam generator are carried out and its structural integrity under the given service levels is evaluated per ASME Code rule. The design loads according to design condition and normal operating steady condition are classified and stresses calculated from stress analyses are linearized and summarized in their stress components. As a result, the SG structure satisfies with design criteria for both service levels. Though the steam header is designed as a thick hemisphere, its design margin is not so high in spite of just steady state condition. Thus, additional evaluation by considering various operating events will be followed.

  18. Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific heterozygous mutation in CHMP2B

    DEFF Research Database (Denmark)

    Zhang, Yu; Schmid, Benjamin; Nielsen, Troels Tolstrup;

    2016-01-01

    have generated an induced pluripotent stem cell (iPSC) line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into one of the alleles, resulting in aberrant splicing. This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3....

  19. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1

    DEFF Research Database (Denmark)

    Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A.;

    2016-01-01

    Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSC swere established by co-electroporation...

  20. 207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM.

    Science.gov (United States)

    Pinzon, C A; Snyder, M; Pryor, J; Thompson, B; Golding, M; Long, C

    2016-01-01

    The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of -1577, -689, -555, and -116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1=sgRNA 1-4; Set 2=sgRNA 2-3). Cells were exposed to puromycin (0.2µgmL(-1)) for 72h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes

  1. A case of congenital central hypoventilation syndrome in a three-generation family with non-polyalanine repeat PHOX2B mutation.

    Science.gov (United States)

    Low, K J; Turnbull, A R; Smith, K R; Hilliard, T N; Hole, L J; Meecham Jones, D J; Williams, M M; Donaldson, A

    2014-10-01

    We describe a three generation family in whom multiple individuals are variably affected due to a PHOX2B non-polyalanine repeat mutation. This family demonstrates extreme phenotypic variability and autosomal dominant transmission over three generations not previously reported in the wider literature. Novel findings also inclue a history of recurrent second trimester miscarriage. Pediatr Pulmonol. 2014; 49:E140-E143. © 2014 Wiley Periodicals, Inc.

  2. Molecular spectrum of TP53 mutations in plasma cell dyscrasias by next generation sequencing: an Italian cohort study and overview of the literature.

    Science.gov (United States)

    Lionetti, Marta; Barbieri, Marzia; Manzoni, Martina; Fabris, Sonia; Bandini, Cecilia; Todoerti, Katia; Nozza, Filomena; Rossi, Davide; Musto, Pellegrino; Baldini, Luca; Neri, Antonino

    2016-04-19

    The prevalence of TP53 mutations greatly varies between tumor types; in multiple myeloma (MM) they were rarely detected at presentation, while increased frequency was reported with disease progression. Using next-generation sequencing, we analyzed TP53 exons 4-9 in a large representative cohort comprising patients with MM at diagnosis and more aggressive forms of plasma cell (PC) dyscrasia, identifying mutations in 4/129 (3%) MM, 6/24 (25%) primary PC leukemia, and 2/10 (20%) secondary PC leukemia cases. A similar increase in prevalence associated with disease aggressiveness (5%, 29.2% and 44%, respectively) was observed for TP53 deletion. Interestingly, in five patients mutations were not concomitant with TP53 deletion. Furthermore, longitudinal analysis revealed the acquisition of TP53 mutations in three of nineteen cases analyzed at relapse. Identified variants were mostly missense mutations concentrated in the DNA binding domain, only partly reflecting the pattern globally observed in human cancers. Our data confirm that TP53 mutations are rare in MM at presentation and rather represent a marker of progression, similarly to del(17p); however, their occurrence even in absence of deletions supports the importance of their assessment in patients with PC dyscrasia, in terms of both risk stratification and therapeutic implications.

  3. Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

    Directory of Open Access Journals (Sweden)

    Wang Bin

    2011-08-01

    Full Text Available Abstract Background Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance. Results Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays. Conclusion This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.

  4. The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions.

    Directory of Open Access Journals (Sweden)

    Marcel H Fens

    Full Text Available Nitric oxide (NO is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

  5. The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions.

    Science.gov (United States)

    Fens, Marcel H; Larkin, Sandra K; Oronsky, Bryan; Scicinski, Jan; Morris, Claudia R; Kuypers, Frans A

    2014-01-01

    Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

  6. Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics

    Directory of Open Access Journals (Sweden)

    Elena Barbon

    2016-01-01

    Full Text Available Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs. However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X to integrate human factor IX (hFIX minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt or mutated (SChFIXex5-2C. In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies.

  7. Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics.

    Science.gov (United States)

    Barbon, Elena; Ferrarese, Mattia; van Wittenberghe, Laetitia; Sanatine, Peggy; Ronzitti, Giuseppe; Collaud, Fanny; Colella, Pasqualina; Pinotti, Mirko; Mingozzi, Federico

    2016-11-29

    Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X) to integrate human factor IX (hFIX) minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt) or mutated (SChFIXex5-2C). In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV) vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies.

  8. Entropy generation of micropolar fluid flow in an inclined porous pipe with convective boundary conditions

    Indian Academy of Sciences (India)

    D SRINIVASACHARYA; K HIMA BINDU

    2017-05-01

    The objective of this paper is to examine the nature of irreversibilities in the form of entropy generation for a micropolar fluid flow through an inclined porous pipe with convective boundary conditions. The governing equations are non-dimensionlized and then linearized using a quasilinearization method. The resulting linearized equations are solved by Chebyshev spectral collocation method. The velocity, microrotation and temperature profiles are presented graphically for various values of governing parameters. Further, these profilesare used to evaluate the entropy generation and Bejan number

  9. Beam conditions for radiation generated by an electromagnetic Hermite-Gaussian model source

    Institute of Scientific and Technical Information of China (English)

    LI Jia; XIN Yu; CHEN Yan-ru

    2011-01-01

    @@ Within the framework of the correlation theory of electromagnetic laser beams, the far field cross-spectral density matrix of the light radiated from an electromagnetic Hermite-Gaussian model source is derived.By utilizing the convergence property of Hermite polynomials, the conditions of the matrices for the source to generate an electromagnetic Hermite-Gaussian beam are obtained.Furthermore, in order to generate a scalar Hermite-Gaussian model beam, it is required that the source should be locally rather coherent in the spatial domain.

  10. Fully Electrical Modeling of Thermoelectric Generators with Contact Thermal Resistance Under Different Operating Conditions

    Science.gov (United States)

    Siouane, Saima; Jovanović, Slaviša; Poure, Philippe

    2017-01-01

    The Seebeck effect is used in thermoelectric generators (TEGs) to supply electronic circuits by converting the waste thermal into electrical energy. This generated electrical power is directly proportional to the temperature difference between the TEG module's hot and cold sides. Depending on the applications, TEGs can be used either under constant temperature gradient between heat reservoirs or constant heat flow conditions. Moreover, the generated electrical power of a TEG depends not only on these operating conditions, but also on the contact thermal resistance. The influence of the contact thermal resistance on the generated electrical power have already been extensively reported in the literature. However, as reported in Park et al. (Energy Convers Manag 86:233, 2014) and Montecucco and Knox (IEEE Trans Power Electron 30:828, 2015), while designing TEG-powered circuit and systems, a TEG module is mostly modeled with a Thévenin equivalent circuit whose resistance is constant and voltage proportional to the temperature gradient applied to the TEG's terminals. This widely used simplified electrical TEG model is inaccurate and not suitable under constant heat flow conditions or when the contact thermal resistance is considered. Moreover, it does not provide realistic behaviour corresponding to the physical phenomena taking place in a TEG. Therefore, from the circuit designer's point of view, faithful and fully electrical TEG models under different operating conditions are needed. Such models are mainly necessary to design and evaluate the power conditioning electronic stages and the maximum power point tracking algorithms of a TEG power supply. In this study, these fully electrical models with the contact thermal resistance taken into account are presented and the analytical expressions of the Thévenin equivalent circuit parameters are provided.

  11. Fully Electrical Modeling of Thermoelectric Generators with Contact Thermal Resistance Under Different Operating Conditions

    Science.gov (United States)

    Siouane, Saima; Jovanović, Slaviša; Poure, Philippe

    2016-09-01

    The Seebeck effect is used in thermoelectric generators (TEGs) to supply electronic circuits by converting the waste thermal into electrical energy. This generated electrical power is directly proportional to the temperature difference between the TEG module's hot and cold sides. Depending on the applications, TEGs can be used either under constant temperature gradient between heat reservoirs or constant heat flow conditions. Moreover, the generated electrical power of a TEG depends not only on these operating conditions, but also on the contact thermal resistance. The influence of the contact thermal resistance on the generated electrical power have already been extensively reported in the literature. However, as reported in Park et al. (Energy Convers Manag 86:233, 2014) and Montecucco and Knox (IEEE Trans Power Electron 30:828, 2015), while designing TEG-powered circuit and systems, a TEG module is mostly modeled with a Thévenin equivalent circuit whose resistance is constant and voltage proportional to the temperature gradient applied to the TEG's terminals. This widely used simplified electrical TEG model is inaccurate and not suitable under constant heat flow conditions or when the contact thermal resistance is considered. Moreover, it does not provide realistic behaviour corresponding to the physical phenomena taking place in a TEG. Therefore, from the circuit designer's point of view, faithful and fully electrical TEG models under different operating conditions are needed. Such models are mainly necessary to design and evaluate the power conditioning electronic stages and the maximum power point tracking algorithms of a TEG power supply. In this study, these fully electrical models with the contact thermal resistance taken into account are presented and the analytical expressions of the Thévenin equivalent circuit parameters are provided.

  12. Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.

    Science.gov (United States)

    Novak, Atara; Barad, Lili; Lorber, Avraham; Gherghiceanu, Mihaela; Reiter, Irina; Eisen, Binyamin; Eldor, Liron; Itskovitz-Eldor, Joseph; Eldar, Michael; Arad, Michael; Binah, Ofer

    2015-08-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca(2+) handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation-contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca(²+) abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2(R420Q) and CASQ2(D307H) mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca(2+) ]i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca(2+) ]i . Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.

  13. Conditional mutations in Drosophila melanogaster: On the occasion of the 150th anniversary of G. Mendel's report in Brünn.

    Science.gov (United States)

    Chadov, Boris F; Fedorova, Nina B; Chadova, Eugenia V

    2015-01-01

    The basis for modern genetics was laid by Gregor Mendel. He proposed that traits belonging to the intraspecific variability class be studied. However, individuals of one species possess traits of another class. They are related to intraspecific similarity. Individuals never differ from each other in these traits. By analogy with traits varying within a species and determined by genes, it is conjectured that intraspecific similarity is determined by genes, too. If so, mutations in these genes can be obtained. This paper provides a review of works published in 2000-2014 that: (1) propose breeding methods for detection of mutations in Drosophila melanogaster genes that lead intraspecific similarity; these mutations were called conditional; (2) describe collections of conditional mutations in chromosomes X, 2, and 3 of Drosophila; (3) show unusual features of epigenetic nature in the mutants; and (4) analyze these features of the mutants. Based on the peculiarities of manifestation it is supposed that the recognized conditional mutations occur in genes responsible for intraspecific similarity. The genes presumably belong to the so-called regulatory network of the Drosophila genome. This approach expands the scope of breeding analysis introduced by G. Mendel for heredity studies 150 years ago. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. A knockin mouse model for human ATP4aR703C mutation identified in familial gastric neuroendocrine tumors recapitulates the premalignant condition of the human disease and suggests new therapeutic strategies

    Directory of Open Access Journals (Sweden)

    Oriol Calvete

    2016-09-01

    Full Text Available By whole exome sequencing, we recently identified a missense mutation (p.R703C in the human ATP4a gene, which encodes the proton pump responsible for gastric acidification. This mutation causes an aggressive familial type I gastric neuroendocrine tumor in homozygous individuals. Affected individuals show an early onset of the disease, characterized by gastric hypoacidity, hypergastrinemia, iron-deficiency anemia, gastric intestinal metaplasia and, in one case, an associated gastric adenocarcinoma. Total gastrectomy was performed as the definitive treatment in all affected individuals. We now describe the generation and characterization of a knockin mouse model for the ATP4aR703C mutation to better understand the tumorigenesis process. Homozygous mice recapitulated most of the phenotypical alterations that were observed in human individuals, strongly suggesting that this mutation is the primary alteration responsible for disease development. Homozygous mice developed premalignant condition with severe hyperplasia, dysplasia and glandular metaplasia in the stomach. Interestingly, gastric acidification in homozygous mice, induced by treatment with 3% HCl acid in the drinking water, prevented (if treated from birth or partially reverted (if treated during adulthood the development of glandular metaplasia and dysplasia in the stomach and partially rescued the abnormal biochemical parameters. We therefore suggest that, in this model, achlorhydria contributes to tumorigenesis to a greater extent than hypergastrinemia. Furthermore, our mouse model represents a unique and novel tool for studying the pathologies associated with disturbances in gastric acid secretion.

  15. Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific homozygous mutation in CHMP2B

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2016-07-01

    Full Text Available Frontotemporal dementia (FTD is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B on chromosome 3 (FTD3, a component of the endosomal sorting complex required for transport III (ESCRT-III. We have generated an induced pluripotent stem cell (iPSC line of a healthy individual and inserted the CHMP2B IVS5AS G-C gene mutation into both alleles, resulting in aberrant splicing. This human iPSC line provides an ideal model to study CHMP2B-dependent phenotypes of FTD3.

  16. Generation of induced pluripotent stem cells (iPSCs from a retinoblastoma patient carrying a c.2663G>A mutation in RB1 gene

    Directory of Open Access Journals (Sweden)

    Sicong Zeng

    2016-09-01

    Full Text Available Skin fibroblasts were obtained from a male patient diagnosed with retinoblastoma (RB carrying a c.2663G>A mutation in the 25 exon of RB1 gene. RB-iPS cells was generated via delivered four reprogramming factors (OCT4, SOX2, NANOG and LIN28 into these skin fibroblasts. The RB-iPS cells retained the RB1 heterozygous mutation resulted in a truncated RB1 mRNA. Characteristic tests proved that the iPSC line presented typical markers of pluripotency and had the capability to form the three germ layers in vitro.

  17. Distinguishing HIV-1 drug resistance, accessory, and viral fitness mutations using conditional selection pressure analysis of treated versus untreated patient samples

    Directory of Open Access Journals (Sweden)

    Lee Christopher

    2006-05-01

    Full Text Available Abstract Background HIV can evolve drug resistance rapidly in response to new drug treatments, often through a combination of multiple mutations 123. It would be useful to develop automated analyses of HIV sequence polymorphism that are able to predict drug resistance mutations, and to distinguish different types of functional roles among such mutations, for example, those that directly cause drug resistance, versus those that play an accessory role. Detecting functional interactions between mutations is essential for this classification. We have adapted a well-known measure of evolutionary selection pressure (Ka/Ks and developed a conditional Ka/Ks approach to detect important interactions. Results We have applied this analysis to four independent HIV protease sequencing datasets: 50,000 clinical samples sequenced by Specialty Laboratories, Inc.; 1800 samples from patients treated with protease inhibitors; 2600 samples from untreated patients; 400 samples from untreated African patients. We have identified 428 mutation interactions in Specialty dataset with statistical significance and we were able to distinguish primary vs. accessory mutations for many well-studied examples. Amino acid interactions identified by conditional Ka/Ks matched 80 of 92 pair wise interactions found by a completely independent study of HIV protease (p-value for this match is significant: 10-70. Furthermore, Ka/Ks selection pressure results were highly reproducible among these independent datasets, both qualitatively and quantitatively, suggesting that they are detecting real drug-resistance and viral fitness mutations in the wild HIV-1 population. Conclusion Conditional Ka/Ks analysis can detect mutation interactions and distinguish primary vs. accessory mutations in HIV-1. Ka/Ks analysis of treated vs. untreated patient data can distinguish drug-resistance vs. viral fitness mutations. Verification of these results would require longitudinal studies. The result

  18. Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

    Science.gov (United States)

    Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  19. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP and generation of a mutant library with diverse phenotypes.

    Directory of Open Access Journals (Sweden)

    Mingyue Fang

    Full Text Available In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

  20. Evaluation of power conditioning architectures for energy production enhancement in thermoelectric generator systems

    DEFF Research Database (Denmark)

    Wu, Hongfei; Sun, Kai; Chen, Min

    2014-01-01

    A large-scale thermoelectric generator (TEG) system has an unbalanced temperature distribution among the TEG modules, which leads to power mismatch among the modules and decreases the power output of the TEG system. To maximize the power output and minimize the power conversion loss, a centralized......- distributed hybrid power conditioning architecture is presented, analyzed, and evaluated for a TEG system. The novel architecture is a combination of a conventional centralized architecture and a fully distributed architecture. By using the proposed architecture, most of the harvested power is processed...... of implementing high MPPT efficiency and high conversion efficiency simultaneously. A 50-W TEG system composed of two TEG modules is built and tested. Experimental results show that the proposed hybrid power conditioning architecture generates up to 5% more energy for a temperature difference between the two...

  1. Prediction of structural integrity of steam generator tubes under severe accident conditions

    Energy Technology Data Exchange (ETDEWEB)

    Majumdar, S. [Argonne National Lab., IL (United States)

    1999-11-01

    Available models for predicting failure of flawed and unflawed steam generator tubes under normal operating and design-basis accident conditions are reviewed. These rate-independent flow stress models are inadequate for predicting failure of steam generator tubes under severe accident conditions because the temperature of the tubes during such accidents can reach as high as 800 C where creep effects become important. Therefore, a creep rupture model for predicting failure was developed and validated by tests on unflawed and flawed specimens containing axial and circumferential flaws and loaded by constant as well as ramped temperature and pressure loadings. Finally, tests were conducted using pressure and temperature histories that are calculated to occur during postulated severe accidents. In all cases, the creep rupture model predicted the failure temperature and time more accurately than the flow stress models. (orig.)

  2. Design and Generation of MLPA Probe Sets for Combined Copy Number and Small-Mutation Analysis of Human Genes: EGFR as an Example

    Directory of Open Access Journals (Sweden)

    Malgorzata Marcinkowska

    2010-01-01

    Full Text Available Multiplex ligation-dependent probe amplification (MLPA is a multiplex copy number analysis method that is routinely used to identify large mutations in many clinical and research labs. One of the most important drawbacks of the standard MLPA setup is a complicated, and therefore expensive, procedure of generating long MLPA probes. This drawback substantially limits the applicability of MLPA to those genomic regions for which ready-to-use commercial kits are available. Here we present a simple protocol for designing MLPA probe sets that are composed entirely of short oligonucleotide half-probes generated through chemical synthesis. As an example, we present the design and generation of an MLPA assay for parallel copy number and small-mutation analysis of the EGFR gene.

  3. Performance Characterization and Validation of Saliva as an Alternative Specimen Source for Detecting Hereditary Breast Cancer Mutations by Next Generation Sequencing

    Science.gov (United States)

    Nahire, Rahul

    2016-01-01

    Identification of pathogenic germline mutations by next generation sequencing is a widely accepted tool for predicting the risk of hereditary cancer development. Blood is the most common source of DNA for such tests. However, blood as a sample type has many drawbacks, including the invasive collection method, poor sample stability, and a relatively high cost of collection. Therefore, in the current study we have assessed the suitability of saliva as an alternative source of genomic DNA for the identification of germline mutations in the BRCA1/2 genes by next generation sequencing (NGS). Our results show that all of the samples yielded DNA concentrations sufficient for library preparation. The concentrations of the final libraries, which were generated by PCR using target specific primers, fall into the expected range with no notable difference between libraries generated from DNA derived from saliva or blood. Quality parameters indicate that sequencing performance is comparable across sample source. An average of (98 ± 0.02)% variant calling concordance was obtained between the two specimen sources. Our data recommends saliva as a potential alternative for detecting germline mutation by next generation sequencing.

  4. Inhibition of sulfide generation by dosing formaldehyde and its derivatives in sewage under anaerobic conditions.

    Science.gov (United States)

    Zhang, L; Mendoza, L; Marzorati, M; Verstraete, W

    2008-01-01

    Hydrogen sulfide emission in sewers is associated with toxicity, corrosion, odor nuisance and a lot of costs. The possibility to inhibit sulfide generation by formaldehyde and its derivatives (paraformaldehyde and urea formaldehyde) has been evaluated under anaerobic conditions. The impact of formaldehyde on an activated sludge system and an appraisal of the economic aspects are also presented. The optimum dosage to inhibit sulfide generation in sewage was 12-19 mg L(-1) formaldehyde. The dosages of 32 mg L(-1) paraformaldehyde or 100 mg L(-1) urea formaldehyde were not capable of inhibiting sulfide generation in sewage. The impact of 19 mg L(-1) formaldehyde on activated sludge system was negligible in terms of COD removal, nitrification rate and oxygen uptake rate.

  5. Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing

    DEFF Research Database (Denmark)

    Rockenbauer, Eszter; Hansen, Stine; Mikkelsen, Martin

    2014-01-01

    . In 18 of the confirmed trios, a genetic inconsistency was observed between one of the parents and the child at the D21S11 locus. NGS of the D21S11 locus revealed which allele had mutated from which parent to the child in 13 of these trios. All characterized mutations could be explained by single...

  6. Human aging and somatic point mutations in mtDNA: a comparative study of generational differences (grandparents and grandchildren

    Directory of Open Access Journals (Sweden)

    Anderson Nonato do Rosário Marinho

    2011-01-01

    Full Text Available The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years and their 62 grandchildren (mean age: 15 ± 4.1 years, the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old mutations (homoplasia and heteroplasmy. It is possible that both of these situations (homoplasia and heteroplasmy were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.

  7. An assessment of the geotemperature conditions of Bazhenov oil generation (Koltogor mezodepression and its framing structures)

    Science.gov (United States)

    Stotskiy, V. V.; Isaev, V. I.; Fomin, M. A.

    2016-09-01

    The thermal history of oil source rocks of Bazhenov deposits was reconstructed in 8 cross-sections of representative wells of Koltogor mezodepression and the structures framing it. The tectonic history and geotemperature were reconstructed for well profiles, some located in the depression zone and others in the positive structures. A comparative analysis of the geotemperature conditions accompanying the generation of Bazhenov oils was performed.

  8. Conditions for Multi-functionality in a Rhythm Generating Network Inspired by Turtle Scratching.

    Science.gov (United States)

    Snyder, Abigail C; Rubin, Jonathan E

    2015-12-01

    Rhythmic behaviors such as breathing, walking, and scratching are vital to many species. Such behaviors can emerge from groups of neurons, called central pattern generators, in the absence of rhythmic inputs. In vertebrates, the identification of the cells that constitute the central pattern generator for particular rhythmic behaviors is difficult, and often, its existence has only been inferred. For example, under experimental conditions, intact turtles generate several rhythmic scratch motor patterns corresponding to non-rhythmic stimulation of different body regions. These patterns feature alternating phases of motoneuron activation that occur repeatedly, with different patterns distinguished by the relative timing and duration of activity of hip extensor, hip flexor, and knee extensor motoneurons. While the central pattern generator network responsible for these outputs has not been located, there is hope to use motoneuron recordings to deduce its properties. To this end, this work presents a model of a previously proposed central pattern generator network and analyzes its capability to produce two distinct scratch rhythms from a single neuron pool, selected by different combinations of tonic drive parameters but with fixed strengths of connections within the network. We show through simulation that the proposed network can achieve the desired multi-functionality, even though it relies on hip unit generators to recruit appropriately timed knee extensor motoneuron activity, including a delay relative to hip activation in rostral scratch. Furthermore, we develop a phase space representation, focusing on the inputs to and the intrinsic slow variable of the knee extensor motoneuron, which we use to derive sufficient conditions for the network to realize each rhythm and which illustrates the role of a saddle-node bifurcation in achieving the knee extensor delay. This framework is harnessed to consider bistability and to make predictions about the responses of the

  9. Analyses of an air conditioning system with entropy generation minimization and entransy theory

    Science.gov (United States)

    Yan-Qiu, Wu; Li, Cai; Hong-Juan, Wu

    2016-06-01

    In this paper, based on the generalized heat transfer law, an air conditioning system is analyzed with the entropy generation minimization and the entransy theory. Taking the coefficient of performance (denoted as COP) and heat flow rate Q out which is released into the room as the optimization objectives, we discuss the applicabilities of the entropy generation minimization and entransy theory to the optimizations. Five numerical cases are presented. Combining the numerical results and theoretical analyses, we can conclude that the optimization applicabilities of the two theories are conditional. If Q out is the optimization objective, larger entransy increase rate always leads to larger Q out, while smaller entropy generation rate does not. If we take COP as the optimization objective, neither the entropy generation minimization nor the concept of entransy increase is always applicable. Furthermore, we find that the concept of entransy dissipation is not applicable for the discussed cases. Project supported by the Youth Programs of Chongqing Three Gorges University, China (Grant No. 13QN18).

  10. Condition based maintenance optimization for wind power generation systems under continuous monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Zhigang; Wu, Bairong; Ding, Fangfang [Concordia Institute for Information Systems Engineering, Concordia University, 1515 Ste-Catherine Street, West EV-7.637, Montreal (Canada); Jin, Tongdan [Ingram School of Engineering, Texas State University (United States)

    2011-05-15

    By utilizing condition monitoring information collected from wind turbine components, condition based maintenance (CBM) strategy can be used to reduce the operation and maintenance costs of wind power generation systems. The existing CBM methods for wind power generation systems deal with wind turbine components separately, that is, maintenance decisions are made on individual components, rather than the whole system. However, a wind farm generally consists of multiple wind turbines, and each wind turbine has multiple components including main bearing, gearbox, generator, etc. There are economic dependencies among wind turbines and their components. That is, once a maintenance team is sent to the wind farm, it may be more economical to take the opportunity to maintain multiple turbines, and when a turbine is stopped for maintenance, it may be more cost-effective to simultaneously replace multiple components which show relatively high risks. In this paper, we develop an optimal CBM solution to the above-mentioned issues. The proposed maintenance policy is defined by two failure probability threshold values at the wind turbine level. Based on the condition monitoring and prognostics information, the failure probability values at the component and the turbine levels can be calculated, and the optimal CBM decisions can be made accordingly. A simulation method is developed to evaluate the cost of the CBM policy. A numerical example is provided to illustrate the proposed CBM approach. A comparative study based on commonly used constant-interval maintenance policy demonstrates the advantage of the proposed CBM approach in reducing the maintenance cost. (author)

  11. Effects of microbial processes on gas generation under expected WIPP repository conditions: Annual report through 1992

    Energy Technology Data Exchange (ETDEWEB)

    Francis, A.J.; Gillow, J.B.

    1993-09-01

    Microbial processes involved in gas generation from degradation of the organic constituents of transuranic waste under conditions expected at the Waste Isolation Pilot Plant (WIPP) repository are being investigated at Brookhaven National Laboratory. These laboratory studies are part of the Sandia National Laboratories -- WIPP Gas Generation Program. Gas generation due to microbial degradation of representative cellulosic waste was investigated in short-term (< 6 months) and long-term (> 6 months) experiments by incubating representative paper (filter paper, paper towels, and tissue) in WIPP brine under initially aerobic (air) and anaerobic (nitrogen) conditions. Samples from the WIPP surficial environment and underground workings harbor gas-producing halophilic microorganisms, the activities of which were studied in short-term experiments. The microorganisms metabolized a variety of organic compounds including cellulose under aerobic, anaerobic, and denitrifying conditions. In long-term experiments, the effects of added nutrients (trace amounts of ammonium nitrate, phosphate, and yeast extract), no nutrients, and nutrients plus excess nitrate on gas production from cellulose degradation.

  12. C.57 C > T Mutation in MIR 184 is Responsible for Congenital Cataracts and Corneal Abnormalities in a Five-generation Family from Galicia, Spain.

    Science.gov (United States)

    Bykhovskaya, Yelena; Caiado Canedo, Ana L; Wright, Kenneth W; Rabinowitz, Yaron S

    2015-01-01

    A c.57 C > T mutation in the seed region of MIR184 located at the 15q25.1 chromosomal region has been independently associated with autosomal dominant keratoconus with early-onset anterior polar cataract in the Northern Irish family and with autosomal dominant EDICT (Endothelial Dystrophy, Iris hypoplasia, Congenital cataracts, and stromal Thinning) syndrome. In this study we report a five-generation family originating in Galicia, Spain with early onset cataracts and variable corneal abnormalities which include non-ectatic corneal thinning and severe early-onset keratoconus. We identified a heterozygous c.57 C > T mutation in miR-184 in the proband and two additional affected relatives on the maternal side. This finding represents a third independent occurrence of this mutation in familiar ocular disease thus strengthening the link between miR-184 abnormalities and inherited eye defects.

  13. A method for aggregating external operating conditions in multi-generation system optimization models

    DEFF Research Database (Denmark)

    Lythcke-Jørgensen, Christoffer Ernst; Münster, Marie; Ensinas, Adriano Viana

    2016-01-01

    This paper presents a novel, simple method for reducing external operating condition datasets to be used in multi-generation system optimization models. The method, called the Characteristic Operating Pattern (CHOP) method, is a visually-based aggregation method that clusters reference data based...... on parameter values rather than time of occurrence, thereby preserving important information on short-term relations between the relevant operating parameters. This is opposed to commonly used methods where data are averaged over chronological periods (months or years), and extreme conditions are hidden...... in the averaged values. The CHOP method is tested in a case study where the operation of a fictive Danish combined heat and power plant is optimized over a historical 5-year period. The optimization model is solved using the full external operating condition dataset, a reduced dataset obtained using the CHOP...

  14. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Directory of Open Access Journals (Sweden)

    Giulia Breveglieri

    2015-01-01

    Full Text Available Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6 carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a the transgenic integration region is located in mouse chromosome 7; (b the expression of the transgene is tissue specific; (c as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin αmu-globin2/βhu-globin2 and, more importantly, (d the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia.

  15. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Science.gov (United States)

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  16. Phenotypic variability in a seven-generation Swedish family segregating autosomal dominant hearing impairment due to a novel EYA4 frameshift mutation.

    Science.gov (United States)

    Frykholm, Carina; Klar, Joakim; Arnesson, Hanna; Rehnman, Anna-Carin; Lodahl, Marianne; Wedén, Ulla; Dahl, Niklas; Tranebjærg, Lisbeth; Rendtorff, Nanna D

    2015-05-25

    Linkage to an interval overlapping the DFNA10 locus on chromosome 6q22-23 was found through genome wide linkage analysis in a seven-generation Swedish family segregating postlingual, autosomal dominant nonsyndromic sensorineural hearing impairment. A novel heterozygous frame-shift mutation (c.579_580insTACC, p.(Asp194Tyrfs*52)) in EYA4 was identified that truncates the so-called variable region of the protein. The mutation is predicted to result in haploinsufficiency of the EYA4 product. No evidence for dilated cardiomyopathy was found in the family, contrasting to a previous family with a deletion resulting in a similar truncation in the variable region. A highly variable age of onset was seen in the mutation carriers. For assessment of the aetiology of this variability, clinical and audiometric data analyses were performed. The affected family members all had similar cross-sectional and longitudinal deterioration of pure tone average (PTA) once the process of hearing deterioration had started, and no gender, parent-of-origin or family branch differences on PTA could be found. Age at onset varied between the family branches. In summary, this is the ninth published genetically verified DFNA10 family. The results imply that unidentified factors, genetic or environmental, other than the EYA4 mutation, are of importance for the age at onset of DFNA10, and that mutation early in the variable region of the EYA4 protein can occur in the absence of dilated cardiomyopathy.

  17. Whacking a mole-cule: clinical activity and mechanisms of resistance to third generation EGFR inhibitors in EGFR mutated lung cancers with EGFR-T790M.

    Science.gov (United States)

    Costa, Daniel B; Kobayashi, Susumu S

    2015-12-01

    Epidermal growth factor receptor (EGFR) mutations, especially EGFR-exon 19 deletions and EGFR-L858R, are the most frequent actionable genomic events in lung adenocarcinomas. Tumors arise due to constitutively activated EGFR signaling and are susceptible to EGFR tyrosine kinase inhibitors (TKIs). First generation EGFR TKIs (gefitinib and erlotinib) and the second generation EGFR TKI afatinib are approved worldwide. Although targeted therapies against EGFR mutants induce dramatic initial responses, acquired resistance (through multiple biological mechanisms) to erlotinib, gefitinib and afatinib emerges within the first 1-2 years of continued monotherapy. EGFR-T790M accounts for more than half of acquired resistance to first or second generation EGFR TKIs by modifying ATP affinity and drug binding kinetics. Two new studies have shown that two covalent pyrimidine inhibitors-AZD9291 and rociletinib of EGFR-T790M (i.e., third generation EGFR TKIs) shown remarkable clinical activity in patients with acquired resistance to erlotinib, gefitinib and afatinib when the tumor carries EGFR-T790M in conjunction with an activating mutation. However, and regrettably, acquired resistance to these third generation EGFR TKIs has already been reported in preclinical models and clinical specimens; such as a tertiary mutation at EGFR-C797S that prevents covalent binding of EGFR TKIs. The experience with sequential EGFR TKI monotherapy highlights tumor heterogeneity and adaptability (i.e., relentless game of whack-a-mole played between TKIs and cancer), and will help shape future clinical development of novel combinatory approaches to manage EGFR mutated lung adenocarcinomas.

  18. Optimum thermal sizing and operating conditions for once through steam generator

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Kunwoo; Ju, Kyongin; Im, Inyoung; Kim, Eunkee [KEPCO Engineering and Construction Company., Inc., Daejeon (Korea, Republic of)

    2014-10-15

    The steam generator is designed to be optimized so as to remove heat and to produce steam vapor. Because of its importance, theoretical and experimental researches have been performed on forced convection boiling heat transfer. The purpose of this study is to predict the thermal behavior and to perform optimum thermal sizing of once through steam generator. To estimate the tube thermal sizing and operating conditions of the steam generator, the analytical modeling is employed on the basis of the empirical correlation equations and theory. The optimized algorithm model, Non-dominated Sorting Genetic Algorithm (NSGA)-II, uses for this analysis. This research is focused on the design of in-vessel steam generator. An one dimensional analysis code is developed to evaluate previous researches and to optimize steam generator design parameters. The results of one-dimensional analysis need to be verified with experimental data. Goals of multi-objective optimization are to minimize tube length, pressure drop and tube number. Feedwater flow rate up to 115.425kg/s is selected so as to have margin of feedwater temperature 20 ..deg. C. For the design of 200MWth once through steam generator, it is evaluated that the tube length shall be over 12.0m for the number of tubes, 2500ea, and the length of the tube shall be over 8.0m for the number of tubes, 4500ea. The parallel coordinates chart can be provided to determine the optimal combination of number of tube, pressure drop, tube diameter and length.

  19. A three generation X-linked family with Kabuki syndrome phenotype and a frameshift mutation in KDM6A.

    Science.gov (United States)

    Lederer, Damien; Shears, Debbie; Benoit, Valérie; Verellen-Dumoulin, Christine; Maystadt, Isabelle

    2014-05-01

    Kabuki syndrome is a rare malformation syndrome characterized by a typical facial appearance, skeletal anomalies, cardiac malformation, and mild to moderate intellectual disability. In 55-80% of patients with Kabuki syndrome, a mutation in MLL2 is identified. Recently, eight patients with Kabuki syndrome and a mutation in KDM6A were described. In this report, we describe two brothers with a mutation in KDM6A inherited from their mother and maternal grandmother. The two boys have Kabuki-like phenotypes whereas the mother and grandmother present with attenuated phenotypes. This family represents the first instance of hereditary X-linked Kabuki syndrome. We present a short literature review of the patients described with a mutation in KDM6A.

  20. Nebulin (NEB) mutations in a childhood onset distal myopathy with rods and cores uncovered by next generation sequencing

    OpenAIRE

    Scoto, Mariacristina; Cullup, Thomas; Cirak, Sebahattin; Yau, Shu; Manzur, Adnan Y.; Feng, Lucy; Jacques, Thomas S; Anderson, Glenn; Abbs, Stephen; Sewry, Caroline; Jungbluth, Heinz; Muntoni, Francesco

    2013-01-01

    Recessive nebulin (NEB) mutations are a common cause of nemaline myopathy (NM), typically characterized by generalized weakness of early-onset and nemaline rods on muscle biopsy. Exceptional adult cases with additional cores and an isolated distal weakness have been reported. The large NEB gene with 183 exons has been an obstacle for the genetic work-up. Here we report a childhood-onset case with distal weakness and a core-rod myopathy, associated with recessive NEB mutations identified by ne...

  1. Conditions for Debris-Background Ion Interactions and Collisionless Shock Wave Generation

    Energy Technology Data Exchange (ETDEWEB)

    Winske, Dan [Los Alamos National Laboratory; Cowee, Misa [Los Alamos National Laboratory

    2012-07-10

    We use hybrid simulations and simple theoretical arguments to determine when debris ions streaming relative to background ions in a collisionless, magnetized plasma couple strongly enough to generate a magnetosonic shock wave. We consider three types of configurations: one-dimensional, the two-dimensional extension of the 1-D case, and a more complex 2-D geometry that contains some effects that would be found in a laser-produced, laboratory plasma. We show that the simulation results as well as previous Russian and LLNL results reduce to a simple condition (R{sub m}/{rho}{sub d} = equal mass radius/debris ion gyroradius {ge} 0.7) for the generation of a shock wave. Strong debris interaction with the background is characterized by the formation of a magnetic pulse that steepens and speeds up as it encounters the debris ions deflected by the magnetic field. The pulse further evolves into a shock. As the earlier work has indicated, the process also involves the generation of a transverse electric field perpendicular to the flow and the magnetic field that accelerates the background ions radially outward, which in turn causes the speedup of the pulse. With electric and magnetic field probes, the UCLA laser experiments should be able to detect these signatures of coupling as well as the generation of the shock wave.

  2. Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease patient iPSC line carrying a A79V mutation in PSEN1

    DEFF Research Database (Denmark)

    Pires, Carlota; Schmid, Benjamin; Petræus, Carina

    2016-01-01

    Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease causing neural cell degeneration and brain atrophy and is considered to be the most common form of dementia. We previously generated an induced pluripotent stem cell (iPSC) line from an AD patient carrying an A79V...... mutation in PSEN1 as an in vitro disease model. Here we generated a gene-corrected version from this hiPSC line by substituting the point mutation with the wild-type sequence. The reported A79V-GC-iPSCs line is a very useful resource in combination with the A79V-iPSC line in order to study pathological...

  3. Novel mutations in NEB cause abnormal nebulin expression and markedly impaired muscle force generation in severe nemaline myopathy

    Directory of Open Access Journals (Sweden)

    Lawlor Michael W

    2011-06-01

    Full Text Available Abstract Background Nemaline myopathy (NM is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB, which is thought to account for roughly 50% of cases. Results We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost. Conclusions Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.

  4. Microbial Gas Generation Under Expected Waste Isolation Pilot Plant Repository Conditions: Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Gillow, J.B.; Francis, A.

    2011-07-01

    Gas generation from the microbial degradation of the organic constituents of transuranic (TRU) waste under conditions expected in the Waste Isolation Pilot Plant (WIPP) was investigated. The biodegradation of mixed cellulosic materials and electron-beam irradiated plastic and rubber materials (polyethylene, polyvinylchloride, hypalon, leaded hypalon, and neoprene) was examined. We evaluated the effects of environmental variables such as initial atmosphere (air or nitrogen), water content (humid ({approx}70% relative humidity, RH) and brine inundated), and nutrient amendments (nitogen phosphate, yeast extract, and excess nitrate) on microbial gas generation. Total gas production was determined by pressure measurement and carbon dioxide (CO{sub 2}) and methane (CH{sub 4}) were analyzed by gas chromatography; cellulose degradation products in solution were analyzed by high-performance liquid chromatography. Microbial populations in the samples were determined by direct microscopy and molecular analysis. The results of this work are summarized.

  5. Development of a system for simultaneously generating triple extreme conditions for neutron scattering experiments

    Energy Technology Data Exchange (ETDEWEB)

    Ichimura, Shigeju [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-10-01

    We have developed new system available for controlling sample environment during the neutron scattering experiments. The system can simultaneously generate triple extreme conditions of low temperature, high magnetic field and high pressure. The system consists of : (1) a liquid-helium cryostat which enables the sample temperature range of 1.7 K to 200 K, (2) a superconducting magnet providing a vertical field up to 5 Tesla with antisymmetric split-coil geometry for polarized-beam experiments, and (3) a non-magnetic clamping high-pressure cell designed with the aim of generating hydrostatic pressure up to 2.5 Gpa. In the workshop, we will report the outline of the system and some results of performance tests using the system at JRR-3M of JAERI. (author)

  6. Hybrid centralized-distributed power conditioning system for thermoelectric generator with high energy efficiency

    DEFF Research Database (Denmark)

    Wu, Hongfei; Sun, Kai; Chen, Min;

    2013-01-01

    The unbalanced temperature distribution influences the power output of thermoelectric generator (TEG) system, which leads to mismatch power among TEG modules. This mismatch power degrades the energy efficiency of TEG systems based on the series-connected TEG modules. A hybrid centralized-distribu......The unbalanced temperature distribution influences the power output of thermoelectric generator (TEG) system, which leads to mismatch power among TEG modules. This mismatch power degrades the energy efficiency of TEG systems based on the series-connected TEG modules. A hybrid centralized...... the proposed system, which benefits for implementing high MPPT efficiency and high conversion efficiency simultaneously. A hybrid MPPT control strategy is proposed for this HCD power conditioning system. The characteristics, circuit implementation and operation principles of the proposed system are presented...

  7. Decreasing sulfide generation in sewage by dosing formaldehyde and its derivatives under anaerobic conditions.

    Science.gov (United States)

    Zhang, L; De Gusseme, B; De Schryver, P; Mendoza, L; Marzorati, M; Verstraete, W

    2009-01-01

    Hydrogen sulfide emission in sewers is associated with toxicity, corrosion, odour nuisance and high costs. In this study, a new method to inhibit sulfide generation by means of formaldehyde and its derivatives has been evaluated under anaerobic conditions. The possible impact of formaldehyde on an activated sludge system and an appraisal of the economic aspects are presented as well. A dosage of 19 mg L(-1) formaldehyde resulted in a decrease of the sulfide production of 90%. Dosing of 32 mg L(-1) paraformaldehyde and addition of 111 mg L(-1) ureaformaldehyde were not sufficient to inhibit the sulfide generation in sewage to the same extent. The impacts of 19 mg L(-1) formaldehyde on activated sludge, in terms of COD removal, nitrification rate and oxygen uptake rates, were negligible. This suggests that formaldehyde dosage is a feasible technique to abate the sulfide problem in sewers.

  8. Bubble Generation in a Continuous Liquid Flow Under Reduced Gravity Conditions

    Science.gov (United States)

    Pais, Salvatore Cezar

    1999-01-01

    The present work reports a study of bubble generation under reduced gravity conditions for both co-flow and cross-flow configurations. Experiments were performed aboard the DC-9 Reduced Gravity Aircraft at NASA Glenn Research Center, using an air-water system. Three different flow tube diameters were used: 1.27, 1.9, and 2.54 cm. Two different ratios of air injection nozzle to tube diameters were considered: 0.1 and 0.2. Gas and liquid volumetric flow rates were varied from 10 to 200 ml/s. It was experimentally observed that with increasing superficial liquid velocity, the bubbles generated decreased in size. The bubble diameter was shown to increase with increasing air injection nozzle diameters. As the tube diameter was increased, the size of the detached bubbles increased. Likewise, as the superficial liquid velocity was increased, the frequency of bubble formation increased and thus the time to detach forming bubbles decreased. Independent of the flow configuration (for either single nozzle or multiple nozzle gas injection), void fraction and hence flow regime transition can be controlled in a somewhat precise manner by solely varying the gas and liquid volumetric flow rates. On the other hand, it is observed that uniformity of bubble size can be controlled more accurately by using single nozzle gas injection than by using multiple port injection, since this latter system gives rise to unpredictable coalescence of adjacent bubbles. A theoretical model, based on an overall force balance, is employed to study single bubble generation in the dynamic and bubbly flow regime. Under conditions of reduced gravity, the gas momentum flux enhances bubble detachment; however, the surface tension forces at the nozzle tip inhibits bubble detachment. Liquid drag and inertia can act either as attaching or detaching force, depending on the relative velocity of the bubble with respect to the surrounding liquid. Predictions of the theoretical model compare well with performed

  9. Robust Current Control of Doubly Fed Wind Turbine Generator under Unbalanced Grid Voltage Conditions

    DEFF Research Database (Denmark)

    Wang, Yun; Gong, Wenming; Wu, Qiuwei

    2014-01-01

    This paper presents the design of a H ∞ current controller for doubly fed induction generators (DFIGs) in order to maintain stable operation under unbalanced voltage conditions. The H ∞ current controller has a multi-input and multi-output (MIMO) structure and is designed using the loop shaping...... method. Case studies have been carried out in order to verify the efficacy of the proposed H ∞ current controller for DFIGs. The case study results show that the proposed H ∞ current controller can realize different control objectives, i.e. stable stator current, stable stator active power and stable...

  10. Focus conditioning effects on molecular field-free alignment observed with high-order harmonic generation

    Institute of Scientific and Technical Information of China (English)

    吴家骏; 尉鹏飞

    2012-01-01

    We investigate the focus conditioning effects on molecular field-free alignment observed with high-order harmonic generation (HHG) from CO2 molecules.We also experimentally demonstrate that both the spectral shape and alignment signal of HHG significantly vary with changing focus position.A maximal alignment signal is achieved at a given focus position because of the optimal intensity of the driving laser.This intensity is related to the ionization potential of the molecules.These results indicate that a unique focus position provides an optimal alignment signal for practical applications.

  11. Application of quantitative second-harmonic generation microscopy to dynamic conditions.

    Science.gov (United States)

    Kabir, Mohammad M; Inavalli, V V G Krishna; Lau, Tung-Yuen; Toussaint, Kimani C

    2013-01-01

    We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.

  12. Enhancement of harmonics generation in hysteretic elastic media induced by conditioning

    Science.gov (United States)

    Mechri, C.; Scalerandi, M.; Bentahar, M.

    2017-04-01

    The physical origin of harmonics generation in non classical (hysteretic) elastic media and the mechanisms of energy transfer among harmonics are still not completely understood. Furthermore the well known conditioning effect observed in such materials is known to have a significant influence on the elastic response of consolidated granular media and damaged composites and metals. Here, we show that the elastic non linearity of samples belonging to these two categories increases after having been excited with a relatively low amplitude stress. The observed behaviours could be described by activation features intrinsically present in phenomenological multistate models proposed in the literature.

  13. A technique for generating consistent ice sheet initial conditions for coupled ice-sheet/climate models

    Directory of Open Access Journals (Sweden)

    J. G. Fyke

    2013-04-01

    Full Text Available A new technique for generating ice sheet preindustrial 1850 initial conditions for coupled ice-sheet/climate models is developed and demonstrated over the Greenland Ice Sheet using the Community Earth System Model (CESM. Paleoclimate end-member simulations and ice core data are used to derive continuous surface mass balance fields which are used to force a long transient ice sheet model simulation. The procedure accounts for the evolution of climate through the last glacial period and converges to a simulated preindustrial 1850 ice sheet that is geometrically and thermodynamically consistent with the 1850 preindustrial simulated CESM state, yet contains a transient memory of past climate that compares well to observations and independent model studies. This allows future coupled ice-sheet/climate projections of climate change that include ice sheets to integrate the effect of past climate conditions on the state of the Greenland Ice Sheet, while maintaining system-wide continuity between past and future climate simulations.

  14. A revised model for Jeffrey nanofluid subject to convective condition and heat generation/absorption.

    Science.gov (United States)

    Hayat, Tasawar; Aziz, Arsalan; Muhammad, Taseer; Alsaedi, Ahmed

    2017-01-01

    Here magnetohydrodynamic (MHD) boundary layer flow of Jeffrey nanofluid by a nonlinear stretching surface is addressed. Heat generation/absorption and convective surface condition effects are considered. Novel features of Brownian motion and thermophoresis are present. A non-uniform applied magnetic field is employed. Boundary layer and small magnetic Reynolds number assumptions are employed in the formulation. A newly developed condition with zero nanoparticles mass flux is imposed. The resulting nonlinear systems are solved. Convergence domains are explicitly identified. Graphs are analyzed for the outcome of sundry variables. Further local Nusselt number is computed and discussed. It is observed that the effects of Hartman number on the temperature and concentration distributions are qualitatively similar. Both temperature and concentration distributions are enhanced for larger Hartman number.

  15. A power conditioning system for thermoelectric generator based on interleaved Boost converter with MPPT control

    DEFF Research Database (Denmark)

    Ni, L.-X; Sun, K.; Zhang, L.

    2011-01-01

    The thermoelectric generation (TEG) system has its special charactristics of high stablility, low voltage and high current output, which is different from PV modules. The power conditioning system and control schemes used in PV applications cannot be directly applied to TEG applications. A power...... conditioning system for TEG based on interleaved Boost converter with maximum power point tracking (MPPT) control is investigated in this paper. Since an internal resistance exists inside TEG modules, an improved perturbation and observation (P&O) MPPT control scheme with power limit is proposed to extract...... maximum power from TEG by matching the load with internal resistance. Since the battery is usually employed as the load for TEG systems, the interleaved Boost converter operates in two different modes for battery charging: before the battery is fully charged, the system outputs the maximum power (MPPT...

  16. How mutation affects evolutionary games on graphs.

    Science.gov (United States)

    Allen, Benjamin; Traulsen, Arne; Tarnita, Corina E; Nowak, Martin A

    2012-04-21

    Evolutionary dynamics are affected by population structure, mutation rates and update rules. Spatial or network structure facilitates the clustering of strategies, which represents a mechanism for the evolution of cooperation. Mutation dilutes this effect. Here we analyze how mutation influences evolutionary clustering on graphs. We introduce new mathematical methods to evolutionary game theory, specifically the analysis of coalescing random walks via generating functions. These techniques allow us to derive exact identity-by-descent (IBD) probabilities, which characterize spatial assortment on lattices and Cayley trees. From these IBD probabilities we obtain exact conditions for the evolution of cooperation and other game strategies, showing the dual effects of graph topology and mutation rate. High mutation rates diminish the clustering of cooperators, hindering their evolutionary success. Our model can represent either genetic evolution with mutation, or social imitation processes with random strategy exploration.

  17. Horizontal steam generator thermal hydraulic simulation in typical steady and transient conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rabiee, Ataollah, E-mail: rabiee@shirazu.ac.ir; Kamalinia, Amir Hossein; Haddad, Kamal

    2016-08-15

    Highlights: • Simulation of the horizontal steam generator with the available code in typical normal and transient operations. • Replacement of tube bundle with a porous media due to the complexity of the SG geometry. • Simulation of typical transient mode of the VVER 440 steam generator, loss of feed water accident. - Abstract: Thermal hydraulic analysis of the steam generators as one of the main components of the power cycle in pressurized water reactor (PWR) is crucial in the design and safety of the nuclear power plants. Two phase flow field simulation near the tube bundles is important in obtaining logical numerical results however the complexity of the tube bundles due to geometry and arrangement makes the numerical analysis complicated. In this research tube bundle has been assumed as the porous media and the outlet boundary condition as the one of the main challenge in these kind of simulations has been optimized according to similar researches. In order to adjust and tune the available computational fluid dynamic (CFD) code, pressure drop of the typical kettle reboiler tube bundle in two various heat fluxes and vapor volume fraction distribution in VVER 1000 steam generator in normal operation have been investigated. The typical transient mode of the VVER 440 steam generator, loss of feed water accident, has been studied eventually. It was observed that obtained vapor volume fraction can predict experimental data with more accuracy than the similar researches and would be increased with the elevation during the accident. On the other hand, pressure drop and level of the feed water value reduces through time and show good adoption with the measurements.

  18. Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

    Science.gov (United States)

    Zhang, Liangxuan; Chen, Liangjing; Sah, Sachin; Latham, Gary J; Patel, Rajesh; Song, Qinghua; Koeppen, Hartmut; Tam, Rachel; Schleifman, Erica; Mashhedi, Haider; Chalasani, Sreedevi; Fu, Ling; Sumiyoshi, Teiko; Raja, Rajiv; Forrest, William; Hampton, Garret M; Lackner, Mark R; Hegde, Priti; Jia, Shidong

    2014-04-01

    The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

  19. Conditions for the generation of beta oscillations in the subthalamic nucleus-globus pallidus network.

    Science.gov (United States)

    Holgado, Alejo J Nevado; Terry, John R; Bogacz, Rafal

    2010-09-15

    The advance of Parkinson's disease is associated with the existence of abnormal oscillations within the basal ganglia with frequencies in the beta band (13-30 Hz). While the origin of these oscillations remains unknown, there is some evidence suggesting that oscillations observed in the basal ganglia arise due to interactions of two nuclei: the subthalamic nucleus (STN) and the globus pallidus pars externa (GPe). To investigate this hypothesis, we develop a computational model of the STN-GPe network based upon anatomical and electrophysiological studies. Significantly, our study shows that for certain parameter regimes, the model intrinsically oscillates in the beta range. Through an analytical study of the model, we identify a simple set of necessary conditions on model parameters that guarantees the existence of beta oscillations. These conditions for generation of oscillations are described by a set of simple inequalities and can be summarized as follows: (1) The excitatory connections from STN to GPe and the inhibitory connections from GPe to STN need to be sufficiently strong. (2) The time required by neurons to react to their inputs needs to be short relative to synaptic transmission delays. (3) The excitatory input from the cortex to STN needs to be high relative to the inhibition from striatum to GPe. We confirmed the validity of these conditions via numerical simulation. These conditions describe changes in parameters that are consistent with those expected as a result of the development of Parkinson's disease, and predict manipulations that could inhibit the pathological oscillations.

  20. Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

    Science.gov (United States)

    Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L.; van Lier, Christina J.; Sha, Jian; Yeager, Linsey A.; Chopra, Ashok K.; Rao, Venigalla B.

    2013-01-01

    Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines. PMID:23853602

  1. Active fiber polymer cladding temperature measurement under conditions of laser generation and amplification

    Science.gov (United States)

    Sypin, V. E.; Prusakov, K. Y.; Ryabushkin, O. A.

    2016-04-01

    Polymer cladding temperature of active fiber in lasing regime is important parameter as it allows determination of fiber core temperature that in turn effects laser generation and amplification efficiency. Besides polymer cladding has much lower temperature damage threshold comparing to fused silica. For example, 200 degrees Kelvin overheating of the polymer cladding can result in fiber degradation. In present paper we introduce novel and simple method for precise temperature measurement of active fibers cladding under conditions of laser generation and amplification. Dependence of longitudinal temperature distribution along active fibers on optical pump power can be determined. This method employs measurement of temperature dependent electrical resistance of the metal wire being in thermal contact with fiber polymer cladding. The wire is reeled on the active fiber segment. Under lasing or amplification conditions the polymer cladding of the active fiber is heated together with coiled metal wire resulting in its electrical resistance change. By measuring resistance variation one can determine the temperature of the given fiber section.

  2. Generation of Turbulent Inflow Conditions for Pipe Flow via an Annular Ribbed Turbulator

    Science.gov (United States)

    Moallemi, Nima; Brinkerhoff, Joshua

    2016-11-01

    The generation of turbulent inflow conditions adds significant computational expense to direct numerical simulations (DNS) of turbulent pipe flows. Typical approaches involve introducing boxes of isotropic turbulence to the velocity field at the inlet of the pipe. In the present study, an alternative method is proposed that incurs a lower computational cost and allows the anisotropy observed in pipe turbulence to be physically captured. The method is based on a periodic DNS of a ribbed turbulator upstream of the inlet boundary of the pipe. The Reynolds number based on the bulk velocity and pipe diameter is 5300 and the blockage ratio (BR) is 0.06 based on the rib height and pipe diameter. The pitch ratio is defined as the ratio of rib streamwise spacing to rib height and is varied between 1.7 and 5.0. The generation of turbulent flow structures downstream of the ribbed turbulator are identified and discussed. Suitability of this method for accurate representation of turbulent inflow conditions is assessed through comparison of the turbulent mean properties, fluctuations, Reynolds stress profiles, and spectra with published pipe flow DNS studies. The DNS results achieve excellent agreement with the numerical and experimental data available in the literature.

  3. Influence of impact conditions on plasma generation during hypervelocity impact by aluminum projectile

    Science.gov (United States)

    Song, Weidong; Lv, Yangtao; Li, Jianqiao; Wang, Cheng; Ning, Jianguo

    2016-07-01

    For describing hypervelocity impact (relative low-speed as related to space debris and much lower than travelling speed of meteoroids) phenomenon associated with plasma generation, a self-developed 3D code was advanced to numerically simulate projectiles impacting on a rigid wall. The numerical results were combined with a new ionization model which was developed in an early study to calculate the ionized materials during the impact. The calculated results of ionization were compared with the empirical formulas concluded by experiments in references and a good agreement was obtained. Then based on the reliable 3D numerical code, a series of impacts with different projectile configurations were simulated to investigate the influence of impact conditions on hypervelocity impact generated plasma. It was found that the form of empirical formula needed to be modified. A new empirical formula with a critical impact velocity was advanced to describe the velocity dependence of plasma generation and the parameters of the modified formula were ensured by the comparison between the numerical predictions and the empirical formulas. For different projectile configurations, the changes of plasma charges with time are different but the integrals of charges on time almost stayed in the same level.

  4. Generation of mice harbouring a conditional loss-of-function allele of Gata6

    Directory of Open Access Journals (Sweden)

    Duncan Stephen A

    2006-04-01

    Full Text Available Abstract The zinc finger transcription factor GATA6 is believed to have important roles in the development of several organs including the liver, gastrointestinal tract and heart. However, analyses of the contribution of GATA6 toward organogenesis have been hampered because Gata6-/- mice fail to develop beyond gastrulation due to defects in extraembryonic endoderm function. We have therefore generated a mouse line harbouring a conditional loss-of-function allele of Gata6 using Cre/loxP technology. LoxP elements were introduced into introns flanking exon 2 of the Gata6 gene by homologous recombination in ES cells. Mice containing this altered allele were bred to homozygosity and were found to be viable and fertile. To assess the functional integrity of the loxP sites and to confirm that we had generated a Gata6 loss-of-function allele, we bred Gata6 'floxed' mice to EIIa-Cre mice in which Cre is ubiquitously expressed, and to Villin-Cre mice that express Cre in the epithelial cells of the intestine. We conclude that we have generated a line of mice in which GATA6 activity can be ablated in a cell type specific manner by expression of Cre recombinase. This line of mice can be used to establish the role of GATA6 in regulating embryonic development and various aspects of mammalian physiology.

  5. Two-Phase Instability Characteristics of Printed Circuit Steam Generator for the Low Pressure Condition

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Han-Ok; Han, Hun Sik; Kim, Young-In; Kim, Keung Koo [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-05-15

    Reduction of installation space for steam generators can lead to much smaller reactor vessel with resultant decrease of overall manufacturing cost for the components. A PCHE(Printed Circuit Heat Exchanger) is one of the compact types of heat exchangers available as an alternative to conventional shell and tube heat exchangers. Its name is derived from the procedure used to manufacture the flat metal plates that form the core of the heat exchanger, which is done by chemical milling. These plates are then stacked and diffusion bonded, converting the plates into a solid metal block containing precisely engineered fluid flow passages. PCSG(Printed Circuit Steam Generator) is a potential candidate to be applied to the integral reactor with its compactness and mechanical robustness. For the introduction of new steam generator, design requirement for the two-phase flow instability should be considered. This paper describes two-phase flow instability characteristics of PCSG for the low pressure condition. PCSG is a potential candidate to be applied to the integral reactor with its compactness and mechanical robustness. Interconnecting flow path was developed to mitigate the two-phase flow instability in the cold side. The flow characteristics of two-phase flow instability at the PCSG is examined experimentally in this study.

  6. Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions.

    Science.gov (United States)

    Swistowski, Andrzej; Peng, Jun; Liu, Qiuyue; Mali, Prashant; Rao, Mahendra S; Cheng, Linzhao; Zeng, Xianmin

    2010-10-01

    Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs.

  7. Influence of the operating conditions on highly oxidative radicals generation in Fenton's systems.

    Science.gov (United States)

    Ciotti, Cesare; Baciocchi, Renato; Tuhkanen, Tuula

    2009-01-15

    In this work, an indirect method for estimating the total amount and concentration of oxidative radicals in aqueous and slurry-phase Fenton's systems was developed. This method, based on the use of benzoic acid as probe compound, was applied for evaluating the effect of the operating conditions on the radicals amount produced, their production efficiency (i.e. moles of radicals generated per mole H2O2 and their concentration. A Rotatable Central Composite design (RCC) was used to select the operating conditions in order to get a statistically meaningful data set. Hydrogen peroxide and ferrous ion concentrations ranged between 0.2-1mM and 0.2-0.5mM, respectively; humic acid concentration between 0 and 15mg/L, whereas the soil/water weight ratio in slurry-phase systems between 1:10 and 9:10. The probe compound concentration was 9 or 0.1mM in experiments aimed to evaluate the total amount or concentration of oxidative radicals, respectively. The obtained results indicated that the amount of radicals generated in both aqueous and soil slurry Fenton's system increased with higher H2O2 concentration and, more specifically, that their production efficiency increased with increasing Fe(II):H2O2 molar ratio. Addition of dissolved organic compounds as humic acid did not notably affect the oxidative radicals amount and concentration. On the contrary, a one order of magnitude reduction in both radicals amount generated and concentration was observed when soil was added to the reaction environment.

  8. Targeted next generation sequencing of a panel of autism-related genes identifies an EHMT1 mutation in a Kleefstra syndrome patient with autism and normal intellectual performance.

    Science.gov (United States)

    Bock, István; Németh, Krisztina; Pentelényi, Klára; Balicza, Péter; Balázs, Anna; Molnár, Mária Judit; Román, Viktor; Nagy, József; Lévay, György; Kobolák, Julianna; Dinnyés, András

    2016-12-31

    Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with unknown genetic and environmental causation in most of the affected individuals. On the other hand, there are a growing number of ASD-associated syndromes, where the exact genetic origin can be revealed. Here we report a method, which included the targeted next generation sequencing (NGS) and filtering of 101 ASD associated genes, followed by database search. Next, RNA sequencing was used to study the region of interest at the transcriptional level. Using this workflow, we identified a de novo mutation in the euchromatic histone-lysine N-methyltransferase 1 gene (EHMT1) of an autistic patient with dysmorphisms. Sequencing of EHMT1 transcripts showed that the premature termination codon (Trp1138Ter) created by a single nucleotide change elicited nonsense-mediated mRNA decay, which led to haploinsufficiency already at the transcriptional level. Database and literature search provided evidence that this mutation caused Kleefstra syndrome (KS), which was confirmed by the presence of the disorder-specific phenotype in the patient. We provide a proof of principle that the implemented method is capable to elucidate the genetic etiology of individuals with syndromic autism. The novel mutation detected in the EHMT1 gene is responsible for KS's symptoms. In addition, further genetic factors might be involved in the ASD pathogenesis of the patient including a missense DPP6 mutation (Arg322Cys), which segregated with the autistic phenotype within the family.

  9. Lampe1: an ENU-germline mutation causing spontaneous hepatosteatosis identified through targeted exon-enrichment and next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Rachel Sheridan

    Full Text Available Using a small scale ENU mutagenesis approach we identified a recessive germline mutant, designated Lampe1 that exhibited growth retardation and spontaneous hepatosteatosis. Low resolution mapping based on 20 intercrossed Lampe1 mice revealed linkage to a ∼14 Mb interval on the distal site of chromosome 11 containing a total of 285 genes. Exons and 50 bp flanking sequences within the critical region were enriched with sequence capture microarrays and subsequently analyzed by next-generation sequencing. Using this approach 98.1 percent of the targeted DNA was covered with a depth of 10 or more reads per nucleotide and 3 homozygote mutations were identified. Two mutations represented intronic nucleotide changes whereas one mutation affected a splice donor site in intron 11-12 of Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1, causing skipping of exon 12. Phenotyping of Acox1(Lampe1 mutants revealed a progression from hepatosteatosis to steatohepatitis, and ultimately hepatocellular carcinoma. The current approach provides a highly efficient and affordable method to identify causative mutations induced by ENU mutagenesis and animal models relevant to human pathology.

  10. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  11. Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination.

    Science.gov (United States)

    Butler, James R; Santos, Rafael M N; Martens, Gregory R; Ladowski, Joseph M; Wang, Zheng-Yu; Li, Ping; Tector, Matthew; Tector, A Joseph

    2017-05-15

    Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic

  12. Constrained approximation of effective generators for multiscale stochastic reaction networks and application to conditioned path sampling

    Science.gov (United States)

    Cotter, Simon L.

    2016-10-01

    Efficient analysis and simulation of multiscale stochastic systems of chemical kinetics is an ongoing area for research, and is the source of many theoretical and computational challenges. In this paper, we present a significant improvement to the constrained approach, which is a method for computing effective dynamics of slowly changing quantities in these systems, but which does not rely on the quasi-steady-state assumption (QSSA). The QSSA can cause errors in the estimation of effective dynamics for systems where the difference in timescales between the "fast" and "slow" variables is not so pronounced. This new application of the constrained approach allows us to compute the effective generator of the slow variables, without the need for expensive stochastic simulations. This is achieved by finding the null space of the generator of the constrained system. For complex systems where this is not possible, or where the constrained subsystem is itself multiscale, the constrained approach can then be applied iteratively. This results in breaking the problem down into finding the solutions to many small eigenvalue problems, which can be efficiently solved using standard methods. Since this methodology does not rely on the quasi steady-state assumption, the effective dynamics that are approximated are highly accurate, and in the case of systems with only monomolecular reactions, are exact. We will demonstrate this with some numerics, and also use the effective generators to sample paths of the slow variables which are conditioned on their endpoints, a task which would be computationally intractable for the generator of the full system.

  13. Liver Steatosis and Increased ChREBP Expression in Mice Carrying a Liver Specific SIRT1 Null Mutation under a Normal Feeding Condition

    Directory of Open Access Journals (Sweden)

    Rui-Hong Wang, Cuiling Li, Chu-Xia Deng

    2010-01-01

    Full Text Available SIRT1, a homolog of yeast Sir2, is a type III NAD+ dependent histone and protein deacetylase. Previous studies of mice carrying liver specific deletion of exon 4 of the Sirt1 gene revealed opposite responses of mutant mice to a high-fat diet in terms of fatty liver formation, which obscures the function of SRIT1 in liver development and lipid metabolism. To investigate this, we deleted exons 5 and 6 of Sirt1 in the liver by using a Cre-loxP approach. Western blot using an antibody to N-terminal SIRT1 does not detect a truncated protein in the liver of the mutant mice (Sirt1flox5-6/flox5-6;Alb-Cre, suggesting a null mutation for SIRT1 is generated in the liver. Unlike the previously reported phenotypes, the Sirt1flox5-6/flox5-6;Alb-Cre mice develop fatty liver under a normal feeding condition. The disease starts at two months of age and incidence increases as the animals become older, affecting 78% of them when they are over one year of age. We showed that the steatosis is accompanied by altered expression of a number of genes, including increased expression of ChREBP, which acts as one of the central determinants of lipid synthesis in the liver. This data uncovers an important role of SIRT1 in regulating lipid metabolism in the liver, and the SIRT1 mutant mice may serve as an animal model for studying human fatty liver disease and facilitate the development of effective therapeutic approach for the disease.

  14. Laboratory investigation of spray generation mechanism in wind-wave interaction under strong wind conditions

    Science.gov (United States)

    Kandaurov, Alexander; Troitskaya, Yuliya; Sergeev, Daniil; Ermakova, Olga; Kazakov, Vassily

    2015-04-01

    The sea spray is considered as a possible mechanism of the reduction of sea surface aerodynamic drag coefficient at hurricane conditions [1]. In this paper the mechanism of generation of spray in the near-surface layer of the atmosphere in a strong wind through the mechanism of «bag-breakup instability» was investigated in laboratory conditions with the help of high-speed video shooting. The laboratory experiments were performed on the Thermostratified Wind-Wave Channel of the IAP RAS (length 10 m, cross section of air channel 0.4 x 0.4 m, wind velocity up to 24 m/s) [2]. Experiments were carried out for the wind speeds from 14 to 22 m/s. In this range spray generation characteristics change dramatically from almost no spray generation to so called catastrophic regime with multiple cascade breakups on each crest. Shooting was performed with High-speed digital camera NAC Memrecam HX-3 in two different setups to obtain both statistical data and detailed spray generation mechanism overview. In first setup bright LED spotlight with mate screen the side of a channel was used for horizontal shadow-method shooting. Camera was placed in semi-submerged box on the opposite side of the channel. Shooting was performed at the distance of 7.5 m from the beginning of the working section. Series of short records of the surface evolution were made at 10 000 fps with 55 to 119 µm/px scale revealed the dominant mechanism of spray generation - bag-breakup instability. Sequences of high resolution images allowed investigating the details of this "bags" evolution. Shadow method provided better image quality for such conditions than side illumination and fluorescence methods. To obtain statistical data on "bags" sizes and densities vertical shadow method was used. Submerged light box was created with two 300 W underwater lamps and mate screen places at the fetch of 6.5 m. Long records (up to 8 seconds) were made with 4500 fps at 124-256 µm/px scales. Specially developed software

  15. Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a Danish five-generation family with a novel FAM83H nonsense mutation.

    Science.gov (United States)

    Haubek, Dorte; Gjørup, Hans; Jensen, Lillian G; Juncker, Inger; Nyegaard, Mette; Børglum, Anders D; Poulsen, Sven; Hertz, Jens M

    2011-11-01

    BACKGROUND.  Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI) is a disease with severe dental manifestations. OBJECTIVES.  The aims were by means of a genome-wide linkage scan to search for the gene underlying the ADHCAI phenotype in a Danish five-generation family and to study the phenotypic variation of the enamel in affected family members. RESULTS.  Significant linkage was found to a locus at chromosome 8q24.3 comprising the gene FAM83H identified to be responsible for ADHCAI in other families. Subsequent sequencing of FAM83H in affected family members revealed a novel nonsense mutation, p.Y302X. Limited phenotypic variation was found among affected family members with loss of translucency and discoloration of the enamel. Extensive posteruptive loss of enamel was found in all teeth of affected subjects. The tip of the cusps on the premolars and molars and a zone along the gingival margin seemed resistant to posteruptive loss of enamel. We have screened FAM83H in another five unrelated Danish patients with a phenotype of ADHCAI similar to that in the five-generation family, and identified a de novo FAM83H nonsense mutation, p.Q452X in one of these patients. CONCLUSION.  We have identified a FAM83H mutation in two of six unrelated families with ADHCAI and found limited phenotypic variation of the enamel in these patients.

  16. Use of targeted next-generation sequencing for molecular diagnosis of craniosynostosis: Identification of a novel de novo mutation of EFNB1.

    Science.gov (United States)

    Yamamoto, Toshiyuki; Igarashi, Naru; Shimojima, Keiko; Sangu, Noriko; Sakamoto, Yuko; Shimoji, Kazuaki; Niijima, Shinichi

    2016-03-01

    Craniofrontonasal syndrome (CFNS; MIM#304110) is characterized by asymmetric facial features with hypertelorism and a broad bifid nose due to synostosis of the coronal suture. CFNS shows a unique X-linked inheritance pattern (most affected patients are female and obligate male carriers exhibit a mild manifestation or no typical features at all) associated with the ephrin-B1 gene (EFNB1) located in the Xq13.1 region. In this study, we performed targeted, massively parallel sequencing using a next-generation sequencer, and identified a novel EFNB1 mutation, c.270_271delCA, in a Japanese female patient with craniosynostosis. Because subsequent Sanger sequencing identified no mutation in either parent, this mutation was determined to be de novo in origin. After obtaining molecular diagnosis, a retrospective clinical evaluation confirmed the clinical diagnosis of CFNS in this patient. Comprehensive molecular diagnosis using a next-generation sequencer would be beneficial for early diagnosis of the patients with undiagnosed craniosynostosis.

  17. Mutations at three sites in the Escherichia coli 23S ribosomal RNA binding region for protein L11 cause UGA-specific suppression and conditional lethality.

    Science.gov (United States)

    Murgola, E J; Xu, W; Arkov, A L

    1995-01-01

    A single nucleotide change, G to A, at nucleotide position 1093 of E. coli 23S ribosomal RNA was found to cause UGA-specific suppression (D.K. Jemiolo, F.T. Pagel and E.J. Murgola, Proc. Natl. Acad. Sci. USA, in press). To obtain new kinds of UGA-specific suppressors in 23S rRNA, we used segment-directed mutagenic PCR, and targeted first the 1405 nucleotide SnaBI/I-CeuI segment, which includes position 1093, of the rrnB operon cloned into a multicopy plasmid. The mutagenized fragments were subcloned into the plasmid vector and used to transform to ampicillin resistance (Ampr) a recipient strain containing a UGA mutation in trpA. The Ampr transformants were then screened for suppression of UGA. After purification, Trp+ transformants were tested for association of the suppressor phenotype first with the plasmid and then specifically with the SnaBI/I-CeuI fragment. In one screening, four different kinds of mutational change were found, all at three sites within a highly conserved hexanucleotide loop in domain II of 23S rRNA. This region is part of the site for binding of the large subunit protein L11, which has been shown to be involved in peptide chain termination in a specific way. All of the mutants (G1093A, G1093 delta, A1095 delta, and U1097 delta) suppress UGA mutations, but not UAA or UAG mutations, and all four types exhibit high-temperature conditional lethality when highly expressed. Several mechanisms can be suggested for the UGA-specific suppression exhibited by these mutants, including altered interaction with protein L11, Second-site mutations that overcome the conditional lethality of G1093A indicate that intramolecular interactions within 23S rRNA may play a role in peptide chain termination at the UGA stop codon.

  18. Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Anita Kloss-Brandstätter

    Full Text Available Oral squamous cell carcinoma (OSCC is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA mutations in primary oral tumors, recurrences and metastases.We performed an in-depth validation of mtDNA next-generation sequencing (NGS on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base.We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.

  19. A Next-Generation Sequencing Approach to Identify Gene Mutations in Early- and Late-Onset Hypertrophic Cardiomyopathy Patients of an Italian Cohort.

    Science.gov (United States)

    Rubattu, Speranza; Bozzao, Cristina; Pennacchini, Ermelinda; Pagannone, Erika; Musumeci, Beatrice Maria; Piane, Maria; Germani, Aldo; Savio, Camilla; Francia, Pietro; Volpe, Massimo; Autore, Camillo; Chessa, Luciana

    2016-07-30

    Sequencing of sarcomere protein genes in patients fulfilling the clinical diagnostic criteria for hypertrophic cardiomyopathy (HCM) identifies a disease-causing mutation in 35% to 60% of cases. Age at diagnosis and family history may increase the yield of mutations screening. In order to assess whether Next-Generation Sequencing (NGS) may fulfil the molecular diagnostic needs in HCM, we included 17 HCM-related genes in a sequencing panel run on PGM IonTorrent. We selected 70 HCM patients, 35 with early (≤25 years) and 35 with late (≥65 years) diagnosis of disease onset. All samples had a 98.6% average of target regions, with coverage higher than 20× (mean coverage 620×). We identified 41 different mutations (seven of them novel) in nine genes: MYBPC3 (17/41 = 41%); MYH7 (10/41 = 24%); TNNT2, CAV3 and MYH6 (3/41 = 7.5% each); TNNI3 (2/41 = 5%); GLA, MYL2, and MYL3 (1/41=2.5% each). Mutation detection rate was 30/35 (85.7%) in early-onset and 8/35 (22.9%) in late-onset HCM patients, respectively (p < 0.0001). The overall detection rate for patients with positive family history was 84%, and 90.5% in patients with early disease onset. In our study NGS revealed higher mutations yield in patients with early onset and with a family history of HCM. Appropriate patient selection can increase the yield of genetic testing and make diagnostic testing cost-effective.

  20. Generation of induced pluripotent stem cell (iPSC line from a 36-year-old Charcot-Marie-Tooth disease patient with GJB1 mutation (CMTX

    Directory of Open Access Journals (Sweden)

    Daryeon Son

    2017-05-01

    Full Text Available Charcot-Marie-Tooth disease (CMTX is inherited neurological disorder caused by gap junction beta 1 gene (GJB1 mutation. We generated induced pluripotent stem cell (iPSC line from 36-year-old CMTX disease patient by electroporation of skin fibroblasts with episomal vectors encoding OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA-p53. Established iPSCs expressed various pluripotency markers, had differentiation potential of three germ layers in vitro, had normal karyotype and retained GJB1 mutation. This CMT patient-derived iPSC line could be useful in vitro tool for CMTX research as disease modeling and drug development.

  1. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a L150P mutation in PSEN-1.

    Science.gov (United States)

    Tubsuwan, Alisa; Pires, Carlota; Rasmussen, Mikkel A; Schmid, Benjamin; Nielsen, Jørgen E; Hjermind, Lena E; Hall, Vanessa; Nielsen, Troels T; Waldemar, Gunhild; Hyttel, Poul; Clausen, Christian; Kitiyanant, Narisorn; Freude, Kristine K; Holst, Bjørn

    2016-01-01

    Induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts isolated from a 58-year old male with a L150P mutation in the presenilin 1 (PSEN-1) gene, which is responsible for the majority of familial cases of Alzheimer's disease (AD). The iPSCs were established by co-electroporation with episomal plasmids containing hOCT4, hSOX2, hL-MYC, hKLF4, hNANOG, hLIN28, and short hairpin RNA against TP53. The iPSCs contained the specific heterozygous mutation c.449C>T, had normal karyotype, expressed the expected pluripotency genes and displayed in vitro differentiation potential to the three germ layers. The iPSCs may be useful for studying familial AD pathology in vitro. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Phillip N., E-mail: pgray@ambrygen.com; Dunlop, Charles L.M.; Elliott, Aaron M. [Ambry Genetics, 15 Argonaut, Aliso Viejo, CA 92656 (United States)

    2015-07-17

    The molecular characterization of tumors using next generation sequencing (NGS) is an emerging diagnostic tool that is quickly becoming an integral part of clinical decision making. Cancer genomic profiling involves significant challenges including DNA quality and quantity, tumor heterogeneity, and the need to detect a wide variety of complex genetic mutations. Most available comprehensive diagnostic tests rely on primer based amplification or probe based capture methods coupled with NGS to detect hotspot mutation sites or whole regions implicated in disease. These tumor panels utilize highly customized bioinformatics pipelines to perform the difficult task of accurately calling cancer relevant alterations such as single nucleotide variations, small indels or large genomic alterations from the NGS data. In this review, we will discuss the challenges of solid tumor assay design/analysis and report a case study that highlights the need to include complementary technologies (i.e., arrays) and germline analysis in tumor testing to reliably identify copy number alterations and actionable variants.

  3. Generation of induced pluripotent stem cells (iPSCs from a Bernard–Soulier syndrome patient carrying a W71R mutation in the GPIX gene

    Directory of Open Access Journals (Sweden)

    Lourdes Lopez-Onieva

    2016-05-01

    Full Text Available We generated an induced pluripotent stem cell (iPSC line from a Bernard–Soulier Syndrome (BSS patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4. Peripheral blood mononuclear cells (PBMCs were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

  4. First-trimester diagnosis of Meckel-Gruber syndrome by fetal ultrasound with molecular identification of CC2D2A mutations by next-generation sequencing.

    Science.gov (United States)

    Jones, D; Fiozzo, F; Waters, B; McKnight, D; Brown, S

    2014-12-01

    We describe a first-trimester ultrasound examination in which the finding of fetal encephalocele and the cystic appearance of the kidneys raised suspicion of Meckel-Gruber syndrome (MKS). On the basis of sonographic findings, the patient elected termination of pregnancy, and post-termination studies using next-generation sequencing of a gene panel revealed two mutations (one previously described and the other novel) in the gene CC2D2A. Mutations in CC2D2A are known to cause MKS and Joubert syndrome, thus providing molecular confirmation of the clinical suspicion of MKS and opening the possibility for future prenatal diagnosis. This case highlights the ability to detect important anomalies in the first trimester using ultrasound, even in low-risk situations. It also demonstrates the growing role of new sequencing technologies in fetal testing. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.

  5. Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Matyas Flemr

    2015-07-01

    Full Text Available Induction of double-strand DNA breaks (DSBs by engineered nucleases, such as CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs, stimulates knockin of exogenous DNA fragments via homologous recombination (HR. However, the knockin efficiencies reported so far have not allowed more complex in vitro genome modifications such as, for instance, simultaneous integration of a DNA fragment at two distinct genomic sites. We developed a reporter system to enrich for cells with engineered nuclease-assisted HR events. Using this system in mouse embryonic stem cells (mESCs, we achieve single-step biallelic and seamless integration of two loxP sites for Cre recombinase-mediated inducible gene knockout, as well as biallelic endogenous gene tagging with high efficiency. Our approach reduces the time and resources required for conditional knockout mESC generation dramatically.

  6. The 4-D descent trajectory generation techniques under realistic operating conditions

    Science.gov (United States)

    Williams, David H.; Knox, Charles E.

    1990-01-01

    NASA-Langley has been conducting and sponsoring research in airborne energy management for a number of years. During the course of this research, two fundamental techniques for the generation of 4D (fixed time) descent trajectories have emerged as viable candidates for advanced flight management systems. The first technique utilizes speed schedules of constant Mach number transitioning to constant calibrated airspeed chosen empirically to produce minimum fuel usage. The second technique computes cost optimized speed schedules of variable airspeed developed through application of optimal control theory. Both techniques have been found to produce reasonable and flyable descent trajectories. The formulation of the algorithms for each technique is evaluated and their suitability for operations in realistic conditions is discussed. Operational factors considered include: airplace speed, thrust, and altitude rate constaints; wind, temperature, and pressure variations; Air Traffic Control altitude, speed, and time constaints; and pilot interface and guidance considerations. Time flexibility, fuel usage, and airborne computational requirements were the primary performance measures.

  7. Generation and characterization of mice carrying a conditional allele of the Wwox tumor suppressor gene.

    Directory of Open Access Journals (Sweden)

    John H Ludes-Meyers

    Full Text Available WWOX, the gene that spans the second most common human chromosomal fragile site, FRA16D, is inactivated in multiple human cancers and behaves as a suppressor of tumor growth. Since we are interested in understanding WWOX function in both normal and cancer tissues we generated mice harboring a conditional Wwox allele by flanking Exon 1 of the Wwox gene with LoxP sites. Wwox knockout (KO mice were developed by breeding with transgenic mice carrying the Cre-recombinase gene under the control of the adenovirus EIIA promoter. We found that Wwox KO mice suffered from severe metabolic defect(s resulting in growth retardation and all mice died by 3 wk of age. All Wwox KO mice displayed significant hypocapnia suggesting a state of metabolic acidosis. This finding and the known high expression of Wwox in kidney tubules suggest a role for Wwox in acid/base balance. Importantly, Wwox KO mice displayed histopathological and hematological signs of impaired hematopoiesis, leukopenia, and splenic atrophy. Impaired hematopoiesis can also be a contributing factor to metabolic acidosis and death. Hypoglycemia and hypocalcemia was also observed affecting the KO mice. In addition, bone metabolic defects were evident in Wwox KO mice. Bones were smaller and thinner having reduced bone volume as a consequence of a defect in mineralization. No evidence of spontaneous neoplasia was observed in Wwox KO mice. We have generated a new mouse model to inactivate the Wwox tumor suppressor gene conditionally. This will greatly facilitate the functional analysis of Wwox in adult mice and will allow investigating neoplastic transformation in specific target tissues.

  8. Conditions for generation of fire-related debris flows, Capulin Canyon, New Mexico

    Science.gov (United States)

    Cannon, S.H.; Reneau, S.L.

    2000-01-01

    Comparison of the responses of three drainage basins burned by the Dome fire of 1996 in New Mexico is used to identify the hillslope, channel and fire characteristics that indicate a susceptibility specifically to wildfire-related debris flow. Summer thunderstorms generated three distinct erosive responses from each of three basins. The Capulin Canyon basin showed widespread erosive sheetwash and rilling from hillslopes, and severe flooding occurred in the channel; the North Tributary basin exhibited extensive erosion of the mineral soil to a depth of 5 cm and downslope movement of up to boulder-sized material, and at least one debris flow occurred in the channel; negligible surface runoff was observed in the South Tributary basin. The negligible surface runoff observed in the South Tributary basin is attributed to the limited extent and severity of the fire in that basin. The factors that best distinguish between debris-flow producing and flood-producing drainages are drainage basin morphology and lithology. A rugged drainage basin morphology, an average 12 per cent channel gradient, and steep, rough hillslopes coupled with colluvium and soil weathered from volcaniclastic and volcanic rocks promoted the generation of debris flows. A less rugged basin morphology, an average gradient of 5 per cent, and long, smooth slopes mantled with pumice promoted flooding. Flood and debris-flow responses were produced without the presence of water-repellent soils. The continuity and severity of the burn mosaic, the condition of the riparian vegetation, the condition of the fibrous root mat, accumulations of dry ravel and colluvial material in the channel and on hillslopes, and past debris-flow activity, appeared to have little bearing on the distinctive responses of the basins. Published in 2000 by John Wiley and Sons, Ltd.

  9. The generation and its sealing condition of natural gas in the Tadong area

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Focusing on the two natural gas exploration geological problems with abundant source of oil cracking gas in the late stage and the sealing condition of the oil cracking gas reservoir,the kinetics of oil cracking gas and the evaluation parameters of gas cap rock are adopted to the study on the natural gas accumulation conditions in the Tadong area. Both the study on the kinetics of oil cracking gas and the statistical results of reservoir bitumen reveal that the geological formation of oil cracking gas in the Tadong area is located in the top of Cambrian. Two kinds of oil cracking gas geological models at least,namely well Mandong-1’s early rapid generation model(Middle Ordovician-end Silurian) and peak cracking model(with the natural gas conversion rate >90%) ,namely well Yingnan-2’s two-stage generation model of oil cracking gas,have been set up. The oil cracking gas of Yingnan-2 in the late stage is very significant in the evaluation of natural gas exploration in the Tadong area. The evaluation results of the cap rock show that the microscopic parameters of cap rock from the lower assemblage of Cambrian-Ordovician are better than those from the upper assemblage. The former has strong capillary sealing ability and higher cap rock breakthrough pressure than the upper assemblage,with strong sealing ability,so that natural gas dissipates mainly by diffusion. According to the above investigations,the lower assemblage Cambrian-Ordovician natural gas of Kongquehe slope,Tadong low uplift and Yingjisu depression in the Tadong area prospects well.

  10. Age at Death of Creutzfeldt-Jakob disease in subsequent family generation carrying the E200K mutation of the prion protein gene.

    Directory of Open Access Journals (Sweden)

    Maurizio Pocchiari

    Full Text Available BACKGROUND: The E200K mutation of the prion protein gene (PRNP is the most frequent amino acid substitution in genetic Creutzfeldt-Jakob disease and is the only one responsible for the appearance of clustered cases in the world. In the Israel and Slovakian clusters, age of disease onset was reduced in successive generations but the absence of a clear molecular basis raised the possibility that this event was an observational bias. The aim of the present study was to investigate possible selection biases or confounding factors related to anticipation in E200K CJD patients belonging to a cluster in Southern Italy. METHODS: Clinical and demographical data of 41 parent-offspring pairs from 19 pedigrees of the Italian cluster of E200K patients were collected. Age at death of parents was compared with age at death of E200K CJD offspring. Subgroup analyses were performed for controlling possible selection biases, confounding factors, or both. RESULTS: The mean age at death/last follow-up of the parent generation was 71.4 years while that of CJD offspring was 59.3 years with an estimated anticipation of 12.1 years. When the same analysis was performed including only parents with CJD or carrying the E200K mutation (n = 26, the difference between offspring and parents increased to 14.8 years. CONCLUSIONS: These results show that early age at death occurs in offspring of families carrying the E200K PRNP mutation and that this event is not linked to observational biases. Although molecular or environmental bases for this occurrence remain unsettled, this information is important for improving the accuracy of information to give to mutated carriers.

  11. Predictions of structural integrity of steam generator tubes under normal operating, accident, an severe accident conditions

    Energy Technology Data Exchange (ETDEWEB)

    Majumdar, S. [Argonne National Lab., IL (United States)

    1997-02-01

    Available models for predicting failure of flawed and unflawed steam generator tubes under normal operating, accident, and severe accident conditions are reviewed. Tests conducted in the past, though limited, tended to show that the earlier flow-stress model for part-through-wall axial cracks overestimated the damaging influence of deep cracks. This observation was confirmed by further tests at high temperatures, as well as by finite-element analysis. A modified correlation for deep cracks can correct this shortcoming of the model. Recent tests have shown that lateral restraint can significantly increase the failure pressure of tubes with unsymmetrical circumferential cracks. This observation was confirmed by finite-element analysis. The rate-independent flow stress models that are successful at low temperatures cannot predict the rate-sensitive failure behavior of steam generator tubes at high temperatures. Therefore, a creep rupture model for predicting failure was developed and validated by tests under various temperature and pressure loadings that can occur during postulated severe accidents.

  12. CargoCBM - Feature Generation and Classification for a Condition Monitoring System for Freight Wagons

    Science.gov (United States)

    Gericke, C.; Hecht, M.

    2012-05-01

    Despite the fact that rail freight transport is one of the most environmentally friendly matters of transport, its growth has been far behind the growth of freight transport in general. Studies showed that a competitive disadvantage is caused by a low availability of rolling stock, especially freight wagons. Changing from a time based to a condition based maintenance strategy is believed to decrease down times by at least one third. To make condition based maintenance for freight wagons possible the TU Berlin and five industry partners started the research project CargoCBM. One task in this project is to develop algorithms for the automatic on-board diagnosis of wheel flats. The focus of the work is on the process of feature generation and feature selection as well as the application of different classifiers to automatically evaluate the data. Based on the results of measured data, features were selected and tested with different classifiers. Thought advanced classifiers such as neural networks have been analysed in accordance to their classification accuracy. It can be shown that with carefully constructed and selected features comparatively simple classifiers can lead to excellent results.

  13. Characterization of Brillouin fiber generator and amplifier for optimized working condition of distributed sensors

    Science.gov (United States)

    Ravet, Fabien; Bao, Xiaoyi; Snoddy, Jeff; Li, Yun; Chen, Liang

    2009-06-01

    The Brillouin fiber amplifier (BFA) has been used in Brillouin optical time and frequency domain analyzer based sensors. For BFA based distributed sensor the optimized working condition is to have the highest Brillouin gain, i.e., highest SNR, which avoids high pump depletion induced gain saturation. We have found that the optimum working condition for distributed sensor system is associated with the stimulated Brillouin scattering (SBS) threshold for BFA, which can be experimentally determined with Stokes power inflexion and/or Stokes spectrum linewidth minimum methods. This threshold depends on both pump and probe power instead of just the pump power as in Brillouin fiber generator (BFG), as well as on sensing length as confirmed by our experimental results and theoretical simulation. This was achieved by introducing the concept of absorption coefficient of the sensing medium defined as the ratio of the total output power to the total input power. We find that the medium absorption is minimized when input Stokes power is an order of magnitude lower than BFA threshold. This minimum is a signature of the balance between maximum Stokes gain and pump depletion which is also the reason why Stokes spectrum linewidth goes through a minimum.

  14. Global stability of stretched jets: conditions for the generation of monodisperse micro-emulsions using coflows

    CERN Document Server

    Gordillo, José Manuel; Campo-Cortés, Francisco

    2014-01-01

    In this paper we reveal the physics underlying the conditions needed for the generation of emulsions composed of uniformly sized drops of micrometric or submicrometric diameters when two immiscible streams flow in parallel under the so-called tip streaming regime after Suryo & Basaran (2006). Indeed, when inertial effects in both liquid streams are negligible, the inner to outer flow-rate and viscosity ratios are small enough and the capillary number is above an experimentally determined threshold which is predicted by our theoretical results with small relative errors, a steady micron-sized jet is issued from the apex of a conical drop. Under these conditions, the jet disintegrates into drops with a very well defined mean diameter, giving rise to a monodisperse micro-emulsion. Here, we demonstrate that the regime in which uniformly-sized drops are produced corresponds to values of the capillary number for which the cone-jet system is globally stable. Interestingly enough, our general stability theory rev...

  15. Reparameterization techniques for generating reservoir descriptions conditioned to variograms and well-test pressure data

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, A.C.; He, N.; Chu, L. [Univ. of Tulsa, OK (United States)] [and others

    1995-12-31

    Recently, we have shown that reservoir descriptions conditioned to multiwell pressure data and univariate and bivariate statistics for permeability and porosity can be obtained by techniques developed from inverse problem theory. The techniques yield estimates of well skin factors and porosity and permeability fields which honor both the spatial statistics and the pressure data. Imbedded in the methodology is the application of the Gauss-Newton method to construct the maximum a posteriori estimate of the reservoir parameters. If one wishes to determine permeability and porosity values at thousands of grid-blocks for use in a reservoir simulator, then inversion of the Hessian matrix at each iteration of the Gauss-Newton procedure becomes computationally expensive. In this work, we present two methods to reparameterize the reservoir model to improve the computational efficiency. The first method uses spectral (eigenvalue/eigenvector) decomposition of the prior model. The second method uses a subspace method to reduce the size of the matrix problem that must be solved at each iteration of the Gauss-Newton method. It is shown that proper implementation of the reparameterization techniques significantly decreases the computational time required to generate realizations of the reservoir model, i.e., the porosity and permeability fields and well skin factors, conditioned to prior information on porosity and permeability and multiwell pressure data.

  16. Multi-parameter Optimization of a Thermoelectric Power Generator and Its Working Conditions

    Science.gov (United States)

    Zhang, T.

    2016-09-01

    The global optimal working conditions and optimal couple design for thermoelectric (TE) generators with realistic thermal coupling between the heat reservoirs and the TE couple were studied in the current work. The heat fluxes enforced by the heat reservoirs at the hot and the cold junctions of the TE couple were used in combination with parameter normalization to obtain a single cubic algebraic equation relating the temperature differences between the TE couple junctions and between the heat reservoirs, through the electric load resistance ratio, the reservoir thermal conductance ratio, the reservoir thermal conductance to the TE couple thermal conductance ratio, the Thomson to Seebeck coefficient ratio, and the figure of merit (Z) of the material based on the linear TE transport equations and their solutions. A broad reservoir thermal conductance ranging between 0.01 W/K and 100 W/K and TE element length ranging from 10-7 m to 10-3 m were explored to find the global optimal systems. The global optimal parameters related to the working conditions, i.e., reservoir thermal conductance ratio and electric load resistance ratio, and the optimal design parameter related to the TE couple were determined for a given TE material. These results demonstrated that the internal and external electric resistance, the thermal resistance between the reservoirs, the thermal resistance between the reservoir and the TE couple, and the optimal thermoelement length have to be well coordinated to obtain optimal power production.

  17. Chemical characterization and in vitro toxicity of diesel exhaust particulate matter generated under varying conditions

    Science.gov (United States)

    Cox, David P.; Drury, Bertram E.; Gould, Timothy R.; Kavanagh, Terrance J.; Paulsen, Michael H.; Sheppard, Lianne; Simpson, Christopher D.; Stewart, James A.; Larson, Timothy V.; Kaufman, Joel D.

    2014-01-01

    Epidemiologic studies have linked diesel exhaust (DE) to cardiovascular and respiratory morbidity and mortality, as well as lung cancer. DE composition is known to vary with many factors, although it is unclear how this influences toxicity. We generated eight DE atmospheres by applying a 2×2×2 factorial design and altering three parameters in a controlled exposure facility: (1) engine load (27 vs 82 %), (2) particle aging (residence time ~5 s vs ~5 min prior to particle collection), and (3) oxidation (with or without ozonation during dilution). Selected exposure concentrations of both diesel exhaust particles (DEPs) and DE gases, DEP oxidative reactivity via DTT activity, and in vitro DEP toxicity in murine endothelial cells were measured for each DE atmosphere. Cell toxicity was assessed via measurement of cell proliferation (colony formation assay), cell viability (MTT assay), and wound healing (scratch assay). Differences in DE composition were observed as a function of engine load. The mean 1-nitropyrene concentration was 15 times higher and oxidative reactivity was two times higher for low engine load versus high load. There were no substantial differences in measured toxicity among the three DE exposure parameters. These results indicate that alteration of applied engine load shifts the composition and can modify the biological reactivity of DE. While engine conditions did not affect the selected in vitro toxicity measures, the change in oxidative reactivity suggests that toxicological studies with DE need to take into account engine conditions in characterizing biological effects. PMID:26539254

  18. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  19. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    OpenAIRE

    Pawel Bialk; Brett Sansbury; Natalia Rivera-Torres; Kevin Bloh; Dula Man; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  20. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    OpenAIRE

    Pawel Bialk; Brett Sansbury; Natalia Rivera-Torres; Kevin Bloh; Dula Man; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  1. Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination

    Directory of Open Access Journals (Sweden)

    Jung Kirsten

    2008-04-01

    Full Text Available Abstract Background Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Results Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. Conclusion Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.

  2. Targeted next generation sequencing reveals a novel intragenic deletion of the TPO gene in a family with intellectual disability

    NARCIS (Netherlands)

    Iqbal, Z.; Neveling, K.; Razzaq, A.; Shahzad, M.; Zahoor, M.Y.; Qasim, M.; Gilissen, C.; Wieskamp, N.; Kwint, M.P.; Gijsen, S.; Brouwer, A.P. de; Veltman, J.A.; Riazuddin, S.; Bokhoven, J.H.L.M. van

    2012-01-01

    BACKGROUNDS AND AIMS: Next generation sequencing (NGS) approaches have revolutionized the identification of mutations underlying genetic disorders. This technology is particularly useful for the identification of mutations in known and new genes for conditions with extensive genetic heterogeneity. I

  3. Study of Wave Conditions at Kvitsøy Prototype Location of Seawave Slot-Cone Generator

    DEFF Research Database (Denmark)

    Kofoed, Jens Peter; Guinot, Florent

    This report presents the results of a study of the wave conditions at the planned location of the prototype of the wave energy converter (WEC) Seawave Slot-Cone Generator (SSG). SSG is a WEC utilizing wave overtopping in multiple reservoirs.......This report presents the results of a study of the wave conditions at the planned location of the prototype of the wave energy converter (WEC) Seawave Slot-Cone Generator (SSG). SSG is a WEC utilizing wave overtopping in multiple reservoirs....

  4. A novel PIK3CD C896T mutation detected in bilateral sudden sensorineural hearing loss using next generation sequencing:An indication of primary immunodeficiency

    Institute of Scientific and Technical Information of China (English)

    Jing Zou; Xiangqiang Duan; Guiliang Zheng; Zhen Zhao; Shiyue Chen; Pu Dai; Hongliang Zheng

    2016-01-01

    Objective:To investigate immune-related genetic background in bilateral sudden sensorineural hearing loss (SSNHL). Case report and methods: The case is a 45-year-old man presenting with a 7-year history of bilateral profound SSNHL. Blood biochemical testing demonstrated increased levels of total cholesterol (5.88 mmol/L). Tests for hepatitis B showed a positive antibody against the hepatitis B core antigen. Complement C3 was below the normal value, and complement C4 and IgG were in the lower range of normal values. CT images showed a normal inner ear and vestibular aqueduct but round window membranous ossification on both sides. A total number of 232 immune-associated genes were sequenced using the next generation sequencing technique. Results: Mutations were detected in 5 genes, including the phosphoinositide 3-kinase catalytic subunit delta (PIK3CD), caspase recruitment domain-containing protein 9 (CARD9), complement factor H-related (CFHR2), immunoglobulin lambda-like polypeptide 1 Protein (IGLL1), and transmembrane channel-like gene family 8 (TMC8). In the PIK3CD gene, a C896T substitute in exon 7 was detected. This mutation causes primary immunodeficiency and is an autosomal dominant disease. Conclusion: The PIK3CD C896T mutation responsible for primary immunodeficiency may contribute to the onset of bilateral SSNHL with subsequent rapid progression.

  5. Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms.

    Science.gov (United States)

    Yu, Jun; Sadakari, Yoshihiko; Shindo, Koji; Suenaga, Masaya; Brant, Aaron; Almario, Jose Alejandro Navarro; Borges, Michael; Barkley, Thomas; Fesharakizadeh, Shahriar; Ford, Madeline; Hruban, Ralph H; Shin, Eun Ji; Lennon, Anne Marie; Canto, Marcia Irene; Goggins, Michael

    2017-09-01

    Secretin-stimulated pancreatic juice contains DNA shed from cells lining the pancreatic ducts. Genetic analysis of this fluid may form a test to detect pancreatic ductal neoplasia. We employed digital next-generation sequencing ('digital NGS') to detect low-abundance mutations in secretin-stimulated juice samples collected from the duodenum of subjects enrolled in Cancer of the Pancreas Screening studies at Johns Hopkins Hospital. For each juice sample, digital NGS necessitated 96 NGS reactions sequencing nine genes. The study population included 115 subjects (53 discovery, 62 validation) (1) with pancreatic ductal adenocarcinoma (PDAC), (2) intraductal papillary mucinous neoplasm (IPMN), (3) controls with non-suspicious pancreata. Cases with PDAC and IPMN were more likely to have mutant DNA detected in pancreatic juice than controls (both pcancer despite close surveillance had SMAD4/TP53 mutations from their cancer detected in juice samples collected over 1 year prior to their pancreatic cancer diagnosis when no suspicious pancreatic lesions were detected by imaging. The detection in pancreatic juice of mutations important for the progression of low-grade dysplasia to high-grade dysplasia and invasive pancreatic cancer may improve the management of patients undergoing pancreatic screening and surveillance. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  6. Identification of a Novel Heterozygous Missense Mutation in the CACNA1F Gene in a Chinese Family with Retinitis Pigmentosa by Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Qi Zhou

    2015-01-01

    Full Text Available Background. Retinitis pigmentosa (RP is an inherited retinal degenerative disease, which is clinically and genetically heterogeneous, and the inheritance pattern is complex. In this study, we have intended to study the possible association of certain genes with X-linked RP (XLRP in a Chinese family. Methods. A Chinese family with RP was recruited, and a total of seven individuals were enrolled in this genetic study. Genomic DNA was isolated from peripheral leukocytes, and used for the next generation sequencing (NGS. Results. The affected individual presented the clinical signs of XLRP. A heterozygous missense mutation (c.1555C>T, p.R519W was identified by NGS in exon 13 of the CACNA1F gene on X chromosome, and was confirmed by Sanger sequencing. It showed perfect cosegregation with the disease in the family. The mutation at this position in the CACNA1F gene of RP was found novel by database searching. Conclusion. By using NGS, we have found a novel heterozygous missense mutation (c.1555C>T, p.R519W in CACNA1F gene, which is probably associated with XLRP. The findings might provide new insights into the cause and diagnosis of RP, and have implications for genetic counseling and clinical management in this family.

  7. Characterization of acoustic droplet vaporization for control of bubble generation under flow conditions.

    Science.gov (United States)

    Kang, Shih-Tsung; Huang, Yi-Luan; Yeh, Chih-Kuang

    2014-03-01

    This study investigated the manipulation of bubbles generated by acoustic droplet vaporization (ADV) under clinically relevant flow conditions. Optical microscopy and high-frequency ultrasound imaging were used to observe bubbles generated by 2-MHz ultrasound pulses at different time points after the onset of ADV. The dependence of the bubble population on droplet concentration, flow velocity, fluid viscosity and acoustic parameters, including acoustic pressure, pulse duration and pulse repetition frequency, was investigated. The results indicated that post-ADV bubble growth spontaneously driven by air permeation markedly affected the bubble population after insonation. The bubbles can grow to a stable equilibrium diameter as great as twice the original diameter in 0.5-1 s, as predicted by the theoretical calculation. The growth trend is independent of flow velocity, but dependent on fluid viscosity and droplet concentration, which directly influence the rate of gas uptake by bubbles and the rate of gas exchange across the wall of the semipermeable tube containing the bubbles and, hence, the gas content of the host medium. Varying the acoustic pressure does not markedly change the formation of bubbles as long as the ADV thresholds of most droplets are reached. Varying pulse duration and pulse repetition frequency markedly reduces the number of bubbles. Lengthening pulse duration favors the production of large bubbles, but reduces the total number of bubbles. Increasing the PRF interestingly provides superior performance in bubble disruption. These results also suggest that an ADV bubble population cannot be assessed simply on the basis of initial droplet size or enhancement of imaging contrast by the bubbles. Determining the optimal acoustic parameters requires careful consideration of their impact on the bubble population produced for different application scenarios.

  8. Early stages of wind wave and drift current generation under non-stationary wind conditions.

    Science.gov (United States)

    Robles-Diaz, Lucia; Ocampo-Torres, Francisco J.; Branger, Hubert

    2016-04-01

    Generation and amplification mechanisms of ocean waves are well understood under constant wind speed or limited fetch conditions. Under these situations, the momentum and energy transfers from air to water are also quite well known. However during the wind field evolution over the ocean, we may observe sometime high wind acceleration/deceleration situations (e.g. Mexican Tehuano or Mediterranean Mistral wind systems). The evolution of wave systems under these conditions is not well understood. The purpose of these laboratory experiments is to better understand the early stages of water-waves and surface-drift currents under non-stationary wind conditions and to determine the balance between transfers creating waves and surface currents during non-equilibrium situations. The experiments were conducted in the Institut Pythéas wind-wave facility in Marseille-France. The wave tank is 40 m long, 2.7 m wide and 1 m deep. The air section is 50 m long, 3 m wide and 1.8 m height. We used 11 different resistive wave-gauges located along the tank. The momentum fluxes in the air column were estimated from single and X hot-film anemometer measurements. The sampling frequency for wind velocity and surface displacement measurements was 256 Hz. Water-current measurements were performed with a profiling velocimeter. This device measures the first 3.5 cm of the water column with a frequency rate of 100Hz. During the experiments, the wind intensity was abruptly modified with a constant acceleration and deceleration over time. We observed that wind drag coefficient values for accelerated wind periods are lower than the ones reported in previous studies for constant wind speed (Large and Pond 1981; Ocampo-Torres et al. 2010; Smith 1980; Yelland and Taylor 1996). This is probably because the turbulent boundary layer is not completely developed during the increasing-wind sequence. As it was reported in some theoretical studies (Miles 1957; Phillips 1957; Kahma and Donelan 1988), we

  9. SRY mutation analysis by next generation (deep sequencing in a cohort of chromosomal Disorders of Sex Development (DSD patients with a mosaic karyotype

    Directory of Open Access Journals (Sweden)

    Hersmus Remko

    2012-11-01

    Full Text Available Abstract Background The presence of the Y-chromosome or Y chromosome-derived material is seen in 4-60% of Turner syndrome patients (Chromosomal Disorders of Sex Development (DSD. DSD patients with specific Y-chromosomal material in their karyotype, the GonadoBlastoma on the Y-chromosome (GBY region, have an increased risk of developing type II germ cell tumors/cancer (GCC, most likely related to TSPY. The Sex determining Region on the Y gene (SRY is located on the short arm of the Y-chromosome and is the crucial switch that initiates testis determination and subsequent male development. Mutations in this gene are responsible for sex reversal in approximately 10-15% of 46,XY pure gonadal dysgenesis (46,XY DSD cases. The majority of the mutations described are located in the central HMG domain, which is involved in the binding and bending of the DNA and harbors two nuclear localization signals. SRY mutations have also been found in a small number of patients with a 45,X/46,XY karyotype and might play a role in the maldevelopment of the gonads. Methods To thoroughly investigate the presence of possible SRY gene mutations in mosaic DSD patients, we performed next generation (deep sequencing on the genomic DNA of fourteen independent patients (twelve 45,X/46,XY, one 45,X/46,XX/46,XY, and one 46,XX/46,XY. Results and conclusions The results demonstrate that aberrations in SRY are rare in mosaic DSD patients and therefore do not play a significant role in the etiology of the disease.

  10. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

    Science.gov (United States)

    Lanman, Richard B.; Mortimer, Stefanie; Zill, Oliver A.; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-01-01

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy. PMID:26452027

  11. Clinical Genotyping of Non–Small Cell Lung Cancers Using Targeted Next-Generation Sequencing: Utility of Identifying Rare and Co-mutations in Oncogenic Driver Genes

    Directory of Open Access Journals (Sweden)

    Laura J. Tafe

    2016-09-01

    Full Text Available Detection of somatic mutations in non–small cell lung cancers (NSCLCs, especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gene AmpliSeq Cancer Hotspot Panel v2 from May 2013 to July 2015. Using 10 ng of DNA, up to 11 samples were simultaneously sequenced on the Ion Torrent PGM (316 and 318 chips. We identified variants with the Ion Torrent Variant Caller Plugin, and Golden Helix's SVS software was used for annotation and prediction of the significance of the variants. Three hundred ninety-eight samples were successfully sequenced (12.1% failure rate. In all, 633 variants in 41 genes were detected with a median of 2 (range of 0 to 7 variants per sample. Mutations detected in BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA were considered potentially actionable and were identified in 237 samples, most commonly in KRAS (37.9%, EGFR (11.1%, BRAF (4.8%, and PIK3CA (4.3%. In our patient population, all mutations in EGFR, KRAS, and BRAF were mutually exclusive. The Ion Torrent Ampliseq technology can be utilized on small biopsy and cytology specimens, requires very little input DNA, and can be applied in clinical laboratories for genotyping of NSCLC. This targeted next-generation sequencing approach allows for detection of common and also rare mutations that are clinically actionable in multiple patients simultaneously.

  12. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients.

    Science.gov (United States)

    Kim, Seung Tae; Lee, Won-Suk; Lanman, Richard B; Mortimer, Stefanie; Zill, Oliver A; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-11-24

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

  13. Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

    Directory of Open Access Journals (Sweden)

    Cristian Miere

    2016-03-01

    Full Text Available The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  14. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    Directory of Open Access Journals (Sweden)

    Laureen Jacquet

    2016-03-01

    Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  15. A High Temperature Experimental Characterization Procedure for Oxide-Based Thermoelectric Generator Modules under Transient Conditions

    Directory of Open Access Journals (Sweden)

    Elena Anamaria Man

    2015-11-01

    Full Text Available The purpose of this study is to analyze the steady-state and transient behavior of the electrical and thermal parameters of thermoelectric generators (TEGs. The focus is on the required wait-time between measurements in order to reduce measurement errors which may appear until the system reaches steady-state. By knowing this waiting time, the total characterization time can also be reduced. The experimental characterization process is performed on a test rig known as TEGeta, which can be used to assess the output characteristics of TEG modules under different load values and temperature conditions. The setup offers the possibility to control the hot side temperature up to 1000 °C with a load variation range value between 0.22–8.13 Ω. A total of ten thermocouples are placed in the setup with the purpose of measuring the temperature in specific points between the heater and the heat sink. Based on the readings, the temperature on the hot and cold side of the modules can be extrapolated. This study provides quantitative data on the minimum waiting time of the temperatures in the surrounding system to reach equilibrium. Laboratory tests are performed on a calcium-manganese oxide module at temperatures between 400 and 800 °C to explore the high temperatures features of the setup.

  16. Investigation of isolated substorms: Generation conditions and characteristics of different phases

    Science.gov (United States)

    Vorobjev, V. G.; Yagodkina, O. I.; Zverev, V. L.

    2016-11-01

    Characteristics of isolated substorms selected by variations in the 1-min values of the AL index are analyzed. The substorms were divided into several types with respect to the behavior of the Bz component of the interplanetary magnetic field (IMF) during the expansion phase. The probability of observations of substorms associated with the northward turn of the Bz component of IMF was 19%, while the substorms taking place at Bz 30 min) period of the southward IMF and a following sharp turn of the Bz component of IMF before the north was detected. The data suggest that a northward IMF turn is neither a necessary nor sufficient condition for generating substorms. It has been shown for substorms of the both types that the average duration of the southward IMF to moment T 0 and the average intensity of the magnetic perturbation in the maximum are approximately the same and amount to 80 min and-650 nT, respectively. However, for substorms at Bz loading-unloading processes in the magnetosphere in the periods of magnetospheric substorms were investigated with different functions that determine the efficiency of the energy transfer from the solar wind to the magnetosphere. It has been shown that the highest correlation coefficient ( r = 0.84) is observed when the function suggested by Newell et al. (2007) is used. It has been detected that a simple function VB S yields a high correlation coefficient ( r = 0.75).

  17. DC Link Current Estimation in Wind-Double Feed Induction Generator Power Conditioning System

    Directory of Open Access Journals (Sweden)

    MARIAN GAICEANU

    2010-12-01

    Full Text Available In this paper the implementation of the DC link current estimator in power conditioning system of the variable speed wind turbine is shown. The wind turbine is connected to double feed induction generator (DFIG. The variable electrical energy parameters delivered by DFIG are fitted with the electrical grid parameters through back-to-back power converter. The bidirectional AC-AC power converter covers a wide speed range from subsynchronous to supersynchronous speeds. The modern control of back-to-back power converter involves power balance concept, therefore its load power should be known in any instant. By using the power balance control, the DC link voltage variation at the load changes can be reduced. In this paper the load power is estimated from the dc link, indirectly, through a second order DC link current estimator. The load current estimator is based on the DC link voltage and on the dc link input current of the rotor side converter. This method presents certain advantages instead of using measured method, which requires a low pass filter: no time delay, the feedforward current component has no ripple, no additional hardware, and more fast control response. Through the numerical simulation the performances of the proposed DC link output current estimator scheme are demonstrated.

  18. Conditionally-Sampled Turbulent and Nonturbulent Measurements of Entropy Generation Rate in the Transition Region of Boundary Layers

    Energy Technology Data Exchange (ETDEWEB)

    D. M. McEligot; J. R. Wolf; K. P. Nolan; E. J. Walsh; R. J. Volino

    2006-05-01

    Conditionally-sampled boundary layer data for an accelerating transitional boundary layer have been analyzed to calculate the entropy generation rate in the transition region. By weighing the nondimensional dissipation coefficient for the laminar-conditioned-data and turbulent-conditioned-data with the intermittency factor the average entropy generation rate in the transition region can be determined and hence be compared to the time averaged data and correlations for steady laminar and turbulent flows. It is demonstrated that this method provides, for the first time, an accurate and detailed picture of the entropy generation rate during transition. The data used in this paper have been taken from detailed boundary layer measurements available in the literature. This paper provides, using an intermittency weighted approach, a methodology for predicting entropy generation in a transitional boundary layer.

  19. Conditionally-Sampled Turbulent and Non-turbulent Measurements of Entropy Generation Rate in the Transition Region of Boundary Layers

    Energy Technology Data Exchange (ETDEWEB)

    Edmond J. Walsh; Kevin P. Nolan; Donald M. McEligot; Ralph J. Volino; Adrian Bejan

    2007-05-01

    Conditionally-sampled boundary layer data for an accelerating transitional boundary layer have been analyzed to calculate the entropy generation rate in the transition region. By weighing the nondimensional dissipation coefficient for the laminar-conditioned-data and turbulent-conditioned-data with the intermittency factor the average entropy generation rate in the transition region can be determined and hence be compared to the time averaged data and correlations for steady laminar and turbulent flows. It is demonstrated that this method provides, for the first time, an accurate and detailed picture of the entropy generation rate during transition. The data used in this paper have been taken from detailed boundary layer measurements available in the literature. This paper provides, using an intermittency weighted approach, a methodology for predicting entropy generation in a transitional boundary layer.

  20. Detection of low-abundance KRAS mutations in colorectal cancer using microfluidic capillary electrophoresis-based restriction fragment length polymorphism method with optimized assay conditions.

    Directory of Open Access Journals (Sweden)

    Huidan Zhang

    Full Text Available Constitutively active KRAS mutations have been found to be involved in various processes of cancer development, and render tumor cells resistant to EGFR-targeted therapies. Mutation detection methods with higher sensitivity will increase the possibility of choosing the correct individual therapy. Here, we established a highly sensitive and efficient microfluidic capillary electrophoresis-based restriction fragment length polymorphism (µCE-based RFLP platform for low-abundance KRAS genotyping with the combination of µCE and RFLP techniques. By using our self-built sensitive laser induced fluorescence (LIF detector and a new DNA intercalating dye YOYO-1, the separation conditions of µCE for ΦX174 HaeIII DNA marker were first optimized. Then, a Mav I digested 107-bp KRAS gene fragment was directly introduced into the microfluidic device and analyzed by µCE, in which field amplified sample stacking (FASS technique was employed to obtain the enrichment of the RFLP digestion products and extremely improved the sensitivity. The accurate analysis of KRAS statuses in HT29, LS174T, CCL187, SW480, Clone A, and CX-1 colorectal cancer (CRC cell lines by µCE-based RFLP were achieved in 5 min with picoliter-scale sample consumption, and as low as 0.01% of mutant KRAS could be identified from a large excess of wild-type genomic DNA (gDNA. In 98 paraffin-embedded CRC tissues, KRAS codon 12 mutations were discovered in 28 (28.6%, significantly higher than that obtained by direct sequencing (13, 13.3%. Clone sequencing confirmed these results and showed this system could detect at least 0.4% of the mutant KRAS in CRC tissue slides. Compared with direct sequencing, the new finding of the µCE-based RFLP platform was that KRAS mutations in codon 12 were correlated with the patient's age. In conclusion, we established a sensitive, fast, and cost-effective screening method for KRAS mutations, and successfully detected low-abundance KRAS mutations in clinical

  1. Next-generation sequencing for diagnosis of thoracic aortic aneurysms and dissections: diagnostic yield, novel mutations and genotype phenotype correlations.

    Science.gov (United States)

    Poninska, J K; Bilinska, Z T; Franaszczyk, M; Michalak, E; Rydzanicz, M; Szpakowski, E; Pollak, A; Milanowska, B; Truszkowska, G; Chmielewski, P; Sioma, A; Janaszek-Sitkowska, H; Klisiewicz, A; Michalowska, I; Makowiecka-Ciesla, M; Kolsut, P; Stawinski, P; Foss-Nieradko, B; Szperl, M; Grzybowski, J; Hoffman, P; Januszewicz, A; Kusmierczyk, M; Ploski, R

    2016-05-04

    Thoracic aortic aneurysms and dissections (TAAD) are silent but possibly lethal condition with up to 40 % of cases being hereditary. Genetic background is heterogeneous. Recently next-generation sequencing enabled efficient and cost-effective examination of gene panels. Aim of the study was to define the diagnostic yield of NGS in the 51 TAAD patients and to look for genotype-phenotype correlations within families of the patients with TAAD. 51 unrelated TAAD patients were examined by either whole exome sequencing or TruSight One sequencing panel. We analyzed rare variants in 10 established thoracic aortic aneurysms-associated genes. Whenever possible, we looked for co-segregation in the families. Kaplan-Meier survival curve was constructed to compare the event-free survival depending on genotype. Aortic events were defined as acute aortic dissection or first planned aortic surgery. In 21 TAAD patients we found 22 rare variants, 6 (27.3 %) of these were previously reported, and 16 (73.7 %) were novel. Based on segregation data, functional analysis and software estimations we assumed that three of novel variants were causative, nine likely causative. Remaining four were classified as of unknown significance (2) and likely benign (2). In all, 9 (17.6 %) of 51 probands had a positive result when considering variants classified as causative only and 18 (35.3 %) if likely causative were also included. Genotype-positive probands (n = 18) showed shorter mean event free survival (41 years, CI 35-46) than reference group, i.e. those (n = 29) without any plausible variant identified (51 years, CI 45-57, p = 0.0083). This effect was also found when the 'genotype-positive' group was restricted to probands with 'likely causative' variants (p = 0.0092) which further supports pathogenicity of these variants. The mean event free survival was particularly low (37 years, CI 27-47) among the probands with defects in the TGF beta signaling (p = 0.0033 vs. the

  2. Using dried blood spots collected under field condition to determine HIV-1 diversity and drug resistance mutations in resource limited Tanzania

    Directory of Open Access Journals (Sweden)

    James Kimaro

    2014-11-01

    Full Text Available Introduction: A dried blood spot (DBS on filter paper has been used for different tests globally and has gained popularities in resource limited settings especially during HIV/AIDS epidemic. We assessed the efficiency of molecular characterization of HIV-1 subtypes using DBS collected under field conditions in northern Tanzania. Materials and Methods: In 2011 and 2012, 60 DBS samples were collected under field conditions from exposed and newly diagnosed HIV-1 infected children from Kilimanjaro (n=20, Arusha (n=20, Tanga (n=10 and Manyara (n=10. Results and discussion: Of 60 DBS analyzed at both Protease (PR and Reverse Transcriptase (RT regions, 45 (75% were analyzed, including 17 (85% from Kilimanjaro, 15 (75% from Arusha, 8 (80% from Tanga, and 5 (50% from Manyara region. All 45 DBS characterized had viral load above 1000 copies/mL with mean log10 viral loads of 3.87 copies/mL (SD 0.995. The phylogenetic results indicated presence of subtype and circulating recombinant form (CRF. In which, 24 were subtype A1 (53.33%, 16 were subtype C (35.55%, 3 were subtype D (6.67% and 2 were CRF10_CD (4.35%. All major mutations were detected in the RT region, none from protease (PR region. The mutations detected were Y181C (n=8, K103 (n=4 and G190A (n=1, conferring resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs, and M184V (n=1, conferring resistance to lamivudine and emtricitabine. Conclusions: Our results indicate that DBS collected from field conditions in resource scarcity areas can be used to determine the phylogeny of the virus and drug resistance mutations in areas with diverse HIV-1 group M subtypes.

  3. Microbial gas generation under expected Waste Isolation Pilot Plant repository conditions

    Energy Technology Data Exchange (ETDEWEB)

    Francis, A.J.; Gillow, J.B.; Giles, M.R. [Brookhaven National Lab., Upton, NY (United States). Dept. of Applied Science

    1997-03-01

    Gas generation from the microbial degradation of the organic constituents of transuranic waste under conditions expected at the Waste Isolation Pilot Plant (WIPP) repository was investigated at Brookhaven National Laboratory. The biodegradation of mixed cellulosics (various types of paper) and electron-beam irradiated plastic and rubber materials (polyethylene, polyvinylchloride, neoprene, hypalon, and leaded hypalon) was examined. The rate of gas production from cellulose biodegradation in inundated samples incubated for 1,228 days at 30 C was biphasic, with an initial rapid rate up to approximately 600 days incubation, followed by a slower rate. The rate of total gas production in anaerobic samples containing mixed inoculum was as follows: 0.002 mL/g cellulose/day without nutrients; 0.004 mL/g cellulose/day with nutrients; and 0.01 mL/g cellulose/day in the presence of excess nitrate. Carbon dioxide production proceeded at a rate of 0.009 {micro}mol/g cellulose/day in anaerobic samples without nutrients, 0.05 {micro}mol/g cellulose/day in the presence of nutrients, and 0.2 {micro}mol/g cellulose/day with excess nitrate. Adding nutrients and excess nitrate stimulated denitrification, as evidenced by the accumulation of N{sub 2}O in the headspace (200 {micro}mol/g cellulose). The addition of the potential backfill bentonite increased the rate of CO{sub 2} production to 0.3 {micro}mol/g cellulose/day in anaerobic samples with excess nitrate. Analysis of the solution showed that lactic, acetic, propionic, butyric, and valeric acids were produced due to cellulose degradation. Samples incubated under anaerobic humid conditions for 415 days produced CO{sub 2} at a rate of 0.2 {micro}mol/g cellulose/day in the absence of nutrients, and 1 {micro}mol/g cellulose/day in the presence of bentonite and nutrients. There was no evidence of biodegradation of electron-beam irradiated plastic and rubber.

  4. Generation of Mice with Hepatocyte-Specific Conditional Deletion of Notum.

    Directory of Open Access Journals (Sweden)

    Frédéric Canal

    Full Text Available Fine tuning of the Wnt/β-catenin signaling pathway is essential for the proper development and function of the liver. Aberrant activation of this pathway is observed in 20%-40% of hepatocellular carcinomas (HCC. Notum encodes a secreted Wnt deacylase that inhibits Wnt activity and thereby restricts the zone of activation of Wnt/β-catenin signaling. An important role of NOTUM has been described in development in drosophila, planaria and zebrafish, but its role in the mammalian liver is unknown. Notum is required for spatial control of the Wnt/β-catenin signaling in several animal models and the Wnt/β-catenin pathway contributes to liver patterning involved in metabolic zonation. Therefore, Notum may be involved in the liver patterning induced by the Wnt/β-catenin signaling during the adult stage.We generated a conditional Notum knockout mouse mutant to study the effect of the deletion of Notum in the liver. We show that Notum is a direct target of the Wnt/β-catenin signaling in the liver. Liver-specific deletion of Notum did not modify liver zonation, but Notum deletion had a long-term effect on mouse physiology. In particular, male mutant mice developed metabolic disorders.We show that Notum is not a key actor of Wnt/β-catenin-dependent liver patterning of adult mice, but has role in liver glucose homeostasis. Male mice deficient in Notum specifically in the liver develop metabolic dysfunctions implicating Notum in the development of Type 2 diabetes.

  5. Necessary Conditions for Nonlinear Ultrasonic Modulation Generation Given a Localized Fatigue Crack in a Plate-Like Structure

    Directory of Open Access Journals (Sweden)

    Hyung Jin Lim

    2017-02-01

    Full Text Available It has been shown that nonlinear ultrasonics can be more sensitive to local incipient defects, such as a fatigue crack, than conventional linear ultrasonics. Therefore, there is an increasing interest in utilizing nonlinear ultrasonics for structural health monitoring and nondestructive testing applications. While the conditions, which are the necessary conditions that should be satisfied for the generation of nonlinear harmonic components, are extensively studied for distributed material nonlinearity, little work has been done to understand the necessary conditions at the presence of a localized nonlinear source such as a fatigue crack. In this paper, the necessary conditions of nonlinear ultrasonic modulation generation in a plate-like structure are formulated specifically for a localized nonlinear source. Then, the correctness of the formulated necessary conditions is experimentally verified using ultrasounds obtained from aluminum plates.

  6. [Method for direct generation data for formatted case report forms based on requirement for data authenticity in actual clinical conditions].

    Science.gov (United States)

    Shao, Ming-Yi; Liu, Bao-Yan; He, Li-Yun; Zhang, Run-Shun

    2013-04-01

    Data authenticity is the basic requirement of clinical studies. In actual clinical conditions how to establish formatted case report forms (CRF) in line with the requirement for data authenticity is the key to ensure clinical data quality. On the basis of the characteristics of clinical data in actual clinical conditions, we determined elements for establishing formatted case report forms by comparing differences in data characteristics of CRFs in traditional clinical studies and in actual clinical conditions, and then generated formatted case report forms in line with the requirement for data authenticity in actual clinical conditions. The data of formatted CRFs generated in this study could not only meet the requirement for data authenticity of clinical studies in actual clinical conditions, but also comply with data management practices for clinical studies, thus it is deemed as a progress in technical methods.

  7. A Study on the Optimal Generation Mix Based on Portfolio Theory with Considering the Basic Condition for Power Supply

    Science.gov (United States)

    Kato, Moritoshi; Zhou, Yicheng

    This paper presents a novel method to analyze the optimal generation mix based on portfolio theory with considering the basic condition for power supply, which means that electricity generation corresponds with load curve. The optimization of portfolio is integrated with the calculation of a capacity factor of each generation in order to satisfy the basic condition for power supply. Besides, each generation is considered to be an asset, and risks of the generation asset both in its operation period and construction period are considered. Environmental measures are evaluated through restriction of CO2 emissions, which are indicated by CO2 price. Numerical examples show the optimal generation mix according to risks such as the deviation of capacity factor of nuclear power or restriction of CO2 emissions, the possibility of introduction of clean coal technology (IGCC, CCS) or renewable energy, and so on. The results of this work will be possibly applied as setting the target of the generation mix for the future according to prospects of risks of each generation and restrictions of CO2 emissions.

  8. Helicobacter pylori infection generated gastric cancer through p53-Rb tumor-suppressor system mutation and telomerase reactivation

    Institute of Scientific and Technical Information of China (English)

    Jing Lan; Yong-Yan Xiong; Yi-Xian Lin; Bi-Cheng Wang; Ling-Ling Gong; Hui-Sen Xu; Guang-Song Guo

    2003-01-01

    AIM: To investigate the relationship between Helicobacterpylori (H. pylori) infection and the expressions of the p53,Rb, c-myc, bcl-2 and hTERT mRNA in a series of diseasesfrom chronic gastritis (CG), intestinal metaplasia type Ⅰ or Ⅱ(IMⅠ-Ⅱ), intestinal metaplasia type Ⅲ (IMⅢ), mild or modestdysplasia (DysⅠ-Ⅱ), severe dysplasia (DysⅢ) to gastric cancer(GC) and to elucidate the mechanism of gastriccarcinogenesis relating to H.pyloriinfection.METHODS: 272 cases between 1998 and 2001 wereavailable for the study including 42 cases of CG, 46 cases ofIMⅠ-Ⅱ, 25 cases of IMⅢ, 48 cases of DysⅠ-Ⅱ, 27 cases ofDysⅢ, 84 cases of GC.-H. pyloriinfection and the expressionsof p53, Rb, c-myc, bcl-2 were detected by means ofstreptavidin-peroxidase (SP) immunohistochemical method.HTERT mRNA was detected byin situ hybridization(ISH).RESULTS: The expressions of p53, Rb, c-myc, hTERT mRNAand bcl-2 were higher in the GC than in CG, IN, Dys. Theexpression of c-myc was higher in IMⅢ with-H.pyloriinfection(10/16) than that without infection (1/9) and the positive ratein DysⅠ-Ⅱ and DysⅢ with-H.pyloriinfection was 18/30 and 13/17, respectively, higher than that without infection (4/18 and3/10, respectively). In our experiment mutated p53 had noassociation with H.pyloriinfection, theexpression of Rb wasassociated with-H. pyloriinfection in GC, but the p53-Rb tumor-suppressor system abnormal in DysⅠ-Ⅱ cases, DysⅢⅡ and GCcases with H. pyloriinfection was 21/30, 15/17 and 48/48respecively, higher than non-infection groups (4/18, 3/10, 28/36). Furthermore the level of hTERT mRNA in GC with H. pyloriinfection (47/48) was higher than that without infection (30/36), however the relationship between bcl-2 and H. pyloriwasonly in IMⅢ. C-myc had a close association with hTERT mRNAin DysⅢ and GC (P=0.0 253,0.0 305 respectively).CONCLUSION: In the gastric carcinogenesis, H. pylorimightcause the severe imbalance of proliferation and apoptosisin the

  9. Testing of PLL-based True Random Number Generator in Changing Working Conditions

    Directory of Open Access Journals (Sweden)

    M. Simka

    2011-04-01

    Full Text Available Security of cryptographic systems depends significantly on security of secret keys. Unpredictability of the keys is achieved by their generation by True Random Number Generators (TRNGs. In the paper we analyze behavior of the Phase-Locked Loop (PLL based TRNG in changing working environment. The frequency of signals synthesized by PLL may be naturally influenced by chip temperature. We show what impact the temperature has on the quality of generated random sequence of the PLL-based TRNG. Thank to analysis of internal signals of the generator we are able to prove dependencies between the PLL parameters, statistical parameters of the generated sequence and temperature. Considering the measured results of experiments we form a new requirement in order to improve the robustness of the designed TRNG.

  10. USING OXYGEN-CONSUMING THERMOSET PLASTICS TO GENERATE HYPOXIC CONDITIONS IN MICROFLUIDIC DEVICES FOR POTENTIAL CELL CULTURE APPLICATIONS

    DEFF Research Database (Denmark)

    Sticker, Drago; Rothbauer, Mario; Ehgartner, Josef

    2017-01-01

    The precise control of the oxygen concentration in a cellular environment allows the study of cells under physiologically relevant conditions. This work reports on a novel method for the generation of reduced dissolved oxygen concentrations in microfluidic chambers for cell- and organ......-on-chip applications. Using a thermoset polymeric material (OSTEMERTM), which effectively scavenges dissolved oxygen (DO), microfluidic devices have been fabricated where oxygen was rapidly depleted from the microfluidic chamber. It is shown that hypoxic and anaerobic conditions can be generated through the inherent...

  11. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-09-09

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

  12. Power density improvement of the power conditioning circuit for combined piezoelectric and electrodynamic generators

    Science.gov (United States)

    Zessin, H.; Spies, P.; Mateu, L.

    2016-11-01

    In this study, we report a power management circuit for a combined piezoelectric- electrodynamic generator. A piezoelectric element is bonded to a spring steel cantilever beam and a magnet, used as tip mass, oscillates through a coil. This principle creates the combined generator. A test setup has been created to automate the characterization of the piezoelectric generator and its power management circuit. Three different power management circuits for the piezoelectric part of the combined generator have been analysed: a bridge rectifier, an SSHI circuit with an external inductance and an SSHI circuit which utilizes the coil of the electrodynamic generator as circuit element. The three circuits are compared in terms of their output power, efficiency and power density. The SSHI circuit with an external inductance has the highest output power and efficiency, followed by the SSHI circuit with the electrodynamic generator coil. The power density of the bridge rectifier is the highest but for higher efficiency the power density of the SSHI circuit with the coil of the electromagnetic generator reaches the best results.

  13. Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

    Directory of Open Access Journals (Sweden)

    Wendy W J de Leng

    Full Text Available Targeted Next Generation Sequencing (NGS offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE versus fresh frozen (FF tissue on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

  14. A thrombomodulin mutation that impairs active protein C generation is detrimental in severe pneumonia-derived gram-negative sepsis (melioidosis.

    Directory of Open Access Journals (Sweden)

    Liesbeth M Kager

    2014-04-01

    Full Text Available BACKGROUND: During severe (pneumosepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC into active protein C (APC. APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumosepsis in South-East Asia caused by Burkholderia (B. pseudomallei. METHODOLOGY/PRINCIPAL FINDINGS: (WT mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination. CONCLUSION/SIGNIFICANCE: TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumosepsis.

  15. Evaluation of Mutational Testing of Preneoplastic Barrett's Mucosa by Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Endoscopic Samples for Detection of Concurrent Dysplasia and Adenocarcinoma in Barrett's Esophagus.

    Science.gov (United States)

    Del Portillo, Armando; Lagana, Stephen M; Yao, Yuan; Uehara, Takeshi; Jhala, Nirag; Ganguly, Tapan; Nagy, Peter; Gutierrez, Jorge; Luna, Aesis; Abrams, Julian; Liu, Yang; Brand, Randall; Sepulveda, Jorge L; Falk, Gary W; Sepulveda, Antonia R

    2015-07-01

    Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EAC (N = 13); progressors to HGD or EAC (BIM-P) with BIM and a worse diagnosis of HGD or EAC (N = 15); and four BIM-negative samples. No mutations were detected in the BIM-NP (0 of 13) or BIM-negative samples, whereas the BIM-P samples had mutations in 6 (75%) of 8 cases in TP53, APC, and CDKN2A (P = 0.0005), detected in samples with as low as 20% BIM. We found that next-generation sequencing from routine FFPE nonneoplastic Barrett's esophagus samples can detect multiple mutations in minute areas of BIM with high analytical sensitivity. Next-generation sequencing panels for detection of TP53 and possibly combined mutations in other genes, such as APC and CDKN2A, may be useful in the clinical setting to improve dysplasia and cancer surveillance in patients with Barrett's esophagus.

  16. A probability evaluation method of early deterioration condition for the critical components of wind turbine generator systems

    Science.gov (United States)

    Hu, Yaogang; Li, Hui; Liao, Xinglin; Song, Erbing; Liu, Haitao; Chen, Z.

    2016-08-01

    This study determines the early deterioration condition of critical components for a wind turbine generator system (WTGS). Due to the uncertainty nature of the fluctuation and intermittence of wind, early deterioration condition evaluation poses a challenge to the traditional vibration-based condition monitoring methods. Considering the its thermal inertia and strong anti-interference capacity, temperature characteristic parameters as a deterioration indication cannot be adequately disturbed by the uncontrollable noise and uncertainty nature of wind. This paper provides a probability evaluation method of early deterioration condition for critical components based only on temperature characteristic parameters. First, the dynamic threshold of deterioration degree function was proposed by analyzing the operational data between temperature and rotor speed. Second, a probability evaluation method of early deterioration condition was presented. Finally, two cases showed the validity of the proposed probability evaluation method in detecting early deterioration condition and in tracking their further deterioration for the critical components.

  17. Susceptibility of steam generator tubes in secondary conditions: Effects of lead and sulphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomez Briceno, D.; Garcia, M.S.; Castano, M.L.; Lancha, A.M. [CIEMAT, Madrid (Spain)

    1997-02-01

    IGA/SCC on the secondary side of steam generators is increasing every year, and represents the cause of some steam generator replacements. Until recently, caustic and acidic environments have been accepted as causes of IGA/SCC, particulary in certain environments: in sludge pile on the tube sheet; at support crevices; in free span. Lead and sulfur have been identified as significant impurities. Present thoughts are that some IGA/SCC at support crevices may have occurred in nearly neutral or mildly alkaline environments. Here the authors present experimental work aimed at studying the influence of lead and sulfur on the behaviour of steam generator tube alloys in different water environments typical of steam generators. Most test results ran for at least 2000 hours, and involved visual and detailed surface analysis during and following the test procedures.

  18. Grid Voltage Synchronization for Distributed Generation Systems under Grid Fault Conditions

    DEFF Research Database (Denmark)

    Luna, Alvaro; Rocabert, J.; Candela, I.

    2015-01-01

    The actual grid code requirements for the grid connection of distributed generation systems, mainly wind and PV systems, are becoming very demanding. The Transmission System Operators (TSOs) are especially concerned about the Low Voltage Ride Through requirements. Solutions based on the installat......The actual grid code requirements for the grid connection of distributed generation systems, mainly wind and PV systems, are becoming very demanding. The Transmission System Operators (TSOs) are especially concerned about the Low Voltage Ride Through requirements. Solutions based...

  19. Preliminary report for analysis of genome wide mutations from four ciprofloxacin resistant B. anthracis Sterne isolates generated by Illumina, 454 sequencing and microarrays for DHS

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, Crystal [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vergez, Lisa [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hinckley, Aubree [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Thissen, James [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Gardner, Shea [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jackson, Paul [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Ellingson, Sally [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hauser, Loren [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Brettin, Tom [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Fofanov, Viacheslav [Eureka Genomics, Hercules, CA (United States); Koshinsky, Heather [Eureka Genomics, Hercules, CA (United States); Fofanov, Yuriy [Univ. of Houston, TX (United States)

    2011-06-21

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzed the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.

  20. Generations.

    Science.gov (United States)

    Chambers, David W

    2005-01-01

    Groups naturally promote their strengths and prefer values and rules that give them an identity and an advantage. This shows up as generational tensions across cohorts who share common experiences, including common elders. Dramatic cultural events in America since 1925 can help create an understanding of the differing value structures of the Silents, the Boomers, Gen Xers, and the Millennials. Differences in how these generations see motivation and values, fundamental reality, relations with others, and work are presented, as are some applications of these differences to the dental profession.

  1. Simulation of the transient processes of load rejection under different accident conditions in a hydroelectric generating set

    Science.gov (United States)

    Guo, W. C.; Yang, J. D.; Chen, J. P.; Peng, Z. Y.; Zhang, Y.; Chen, C. C.

    2016-11-01

    Load rejection test is one of the essential tests that carried out before the hydroelectric generating set is put into operation formally. The test aims at inspecting the rationality of the design of the water diversion and power generation system of hydropower station, reliability of the equipment of generating set and the dynamic characteristics of hydroturbine governing system. Proceeding from different accident conditions of hydroelectric generating set, this paper presents the transient processes of load rejection corresponding to different accident conditions, and elaborates the characteristics of different types of load rejection. Then the numerical simulation method of different types of load rejection is established. An engineering project is calculated to verify the validity of the method. Finally, based on the numerical simulation results, the relationship among the different types of load rejection and their functions on the design of hydropower station and the operation of load rejection test are pointed out. The results indicate that: The load rejection caused by the accident within the hydroelectric generating set is realized by emergency distributing valve, and it is the basis of the optimization for the closing law of guide vane and the calculation of regulation and guarantee. The load rejection caused by the accident outside the hydroelectric generating set is realized by the governor. It is the most efficient measure to inspect the dynamic characteristics of hydro-turbine governing system, and its closure rate of guide vane set in the governor depends on the optimization result in the former type load rejection.

  2. Characterization of Wear Particles Generated from CoCrMo Alloy under Sliding Wear Conditions.

    Science.gov (United States)

    Pourzal, R; Catelas, I; Theissmann, R; Kaddick, C; Fischer, A

    2011-07-29

    Biological effects of wear products (particles and metal ions) generated by metal-on-metal (MoM) hip replacements made of CoCrMo alloy remain a major cause of concern. Periprosthetic osteolysis, potential hypersensitivity response and pseudotumour formation are possible reactions that can lead to early revisions. To accurately analyse the biological response to wear particles from MoM implants, the exact nature of these particles needs to be characterized. Most previous studies used energy-dispersive X-ray spectroscopy (EDS) analysis for characterization. The present study used energy filtered transmission electron microscopy (TEM) and electron diffraction pattern analysis to allow for a more precise determination of the chemical composition and to gain knowledge of the crystalline structure of the wear particles.Particles were retrieved from two different test rigs: a reciprocating sliding wear tribometer (CoCrMo cylinder vs. bar) and a hip simulator according to ISO 14242-1 (CoCrMo head vs. CoCrMo cup). All tests were conducted in bovine serum. Particles were retrieved from the test medium using a previously published enzymatic digestion protocol.Particles isolated from tribometer samples had a size of 100 - 500 nm. Diffraction pattern analysis clearly revealed the lattice structure of strain induced hcp ε-martensite. Hip simulator samples revealed numerous particles of 15 - 30 nm and 30 - 80 nm size. Most of the larger particles appeared to be only partially oxidized and exhibited cobalt locally. The smallest particles were Cr(2)O(3) with no trace of cobalt. It optically appeared that these Cr(2)O(3) particles were flaking off the surface of larger particles that depicted a very high intensity of oxygen, as well as chromium, and only background noise of cobalt. The particle size difference between the two test rigs is likely related to the conditions of the two tribosystems, in particular the difference in the sample geometry and in the type of sliding

  3. Effects of inflow conditions on discrete frequency noise generated by small, axial flow fans

    Science.gov (United States)

    Washburn, K. B.; Lauchle, G. C.

    1985-09-01

    Discrete frequency acoustic radiation is generated by subsonic axial flow fans through both steady and unsteady blade loading. Steady loading is a function of pumping requirement, and unsteady loading is generated by spatially periodic inflow distortions. The latter effect is the dominant generation mechanism when small fans are used to cool electronic equipment. Fans mounted to exhaust out of a device ingest distortions created by all objects within the unit. This work represents an empirical survey of noise generated by small axial flow cooling fans in the presence of upstream obstructions and various inlet configurations. The obstructions include a cylinder, a thin rectangle, a thick rectangle, and an electronic card gate model. Each of these represents obstructions found in a typical installation. Simple and modified inlet baffles, finger guards, and honeycomb flow straighteners are investigated. Design recommendations are offered to minimize discrete tone generation. These include aerodynamic shaping of unaviodable obstructions, a minimum axial distance of 0.3 fan radii for obstructions in the inlet, the avoidance of blockage in lateral inflow and the use of an inlet baffle to smooth inlet distortions. Account is taken of the spatial restictions of typical installations.

  4. Next-generation sequencing (NGS) as a fast molecular diagnosis tool for left ventricular noncompaction in an infant with compound mutations in the MYBPC3 gene.

    Science.gov (United States)

    Schaefer, Elise; Helms, Pauline; Marcellin, Luc; Desprez, Philippe; Billaud, Philippe; Chanavat, Valérie; Rousson, Robert; Millat, Gilles

    2014-03-01

    Left ventricular noncompaction (LVNC) is a clinically heterogeneous disorder characterized by a trabecular meshwork and deep intertrabecular myocardial recesses that communicate with the left ventricular cavity. LVNC is classified as a rare genetic cardiomyopathy. Molecular diagnosis is a challenge for the medical community as the condition shares morphologic features of hypertrophic and dilated cardiomyopathies. Several genetic causes of LVNC have been reported, with variable modes of inheritance, including autosomal dominant and X-linked inheritance, but relatively few responsible genes have been identified. In this report, we describe a case of a severe form of LVNC leading to death at 6 months of life. NGS sequencing using a custom design for hypertrophic cardiomyopathy panel allowed us to identify compound heterozygosity in the MYBPC3 gene (p.Lys505del, p.Pro955fs) in 3 days, confirming NGS sequencing as a fast molecular diagnosis tool. Other studies have reported neonatal presentation of cardiomyopathies associated with compound heterozygous or homozygous MYBPC3 mutations. In this family and in families in which parental truncating MYBPC3 mutations are identified, preimplantation or prenatal genetic screening should be considered as these genotypes leads to neonatal mortality and morbidity.

  5. Reliable Grid Condition Detection and Control of Single-Phase Distributed Power Generation Systems

    DEFF Research Database (Denmark)

    Ciobotaru, Mihai

    The constant growth of Distributed Power Generation Systems (DPGS) presents an efficient and economic way of generating electricity closer to the load(s). The DPGS can contribute to an efficient and renewable electricity future by potentially: increasing the use of renewable sources of energy......; improving the efficiency of the electricity system by reducing transmission and distribution losses; improving the security of the electricity supply through increased diversity of supply and reduced vulnerability to simultaneous system failures. However, the new trend of using DPGS comes also with a suite...... of new challenges. One of the challenges is the interaction between the DPGS and the utility grid. As a consequence, grid interconnection requirements applying to distributed generation are continuously updated in order to maintain the quality and the stability of the utility grid. The new upcoming...

  6. A Miniature Condition in Brahman Cattle is Associated with a Single Nucleotide Mutation Within the Growth Hormone Gene

    Science.gov (United States)

    Miniature Brahman cattle at the USDA ARS in Brooksville, FL have normal proportioned growth but are approximately 70% of normal mature height and weight when compared with Brahman cattle in the same heard. Pedigree analyses suggest that the condition is inherited as a recessive allele. The miniature...

  7. Focusing of Rayleigh waves generated by high-speed trains under the condition of ground vibration boom

    CERN Document Server

    Krylov, Victor V

    2015-01-01

    In the present paper, the effects of focusing of Rayleigh waves generated by high speed trains in the supporting ground under the condition of ground vibration boom are considered theoretically. These effects are similar to the effects of focusing of sound waves radiated by aircraft under the condition of sonic boom. In particular, if a railway track has a bend to provide the possibility of changing direction of train movement, the Rayleigh surface waves generated by high-speed trains under the condition of ground vibration boom may become focused. This results in concentration of their energy along a simple caustic line at one side of the track and in the corresponding increase in ground vibration amplitudes. The effect of focusing of Rayleigh waves may occur also if a train moves along a straight line with acceleration and its current speed is higher than Rayleigh wave velocity in the ground. The obtained results are illustrated by numerical calculations.

  8. Hybrid centralized-distributed power conditioning system for thermoelectric generator with high energy efficiency

    DEFF Research Database (Denmark)

    Wu, Hongfei; Sun, Kai; Chen, Min

    2013-01-01

    the proposed system, which benefits for implementing high MPPT efficiency and high conversion efficiency simultaneously. A hybrid MPPT control strategy is proposed for this HCD power conditioning system. The characteristics, circuit implementation and operation principles of the proposed system are presented......-distributed (HCD) power conditioning system for TEG and its control strategy are proposed in this paper. The HCD power conditioning system is composed by a centralized power conversion stage and multiple distributed power conversion stages. Most of the power is processed by the centralized power conversion stage...

  9. [Bipolar ionisation of indoor air through ion generators mountable into inflow ventilation and conditioning].

    Science.gov (United States)

    Dudarev, A A; Spichkin, G L; Denisikhina, D M; Burtsev, S I

    2010-01-01

    Experimental studies and digital modelling of artificial indoor air ionisation through bipolar ionisers mountable into inflow ventilation and conditioning proved possible creation of continuous even bipolar ion background in indoor air, similar to the natural one.

  10. Reasoning in Design: Idea Generation Condition Effects on Reasoning Processes and Evaluation of Ideas

    DEFF Research Database (Denmark)

    Cramer-Petersen, Claus Lundgaard; Ahmed-Kristensen, Saeema

    2015-01-01

    performance of ideas developed. Understanding reasoning in design and its relationship to the performance of ideas generated is important to understand design activity, which can be used to develop tools or methods that can improve the effectiveness of design teams. Protocol analyses were conducted...

  11. Increased power generation from primary sludge by a submersible microbial fuel cell and optimum operational conditions

    DEFF Research Database (Denmark)

    Vologni, Valentina; Kakarla, Ramesh; Angelidaki, Irini;

    2013-01-01

    Microbial fuel cells (MFCs) have received attention as a promising renewable energy technology for waste treatment and energy recovery. We tested a submersible MFC with an innovative design capable of generating a stable voltage of 0.250 ± 0.008 V (with a fixed 470 Ω resistor) directly from primary...

  12. Does Conditionality Generate Heterogeneity and Regressivity in Program Impacts? The Progresa Experience

    OpenAIRE

    Campo, Juan Carlos Chavez-Martin del

    2006-01-01

    We study both empirically and theoretically the consequences of introducing a conditional cash transfer scheme for the distribution of program impacts. Intuitively, if the conditioned-on good is normal, then better-off households tend to receive a larger positive impact. I formalize this insight by means of a simple model of child labor, applying the Nash-Bargaining approach as the solution concept. A series of tests for heterogeneity in program impacts are developed and applied to Progresa, ...

  13. Anditalea andensis ANESC-ST--An Alkaliphilic Halotolerant Bacterium Capable of Electricity Generation under Alkaline-Saline Conditions.

    Directory of Open Access Journals (Sweden)

    Wei Shi

    Full Text Available A great challenge in wastewater bioremediation is the sustained activity of viable microorganisms, which can contribute to the breakdown of waste contaminants, especially in alkaline pH conditions. Identification of extremophiles with bioremediation capability can improve the efficiency of wastewater treatment. Here, we report the discovery of an electrochemically active alkaliphilic halotolerant bacterium, Anditalea andensis ANESC-ST (=CICC10485T=NCCB 100412T, which is capable of generating bioelectricity in alkaline-saline conditions. A. andensis ANESC-ST was shown to grow in alkaline conditions between pH 7.0-11.0 and also under high salt condition (up to 4 wt% NaCl. Electrical output was further demonstrated in microbial fuel cells (MFCs with an average current density of ~0.5 µA/cm2, even under the harsh condition of 4 wt% NaCl and pH 9.0. Subsequent introduction of secreted extracellular metabolites into MFCs inoculated with Escherichia coli or Pseudomonas aeruginosa yielded enhanced electrical output. The ability of A. andensis ANESC-ST to generate energy under alkaline-saline conditions points towards a solution for bioelectricity recovery from alkaline-saline wastewater. This is the first report of A.andensis ANESC-ST producing bioelectricity at high salt concentration and pH.

  14. Transgenerational epimutations induced by multi-generation drought imposition mediate rice plant’s adaptation to drought condition

    Science.gov (United States)

    Zheng, Xiaoguo; Chen, Liang; Xia, Hui; Wei, Haibin; Lou, Qiaojun; Li, Mingshou; Li, Tiemei; Luo, Lijun

    2017-01-01

    Epigenetic mechanisms are crucial mediators of appropriate plant reactions to adverse environments, but their involvement in long-term adaptation is less clear. Here, we established two rice epimutation accumulation lines by applying drought conditions to 11 successive generations of two rice varieties. We took advantage of recent technical advances to examine the role of DNA methylation variations on rice adaptation to drought stress. We found that multi-generational drought improved the drought adaptability of offspring in upland fields. At single-base resolution, we discovered non-random appearance of drought-induced epimutations. Moreover, we found that a high proportion of drought-induced epimutations maintained their altered DNA methylation status in advanced generations. In addition, genes related to transgenerational epimutations directly participated in stress-responsive pathways. Analysis based on a cluster of drought-responsive genes revealed that their DNA methylation patterns were affected by multi-generational drought. These results suggested that epigenetic mechanisms play important roles in rice adaptations to upland growth conditions. Epigenetic variations have morphological, physiological and ecological consequences and are heritable across generations, suggesting that epigenetics can be considered an important regulatory mechanism in plant long-term adaptation and evolution under adverse environments. PMID:28051176

  15. Modeling the spectrum of infrasonic hydroacoustic radiation generated by the sea surface under storm conditions

    Science.gov (United States)

    Zapevalov, A. S.; Pokazeev, K. V.

    2016-09-01

    Generation of infrasonic radiation into a water medium by sea surface waves is analyzed. The analysis is carried out for the situation in which the infrasound is generated by surface waves with frequencies close to those of dominant waves. The presence of two wave systems on the sea surface is assumed: swell and wind waves. It is shown that if the frequencies of spectral peaks of wind waves and swell diverge by 20%, the maximum value of the radiation spectrum decreases by approximately 40% (if the general directions of the two wave systems are oriented strictly towards each other). A deviation of the general directions of the two wave systems from the opposite direction by 45° leads to a decrease in the maximum value of the radiation spectrum by more than two times.

  16. Liquid-phase chemical hydrogen storage: catalytic hydrogen generation under ambient conditions.

    Science.gov (United States)

    Jiang, Hai-Long; Singh, Sanjay Kumar; Yan, Jun-Min; Zhang, Xin-Bo; Xu, Qiang

    2010-05-25

    There is a demand for a sufficient and sustainable energy supply. Hence, the search for applicable hydrogen storage materials is extremely important owing to the diversified merits of hydrogen energy. Lithium and sodium borohydride, ammonia borane, hydrazine, and formic acid have been extensively investigated as promising hydrogen storage materials based on their relatively high hydrogen content. Significant advances, such as hydrogen generation temperatures and reaction kinetics, have been made in the catalytic hydrolysis of aqueous lithium and sodium borohydride and ammonia borane as well as in the catalytic decomposition of hydrous hydrazine and formic acid. In this Minireview we briefly survey the research progresses in catalytic hydrogen generation from these liquid-phase chemical hydrogen storage materials.

  17. Hydrocarbon generation conditions and exploration potential of the Taoudeni Basin, Mauritania

    Institute of Scientific and Technical Information of China (English)

    Gang Wenzhe

    2009-01-01

    The Taoudeni Basin is a typical and steady intracratonic basin in Mauritania, northwest Africa. There are six sets of potential source rocks and five regional unconformable surfaces of the Infracambrian and Paleozoic developed in the basin. We used seismic stratigraphic correlation to recover the denudation thickness of formations at a particular well location. Studies of the hydrocarbon generation history of the basin illustrate that hydrocarbon migration and accumulation occurred in the end of the Carboniferous, and after that, the whole basin suffered denudation for a long period of time. Because there is no thick Mesozoic overburden in the basin, the Silurian source rocks could not generate hydrocarbon in the Mesozoic era for the second time. Consequently, the prospects for successful hydrocarbon exploration in the basin are not good.

  18. Divertor conditions relevant for fusion reactors achieved with linear plasma generator

    NARCIS (Netherlands)

    van Eck, H. J. N.; Kleyn, A. W.; Lof, A.; van der Meiden, H. J.; van Rooij, G. J.; Scholten, J.; van Emmichoven, P. A. Zeijlma

    2012-01-01

    Intense magnetized hydrogen and deuterium plasmas have been produced with electron densities up to 3.6 × 1020 m−3 and electron temperatures up to 3.7 eV with a linear plasma generator. Exposure of a W target has led to average heat and particle flux densities well in excess of 4 MW m−2 and 1024 m−2

  19. Divertor conditions relevant for fusion reactors achieved with linear plasma generator

    NARCIS (Netherlands)

    H.J.N. van Eck; A.W. Kleijn; A. Lof; H.J. van der Meiden; G.J. van Rooij; J. Scholten; P.A. Zeijlmans van Emmichoven

    2012-01-01

    Intense magnetized hydrogen and deuterium plasmas have been produced with electron densities up to 3.6 × 10 20 m−3 and electron temperatures up to 3.7 eV with a linear plasma generator. Exposure of a W target has led to average heat and particle flux densities well in excess of 4 MW m−2 and 10 24 m−

  20. Condition-Based Maintenance Strategy for Production Systems Generating Environmental Damage

    Directory of Open Access Journals (Sweden)

    L. Tlili

    2015-01-01

    Full Text Available We consider production systems which generate damage to environment as they get older and degrade. The system is submitted to inspections to assess the generated environmental damage. The inspections can be periodic or nonperiodic. In case an inspection reveals that the environmental degradation level has exceeded the critical level U, the system is considered in an advanced deterioration state and will have generated significant environmental damage. A corrective maintenance action is then performed to renew the system and clean the environment and a penalty has to be paid. In order to prevent such an undesirable situation, a lower threshold level L is considered to trigger a preventive maintenance action to bring back the system to a state as good as new at a lower cost and without paying the penalty. Two inspection policies are considered (periodic and nonperiodic. For each one of them, a mathematical model and a numerical procedure are developed to determine simultaneously the preventive maintenance (PM threshold L∗ and the inspection sequence which minimize the average long-run cost per time unit. Numerical calculations are performed to illustrate the proposed maintenance policies and highlight their main characteristics with respect to relevant input parameters.

  1. A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Junghwa [Chemical Genomics National Research Lab., Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Jung, Hye Jin [Department of Pharmaceutical Engineering, Sun Moon University, Asansi, Chungnam 330-150 (Korea, Republic of); Jeong, Seung Hun; Kim, Hyoung Kyu; Han, Jin [National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Department of Health Sciences and Technology, Cardiovascular and Metabolic Disease Center, Inje University, Busan (Korea, Republic of); Kwon, Ho Jeong, E-mail: kwonhj@yonsei.ac.kr [Chemical Genomics National Research Lab., Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2014-12-12

    Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.

  2. Validation and Application of a Custom-Designed Targeted Next-Generation Sequencing Panel for the Diagnostic Mutational Profiling of Solid Tumors.

    Science.gov (United States)

    Froyen, Guy; Broekmans, An; Hillen, Femke; Pat, Karin; Achten, Ruth; Mebis, Jeroen; Rummens, Jean-Luc; Willemse, Johan; Maes, Brigitte

    2016-01-01

    The inevitable switch from standard molecular methods to next-generation sequencing for the molecular profiling of tumors is challenging for most diagnostic laboratories. However, fixed validation criteria for diagnostic accreditation are not in place because of the great variability in methods and aims. Here, we describe the validation of a custom panel of hotspots in 24 genes for the detection of somatic mutations in non-small cell lung carcinoma, colorectal carcinoma and malignant melanoma starting from FFPE sections, using 14, 36 and 5 cases, respectively. The targeted hotspots were selected for their present or future clinical relevance in solid tumor types. The target regions were enriched with the TruSeq approach starting from limited amounts of DNA. Cost effective sequencing of 12 pooled libraries was done using a micro flow cell on the MiSeq and subsequent data analysis with MiSeqReporter and VariantStudio. The entire workflow was diagnostically validated showing a robust performance with maximal sensitivity and specificity using as thresholds a variant allele frequency >5% and a minimal amplicon coverage of 300. We implemented this method through the analysis of 150 routine diagnostic samples and identified clinically relevant mutations in 16 genes including KRAS (32%), TP53 (32%), BRAF (12%), APC (11%), EGFR (8%) and NRAS (5%). Importantly, the highest success rate was obtained when using also the low quality DNA samples. In conclusion, we provide a workflow for the validation of targeted NGS by a custom-designed pan-solid tumor panel in a molecular diagnostic lab and demonstrate its robustness in a clinical setting.

  3. SubClonal Hierarchy Inference from Somatic Mutations: Automatic Reconstruction of Cancer Evolutionary Trees from Multi-region Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Noushin Niknafs

    2015-10-01

    Full Text Available Recent improvements in next-generation sequencing of tumor samples and the ability to identify somatic mutations at low allelic fractions have opened the way for new approaches to model the evolution of individual cancers. The power and utility of these models is increased when tumor samples from multiple sites are sequenced. Temporal ordering of the samples may provide insight into the etiology of both primary and metastatic lesions and rationalizations for tumor recurrence and therapeutic failures. Additional insights may be provided by temporal ordering of evolving subclones--cellular subpopulations with unique mutational profiles. Current methods for subclone hierarchy inference tightly couple the problem of temporal ordering with that of estimating the fraction of cancer cells harboring each mutation. We present a new framework that includes a rigorous statistical hypothesis test and a collection of tools that make it possible to decouple these problems, which we believe will enable substantial progress in the field of subclone hierarchy inference. The methods presented here can be flexibly combined with methods developed by others addressing either of these problems. We provide tools to interpret hypothesis test results, which inform phylogenetic tree construction, and we introduce the first genetic algorithm designed for this purpose. The utility of our framework is systematically demonstrated in simulations. For most tested combinations of tumor purity, sequencing coverage, and tree complexity, good power (≥ 0.8 can be achieved and Type 1 error is well controlled when at least three tumor samples are available from a patient. Using data from three published multi-region tumor sequencing studies of (murine small cell lung cancer, acute myeloid leukemia, and chronic lymphocytic leukemia, in which the authors reconstructed subclonal phylogenetic trees by manual expert curation, we show how different configurations of our tools can

  4. A probability evaluation method of early deterioration condition for the critical components of wind turbine generator systems

    DEFF Research Database (Denmark)

    Hu, Y.; Li, H.; Liao, X;

    2016-01-01

    This study determines the early deterioration condition of critical components for a wind turbine generator system (WTGS). Due to the uncertainty nature of the fluctuation and intermittence of wind, early deterioration condition evaluation poses a challenge to the traditional vibration......-based condition monitoring methods. Considering the its thermal inertia and strong anti-interference capacity, temperature characteristic parameters as a deterioration indication cannot be adequately disturbed by the uncontrollable noise and uncertainty nature of wind. This paper provides a probability evaluation...... method of early deterioration condition for critical components based only on temperature characteristic parameters. First, the dynamic threshold of deterioration degree function was proposed by analyzing the operational data between temperature and rotor speed. Second, a probability evaluation method...

  5. Generative period in development of Mammillaria Haw. (Cactaceae Juss. plants in greenhouse conditions

    Directory of Open Access Journals (Sweden)

    Kateryna Baglay

    2013-04-01

    Full Text Available The taxonomic composition of the collection of the genus Mammillariain O.V. Fomin Botanical Garden, as well as the peculiarities of blooming and seed germination have been considered. The list of the plants from the genus Mammillariain this collection which are included into IUCN Red List have been represented. The optimal conditions of seed conservation have been determined.

  6. Dissipative particle dynamics simulation of flow generated by two rotating concentric cylinders: boundary conditions.

    Science.gov (United States)

    Haber, S; Filipovic, N; Kojic, M; Tsuda, A

    2006-10-01

    The dissipative particle dynamics (DPD) method was used to simulate the flow in a system comprised of a fluid occupying the space between two cylinders rotating with equal angular velocities. The fluid, initially at rest, ultimately reaches a steady, linear velocity distribution (a rigid-body rotation). Since the induced flow field is solely associated with the no-slip boundary condition at the walls, we employed this system as a benchmark to examine the effect of bounce-back reflections, specular reflections, and Pivkin-Karniadakis no-slip boundary conditions, upon the steady-state velocity, density, and temperature distributions. An additional advantage of the foregoing system is that the fluid occupies inherently a finite bounded domain so that the results are affected by the prescribed no-slip boundary conditions only. Past benchmark systems such as Couette flow between two infinite parallel plates or Poiseuille flow in an infinitely long cylinder must employ artificial periodic boundary conditions at arbitrary upstream and downstream locations, a possible source of spurious effects. In addition, the effect of the foregoing boundary conditions on the time evolution of the simulated velocity profile was compared with that of the known, time-dependent analytical solution. It was shown that bounce-back reflection yields the best results for the velocity distributions with small fluctuations in density and temperature at the inner fluid domain and larger deviations near the walls. For the unsteady solutions a good fit is obtained if the DPD friction coefficient is proportional to the kinematic viscosity. Based on dimensional analysis and the numerical results a universal correlation is suggested between the friction coefficient and the kinematic viscosity.

  7. Mesenchymal stem cell-conditioned medium triggers neuroinflammation and reactive species generation in organotypic cultures of rat hippocampus.

    Science.gov (United States)

    Horn, Ana Paula; Bernardi, Andressa; Luiz Frozza, Rudimar; Grudzinski, Patrícia Bencke; Hoppe, Juliana Bender; de Souza, Luiz Fernando; Chagastelles, Pedro; de Souza Wyse, Angela Terezinha; Bernard, Elena Aida; Battastini, Ana Maria Oliveira; Campos, Maria Martha; Lenz, Guido; Nardi, Nance Beyer; Salbego, Christianne

    2011-07-01

    Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.

  8. A research on thermoelectric generator's electrical performance under temperature mismatch conditions for automotive waste heat recovery system

    Directory of Open Access Journals (Sweden)

    Z.B. Tang

    2015-03-01

    Full Text Available The thermoelectric generators recover useful energy by the function of thermoelectric modules which can convert waste heat energy into electricity from automotive exhaust. In the actual operation, the electrical connected thermoelectric modules are operated under temperature mismatch conditions and then the problem of decreased power output causes due to the inhomogeneous temperature gradient distribution on heat exchanger surface. In this case study, an individual module test system and a test bench have been carried out to test and analyze the impact of thermal imbalance on the output electrical power at module and system level. Variability of the temperature difference and clamping pressure are also tested in the individual module measurement. The system level experimental results clearly describe the phenomenon of thermoelectric generator's decreased power output under mismatched temperature condition and limited working temperature. This situation is improved with thermal insulation on the modules and proved to be effective.

  9. Generation of a human induced pluripotent stem cell (iPSC line from a patient with family history of diabetes carrying a C18R mutation in the PDX1 gene

    Directory of Open Access Journals (Sweden)

    Xianming Wang

    2016-09-01

    Full Text Available Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4 and Type 2 Diabetes Mellitus (T2DM. To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a C18R heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 C18R iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms.

  10. conditions

    Directory of Open Access Journals (Sweden)

    M. Venkatesulu

    1996-01-01

    Full Text Available Solutions of initial value problems associated with a pair of ordinary differential systems (L1,L2 defined on two adjacent intervals I1 and I2 and satisfying certain interface-spatial conditions at the common end (interface point are studied.

  11. A simple simulation approach to generate complex rainfall fields conditioned by elevation: example of the eastern Mediterranean region

    Science.gov (United States)

    Oriani, Fabio; Ohana-Levi, Noa; Straubhaar, Julien; Renard, Philippe; Karnieli, Arnon; Mariethoz, Grégoire; Morin, Efrat; Marra, Francesco

    2016-04-01

    Stochastically generating realistic rainfall fields is useful to study the uncertainty related to catchment recharge and its propagation to distributed hydrological models. To this end, it is critical to use weather radar images as training data, being the single most informative source for rainfall spatial heterogeneity. Generating realistic simulations is particularly important in regions like the eastern Mediterranean, where the synoptic conditions can lead to rainfall fields presenting various morphology, anisotropy and non-stationarity. The Direct Sampling (DS) technique [Mariethoz2010] is proposed here as a stochastic generator of spatial daily rainfall fields relying on the simulation of radar imagery. The technique is based on resampling of a training data set (in this case, a stack of radar images) and the generation of similar patterns to the ones found in the data. The strong point of DS, which makes it an attractive simulation approach for rainfall, is its capability to preserve the high-order statistical features present in the training image (e.g., rainfall cell shape, spatial non-stationarity) with minimal parameterization. Moreover, factors influencing rainfall, like elevation, can be used as conditioning variables, without the need of a complex statistical dependence model. A DS setup for radar image simulation is presented and tested for the simulation of daily rainfall fields using a 10-year radar-image record from the central region of Israel. Using a synoptic weather classification to train the model, the algorithm can generate realistic spatial fields for different rainfall types, preserving the variability and the covariance structure of the reference reasonably well. Moreover, the simulation is conditioned using the digital elevation model to preserve the complex relation between rainfall intensity and altitude that is characteristic for this region. [Mariethoz2010] G. Mariethoz, P. Renard, and J. Straubhaar. The direct sampling method to

  12. Coordinated control of a DFIG-based wind-power generation system with SGSC under distorted grid voltage conditions

    DEFF Research Database (Denmark)

    Yao, Jun; Li, Qing; Chen, Zhe

    2013-01-01

    currents injected to the grid. Furthermore, the respective PI-R controller in the positive synchronous reference frame for the SGSC voltage control and PGSC current control have been developed to achieve precise and rapid regulation of the corresponding components. Finally, the proposed coordinated control......This paper presents a coordinated control method for a doubly-fed induction generator (DFIG)-based wind-power generation system with a series grid-side converter (SGSC) under distorted grid voltage conditions. The detailed mathematical models of the DFIG system with SGSC are developed...... in the multiple synchronous rotating reference frames. In order to counteract the adverse effects of the voltage harmonics upon the DFIG, the SGSC generates series compensation control voltages to keep the stator voltage sinusoidal and symmetrical, which allows the use of the conventional vector control strategy...

  13. Sufficient conditions for the additivity of stall forces generated by multiple filaments or motors

    Science.gov (United States)

    Bameta, Tripti; Das, Dipjyoti; Das, Dibyendu; Padinhateeri, Ranjith; Inamdar, Mandar M.

    2017-02-01

    Molecular motors and cytoskeletal filaments work collectively most of the time under opposing forces. This opposing force may be due to cargo carried by motors or resistance coming from the cell membrane pressing against the cytoskeletal filaments. Some recent studies have shown that the collective maximum force (stall force) generated by multiple cytoskeletal filaments or molecular motors may not always be just a simple sum of the stall forces of the individual filaments or motors. To understand this excess or deficit in the collective force, we study a broad class of models of both cytoskeletal filaments and molecular motors. We argue that the stall force generated by a group of filaments or motors is additive, that is, the stall force of N number of filaments (motors) is N times the stall force of one filament (motor), when the system is reversible at stall. Conversely, we show that this additive property typically does not hold true when the system is irreversible at stall. We thus present a novel and unified understanding of the existing models exhibiting such non-addivity, and generalise our arguments by developing new models that demonstrate this phenomena. We also propose a quantity similar to thermodynamic efficiency to easily predict this deviation from stall-force additivity for filament and motor collectives.

  14. Secondary current properties generated by wind-induced water waves in experimental conditions

    Directory of Open Access Journals (Sweden)

    Michio Sanjou

    2014-06-01

    Full Text Available Secondary currents such as the Langmuir circulation are of high interest in natural rivers and the ocean because they have striking impacts on scour, sedimentation, and mass transport. Basic characteristics have been well-studied in straight open-channel flows. However, little is known regarding secondary circulation induced by wind waves. The presented study describes the generation properties of wind waves observed in the laboratory tank. Wind-induced water waves are known to produce large scale circulations. The phenomenon is observed together with high-speed and low-speed streaks, convergence and divergence zones, respectively. Therefore, it is important to determine the hydrodynamic properties of secondary currents for wind-induced water waves within rivers and lakes. In this study, using two high-speed CMOS cameras, stereoscopic particle image velocimetry (PIV measurements were conducted in order to reveal the distribution of all three components of velocity vectors. The experiments allowed us to investigate the three-dimensional turbulent structure under water waves and the generation mechanism of large-scale circulations. Additionally, a third CMOS camera was used to measure the spanwise profile of thefree-surface elevation. The time-series of velocity components and the free-surface were obtained simultaneously. From our experiments, free-surface variations were found to influence the instantaneous velocity distributions of the cross-sectional plane. We also considered thegeneration process by the phase analysis related to gravity waves and compared the contribution of the apparent stress.

  15. A High Temperature Experimental Characterization Procedure for Oxide-Based Thermoelectric Generator Modules under Transient Conditions

    DEFF Research Database (Denmark)

    Man, Elena Anamaria; Schaltz, Erik; Rosendahl, Lasse

    2015-01-01

    to 1000 °C with a load variation range value between [0.22 – 8.13] Ω. A total of ten thermocouples are placed in the setup with the purpose of measuring the temperature in specific points between the heater and the heat sink. Based on the readings, the temperature on the hot and cold side of the modules...... will be shown during the transient and steady-state thermal conditions....

  16. Geochemical Processes Controlling the Generation and Environmental Impacts of Acid Mine Drainage in Semi Arid Conditions

    OpenAIRE

    Magombedze, Chris

    2006-01-01

    This study evaluates the geochemical processes that control the geochemistry of acid mine drainage in semi arid conditions. The central objective is to characterise and understand the evolution of acid mine drainage and its potential environmental impacts on the Mazowe River sub-catchment, in north east Zimbabwe. The work is based on a case study at three neighbouring metal sulphide mines, namely Trojan Nickel Mine, Mazowe Gold Mine and Iron Duke Pyrites.The methodology used in this research ...

  17. Comparisons of methods for generating conditional Poisson samples and Sampford samples

    OpenAIRE

    Grafström, Anton

    2005-01-01

    Methods for conditional Poisson sampling (CP-sampling) and Sampford sampling are compared and the focus is on the efficiency of the methods. The efficiency is investigated by simulation in different sampling situations. It was of interest to compare methods since new methods for both CP-sampling and Sampford sampling were introduced by Bondesson, Traat & Lundqvist in 2004. The new methods are acceptance rejection methods that use the efficient Pareto sampling method. They are found to be ...

  18. Heat transfer in a couple stress fluid over a continuous moving surface with internal hat generation and convective boundary conditions

    Energy Technology Data Exchange (ETDEWEB)

    Hayat, Tasawar [Quaid-i-Azam Univ., Islamabad (Pakistan). Dept. of Mathematics; King Saud Univ., Riyadh (Saudi Arabia). Dept. of Physics; Iqbal, Zahid [Quaid-i-Azam Univ., Islamabad (Pakistan). Dept. of Mathematics; Qasim, Muhammad [COMSATS Institute of Information Technology (CIIT), Islamabad (Pakistan). Dept. of Mathematics; Aldossary, Omar M. [King Saud Univ., Riyadh (Saudi Arabia). Dept. of Physics

    2012-05-15

    This investigation reports the boundary layer flow and heat transfer characteristics in a couple stress fluid flow over a continuos moving surface with a parallel free stream. The effects of heat generation in the presence of convective boundary conditions are also investigated. Series solutions for the velocity and temperature distributions are obtained by the homotopy analysis method (HAM). Convergence of obtained series solutions are analyzed. The results are obtained and discussed through graphs for physical parameters of interest. (orig.)

  19. Differential winter stemflow generation under contrasting storm conditions in a southern New England broad-leaved deciduous forest

    Science.gov (United States)

    Levia, Delphis F., Jr.

    2004-04-01

    Despite the importance of stemflow as a hydroecological process, differential winter stemflow generation among and within precipitation types (e.g. snow, rain-to-snow) is poorly understood. The purpose of the present study is to understand winter stemflow generation better under contrasting meteorological conditions in a leafless deciduous forest. It is hypothesized that stemflow volume and production, expressed as a funnelling ratio, will differ significantly among and within precipitation event types. Acceptance of this hypothesis would mean that intra-event meteorological conditions have a significant and discernible effect on stemflow production in deciduous forests, differentially affecting soil moisture, groundwater recharge, soil solution chemistry, and nutrient cycling. Stemflow volumes generated from seven canopy trees were monitored in a leafless deciduous forest of southern New England on a precipitation event basis over two successive winter seasons. Stemflow volume was found to differ significantly among different and same-type precipitation events. A rain event that occurred on 8 December 1998 and snow-to-rain event on 18 January 1999 were of similar duration, magnitude, and intensity, yet produced stemflow volumes that differed by a factor of 4. For two snow-to-rain events, stemflow volumes differed by a factor of 11. Stemflow production even varies widely within a discrete precipitation event as a function of meteorological conditions. Significant differential stemflow yield under contrasting storm conditions obviates generalizations concerning stemflow production as a function of precipitation event type and necessitates a deeper, process-level understanding of winter stemflow generation before modelling exercises are undertaken.

  20. Reactive oxygen species generation-scavenging and signaling during plant-arbuscular mycorrhizal and Piriformospora indica interaction under stress condition

    OpenAIRE

    Manoj Nath; Deepesh Bhatt; Ram Prasad; Sarvajeet Singh Gill; Naser A. Anjum; Narendra Tuteja

    2016-01-01

    A defined balance between the generation and scavenging of reactive oxygen species (ROS) is essential to utilize ROS as an adaptive defense response of plants under biotic and abiotic stress conditions. Moreover, ROS are not only a major determinant of stress response but also acts as signaling molecule that regulates various cellular processes including plant-microbe interaction. In particular, rhizosphere constitutes the biologically dynamic zone for plant–microbe interactions which forms a...

  1. Wind-power generators designed for weak and moderate wind conditions

    Energy Technology Data Exchange (ETDEWEB)

    Spinadel, E.; Spinadel, P.; Gamallo, F. [University of Buenos Aires (Argentina)

    1996-12-31

    The United Nations Organization for Industrial Development (UNIDO), carried out a technical assistance program for transferring technology and for adapting the design of a wind power turbine to be produced in the Republic of Indonesia. The basic purpose for this program aimed at providing electricity to people living in remote areas and/or islands of the Indonesian Archipelago, was that of selecting the appropriate design for a wind power turbine, which would allow for the transference of technology and set the conditions for its local serial production. Two reconnaissance missions were carried out to study the conditions and the possibilities offered by the Indonesian industry, the wind characteristics of the proposed site for installation, and the capability of the people there to carry out the project. A solution was proposed that optimised the requirements, needs, and local technical capacity`s equation. The electricity producing element would have to be a rugged, low cost 500/1000 Watt vertical-axis mill, which at the same time should need to be easy to produce and install, with extremely low maintenance requirements. As such a product is not readily available in the market, the decision was taken to design it in Argentina. A prototype appropriate for the environmental conditions, which was approved by UNIDO and the Indonesian government was produced. (author)

  2. OPTIMIZATION OF THE OPERATING CONDITIONS FOR RHAMNOLIPID PRODUCTION USING SLAUGHTERHOUSE-GENERATED INDUSTRIAL FLOAT AS SUBSTRATE

    Directory of Open Access Journals (Sweden)

    W. S. Borges

    2015-06-01

    Full Text Available AbstractBiosurfactants have a wide range of applications in emulsions, separations, and solubilization because these chemicals reduce the surface tension and viscosity of solutions. This work studied rhamnolipid production using a batch bioreactor with a working volume of 1.5 liters, Pseudomonas aeruginosa ATCC (American Type Culture Collection 10145 strain, and the greasy effluent from the slaughter of poultry and pigs as the substrate. The main goal of this research was to evaluate the level of aeration, agitation speed and inoculum concentration using a Central Composite Design (CCD. Experimental conditions were selected using the surface response technique obtained from the CCD, and the results were validated to test the reproducibility. The following operating conditions were selected: 1.2 vvm level of aeration, 600 rpm agitation speed, and 1.0 g/L biomass inoculum concentration. Under these conditions, the following results were obtained: the rhamnose production, surface tension and emulsifying index were 5.37 g/L, 25.6 dyne/cm and 100%, respectively.

  3. Fuel Savings and Emission Reductions from Next-Generation Mobile Air Conditioning Technology in India

    Energy Technology Data Exchange (ETDEWEB)

    Chaney, L.; Thundiyil, K.; Andersen, S.; Chidambaram, S.; Abbi, Y. P.

    2007-01-01

    Up to 19.4% of vehicle fuel consumption in India is devoted to air conditioning (A/C). Indian A/C fuel consumption is almost four times the fuel penalty in the United States and close to six times that in the European Union because India's temperature and humidity are higher and because road congestion forces vehicles to operate inefficiently. Car A/C efficiency in India is an issue worthy of national attention considering the rate of increase of A/C penetration into the new car market, India's hot climatic conditions and high fuel costs. Car A/C systems originally posed an ozone layer depletion concern. Now that industrialized and many developing countries have moved away from ozone-depleting substances per Montreal Protocol obligations, car A/C impact on climate has captured the attention of policy makers and corporate leaders. Car A/C systems have a climate impact from potent global warming potential gas emissions and from fuel used to power the car A/Cs. This paper focuses on car A/C fuel consumption in the context of the rapidly expanding Indian car market and how new technological improvements can result in significant fuel savings and consequently, emission reductions. A 19.4% fuel penalty is associated with A/C use in the typical Indian passenger car. Car A/C fuel use and associated tailpipe emissions are strong functions of vehicle design, vehicle use, and climate conditions. Several techniques: reducing thermal load, improving vehicle design, improving occupants thermal comfort design, improving equipment, educating consumers on impacts of driver behaviour on MAC fuel use, and others - can lead to reduced A/C fuel consumption.

  4. Double-layer rotor magnetic shield performance analysis in high temperature superconducting synchronous generators under short circuit fault conditions

    Science.gov (United States)

    Hekmati, Arsalan; Aliahmadi, Mehdi

    2016-12-01

    High temperature superconducting, HTS, synchronous machines benefit from a rotor magnetic shield in order to protect superconducting coils against asynchronous magnetic fields. This magnetic shield, however, suffers from exerted Lorentz forces generated in light of induced eddy currents during transient conditions, e.g. stator windings short-circuit fault. In addition, to the exerted electromagnetic forces, eddy current losses and the associated effects on the cryogenic system are the other consequences of shielding HTS coils. This study aims at investigating the Rotor Magnetic Shield, RMS, performance in HTS synchronous generators under stator winding short-circuit fault conditions. The induced eddy currents in different circumferential positions of the rotor magnetic shield along with associated Joule heating losses would be studied using 2-D time-stepping Finite Element Analysis, FEA. The investigation of Lorentz forces exerted on the magnetic shield during transient conditions has also been performed in this paper. The obtained results show that double line-to-ground fault is of the most importance among different types of short-circuit faults. It was revealed that when it comes to the design of the rotor magnetic shields, in addition to the eddy current distribution and the associated ohmic losses, two phase-to-ground fault should be taken into account since the produced electromagnetic forces in the time of fault conditions are more severe during double line-to-ground fault.

  5. Early evaluation and on field conditions of resistance to Mycosphaerella fijiensis Morelet of plants from Grande naine (AAA cultivar, obtained through out tissue culture and mutations induction

    Directory of Open Access Journals (Sweden)

    Lourdes R. García

    2003-04-01

    Full Text Available The present work was carried out in the Plants Biotechnology Institute of the Central University of Las Villas. The plant material from the cv. Grande Naine (AAA was treated with physical mutagenic agents(gamma radiation 60Co source to induce genetic variability. The behaviour of the population to the black Sigatoka was evaluated. A somaclone was selected by its disease resistance and was in vitro multiplied and the plants were acclimatized to evaluate its behaviour facing the disease on greenhouse conditions and in a second cycle of multiplication in the field. The results showed that in the majority of the plants were not found differences respect cv Grande Naine, just one presented similar reaction to cv. ‘FHIA 18’ (AAAB (partially resistant as for the variable evaluated, being obtained a frequency of 0.018% for this character. This plant was named IBP 446. After 60 days of application of the mycelial homogenized of M. fijiensis in micropropagated plants of this somaclone, differences in the respect affectation states were found at susceptible witness in greenhouse conditions. When plants of the IBP 446 were evaluated in a second cycle of multiplication differences were found with the susceptible control only at flowering, while they behaved similar at susceptible control in the crop. Key words: early detection, breeding, mutation, Black Sigatoka

  6. Credit risk metric under the condition of mutative variance%变方差条件下的信用风险度量

    Institute of Scientific and Technical Information of China (English)

    韩立媛; 古志辉; 丁小培

    2012-01-01

    Based on the option pricing model, the paper uses smooth pasting to evaluate the debt value and probability of default under the condition of imitative variance. The conclusion are that the probability of default under conditions of invariable variance is less than under mutative variance. Furthermore, the probability of default will decrease with the risk-free rate, which means that the reduction of interest may lead to the rise of non-performing asset ratio.%以期权定价模型为基础,运用平滑粘贴条件求解了变方差条件下的负债价值及违约概率.研究表明方差不变条件下获得的违约概率低于变方差条件下的违约概率.随着无风险收益率的上升,债务违约概率将会减小,这说明政府降低利息率可能会导致银行不良资产比率上升.

  7. Development of elements of the condition monitoring system of turbo generators of thermal power stations and nuclear power plants

    Science.gov (United States)

    Kumenko, A. I.; Kostyukov, V. N.; Kuz'minykh, N. Yu.; Boichenko, S. N.; Timin, A. V.

    2017-08-01

    The rationale is given for the improvement of the regulatory framework for the use of shaft sensors for the in-service condition monitoring of turbo generators and the development of control systems of shaft surfacing and misalignments of supports. A modern concept and a set of methods are proposed for the condition monitoring of the "shaft line-thrust bearing oil film-turbo generator supports" system elements based on the domestic COMPACS® technology. The system raw data are design, technology, installation, and operating parameters of the turbo generator as well as measured parameters of the absolute vibration of supports and mechanical quantities, relative displacements and relative vibration of the rotor teeth in accordance with GOST R 55263-2012. The precalculated shaft line assembly line in the cold state, the nominal parameters of rotor teeth positions on the dynamic equilibrium curve, the static and dynamic characteristics of the oil film of thrust bearings, and the shaft line stiffness matrix of unit support displacements have been introduced into the system. Using the COMPACS-T system, it is planned to measure positions and oscillations of rotor teeth, to count corresponding static and dynamic characteristics of the oil film, and the static and dynamic loads in the supports in real time. Using the obtained data, the system must determine the misalignments of supports and corrective alignments of rotors of coupling halves, voltages in rotor teeth, welds, and bolts of the coupling halves, and provide automatic conclusion if condition monitoring parameters correspond to standard values. A part of the methodological support for the proposed system is presented, including methods for determining static reactions of supports under load, the method for determining shaft line stiffness matrices, and the method for solving the inverse problem, i.e., the determination of the misalignments of the supports by measurements of rotor teeth relative positions in bearing

  8. Generating and monitoring Schrödinger cats in conditional measurement on a beam splitter

    CERN Document Server

    Dakna, M; Knöll, L; Welsch, D G

    1998-01-01

    Preparation of Schrödinger-cat-like states via conditional output measurement on a beam splitter is studied. In the scheme, a mode prepared in a squeezed vacuum is mixed with a mode prepared in a Fock state and photocounting is performed in one of the output channels of the beam splitter. In this way the mode in the other output channel is prepared in a Schrödinger-cat-like state that is either a photon-subtracted or a photon-added Jacobi polynomial squeezed vacuum state, depending upon the difference between the number of photons in the input Fock state and the number of photons in the output Fock state onto which it is projected. Two possible photocounting schemes are considered, and the problem of monitoring cats that are ``hidden'' in a statistical mixture of states is studied.

  9. Dynamic Analysis of Jacket Substructure for Offshore Wind Turbine Generators under Extreme Environmental Conditions

    Directory of Open Access Journals (Sweden)

    Wen-Jeng Lai

    2016-10-01

    Full Text Available In order to develop dynamic analysis technologies regarding the design of offshore wind turbine generators (OWTGs, a special project called Offshore Code Comparison Collaboration Continuation (OC4 was conducted by IEA (International Energy Agency in 2010. A similar project named INER-OC4 has been performed by the Institute of Nuclear Energy Research (INER to develop the OWTG technologies of Taiwan. Since the jacket substructure will be applied to Taiwan OWTGs before 2020, the INER-OC4 project has been devoted to the design and analysis of jacket support structure. In this work, the preliminary result of INER-OC4 is presented. A simplified analysis procedure for jacket support structure has been proposed. Both of the NREL (National Renewable Energy Laboratory 5 MW OWTG FAST model and OC4 jacket substructure model have been built and analyzed under severe design load cases (DLCs of IEC (International Electrotechnical commission 61400-3. Simulation results of six severe DLCs are performed in this work and the results are in agreement with the requirements of API (American Petroleum Institute and NORSOK (Norwegian Petroleum Industry standards.

  10. The excitation conditions of magnetospheric convection by the electric current generated in the bow shock

    Science.gov (United States)

    Sedykh, P. A.; Ponomarev, E. A.

    The solar wind undergoes the greatest change of its parameters during the passage through the bow shock front Its density in this case increases by the factor of four and gas and magnetic pressure increase more than by an order of magnitude In this paper we re-examine the consequences of the fact of electric current generation at the bow shock front that we considered at an earlier date and the dependence of the direction of this current on the sign of IMF Bz-component The first consequence is the closure of the aforementioned current through the magnetosphere It was found that this process is a two-stage one Initially the electric field penetrates and establishes in the medium a new convective regime After that depending on the degree of flow inhomogeneity a plasma density distribution can be established which corresponds to the electric current equal to the external current The new steady state to which the new convection velocity field and the new plasma pressure field correspond is established within the time of the order of the transit time taken by the magnetosonic wave to propagate through the entire system Also a linkage between the power dissipated inside the magnetosphere and the parameters of plasma convection existing therein is shown

  11. Mice lacking Ras-GRF1 show contextual fear conditioning but not spatial memory impairments: convergent evidence from two independently generated mouse mutant lines

    Directory of Open Access Journals (Sweden)

    Raffaele ed'Isa

    2011-12-01

    Full Text Available Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning the Brambilla’s mice with a third mouse line (GENA53 in which a nonsense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in the Brambilla’s mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdalar functions but possibly to some distinct hippocampal connections specific to

  12. Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1

    Science.gov (United States)

    Robson, Matthew J.; Zhu, Chong-Bin; Quinlan, Meagan A.; Botschner, David A.; Baganz, Nicole L.; Lindler, Kathryn M.; Thome, Jason G.; Hewlett, William A.; Blakely, Randy D.

    2016-01-01

    The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1). Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP), with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV)-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development. PMID:26930558

  13. Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1.

    Directory of Open Access Journals (Sweden)

    Matthew J Robson

    Full Text Available The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1. Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP, with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development.

  14. Comparison study of judged clinical skills competence from standard setting ratings generated under different administration conditions.

    Science.gov (United States)

    Roberts, William L; Boulet, John; Sandella, Jeanne

    2017-02-21

    When the safety of the public is at stake, it is particularly relevant for licensing and credentialing exam agencies to use defensible standard setting methods to categorize candidates into competence categories (e.g., pass/fail). The aim of this study was to gather evidence to support change to the Comprehensive Osteopathic Medical Licensing-USA Level 2-Performance Evaluation standard setting design and administrative process. Twenty-two video recordings of candidates assessed for clinical competence were randomly selected from the 2014-2015 Humanistic domain test score distribution ranging from the highest to lowest quintile of performance. Nineteen panelists convened at the same site to receive training and practice prior to generating judgments of qualified or not qualified performance to each of the twenty videos. At the end of training, one panel remained onsite to complete their judgments and the second panel was released and given 1 week to observe the same twenty videos and complete their judgments offsite. The two one-sided test procedure established equivalence between panel group means at the 0.05 confidence level, controlling for rater errors within each panel group. From a practical cost-effective and administrative resource perspective, results from this study suggest it is possible to diverge from typical panel groups, who are sequestered the entire time onsite, to larger numbers of panelists who can make their judgments offsite with little impact on judged samples of qualified performance. Standard setting designs having panelists train together and then allowing those to provide judgments yields equivalent ratings and, ultimately, similar cut scores.

  15. Generation IV benchmarking of TRISO fuel performance models under accident conditions: Modeling input data

    Energy Technology Data Exchange (ETDEWEB)

    Collin, Blaise P. [Idaho National Laboratory (INL), Idaho Falls, ID (United States)

    2014-09-01

    This document presents the benchmark plan for the calculation of particle fuel performance on safety testing experiments that are representative of operational accidental transients. The benchmark is dedicated to the modeling of fission product release under accident conditions by fuel performance codes from around the world, and the subsequent comparison to post-irradiation experiment (PIE) data from the modeled heating tests. The accident condition benchmark is divided into three parts: the modeling of a simplified benchmark problem to assess potential numerical calculation issues at low fission product release; the modeling of the AGR-1 and HFR-EU1bis safety testing experiments; and, the comparison of the AGR-1 and HFR-EU1bis modeling results with PIE data. The simplified benchmark case, thereafter named NCC (Numerical Calculation Case), is derived from ''Case 5'' of the International Atomic Energy Agency (IAEA) Coordinated Research Program (CRP) on coated particle fuel technology [IAEA 2012]. It is included so participants can evaluate their codes at low fission product release. ''Case 5'' of the IAEA CRP-6 showed large code-to-code discrepancies in the release of fission products, which were attributed to ''effects of the numerical calculation method rather than the physical model''[IAEA 2012]. The NCC is therefore intended to check if these numerical effects subsist. The first two steps imply the involvement of the benchmark participants with a modeling effort following the guidelines and recommendations provided by this document. The third step involves the collection of the modeling results by Idaho National Laboratory (INL) and the comparison of these results with the available PIE data. The objective of this document is to provide all necessary input data to model the benchmark cases, and to give some methodology guidelines and recommendations in order to make all results suitable for comparison

  16. Generation IV benchmarking of TRISO fuel performance models under accident conditions: Modeling input data

    Energy Technology Data Exchange (ETDEWEB)

    Collin, Blaise P. [Idaho National Laboratory (INL), Idaho Falls, ID (United States)

    2014-09-01

    This document presents the benchmark plan for the calculation of particle fuel performance on safety testing experiments that are representative of operational accidental transients. The benchmark is dedicated to the modeling of fission product release under accident conditions by fuel performance codes from around the world, and the subsequent comparison to post-irradiation experiment (PIE) data from the modeled heating tests. The accident condition benchmark is divided into three parts: the modeling of a simplified benchmark problem to assess potential numerical calculation issues at low fission product release; the modeling of the AGR-1 and HFR-EU1bis safety testing experiments; and, the comparison of the AGR-1 and HFR-EU1bis modeling results with PIE data. The simplified benchmark case, thereafter named NCC (Numerical Calculation Case), is derived from ''Case 5'' of the International Atomic Energy Agency (IAEA) Coordinated Research Program (CRP) on coated particle fuel technology [IAEA 2012]. It is included so participants can evaluate their codes at low fission product release. ''Case 5'' of the IAEA CRP-6 showed large code-to-code discrepancies in the release of fission products, which were attributed to ''effects of the numerical calculation method rather than the physical model''[IAEA 2012]. The NCC is therefore intended to check if these numerical effects subsist. The first two steps imply the involvement of the benchmark participants with a modeling effort following the guidelines and recommendations provided by this document. The third step involves the collection of the modeling results by Idaho National Laboratory (INL) and the comparison of these results with the available PIE data. The objective of this document is to provide all necessary input data to model the benchmark cases, and to give some methodology guidelines and recommendations in order to make all results suitable for comparison

  17. Coordinated Control of a DFIG-Based Wind-Power Generation System with SGSC under Distorted Grid Voltage Conditions

    Directory of Open Access Journals (Sweden)

    Aolin Liu

    2013-05-01

    Full Text Available This paper presents a coordinated control method for a doubly-fed induction generator (DFIG-based wind-power generation system with a series grid-side converter (SGSC under distorted grid voltage conditions. The detailed mathematical models of the DFIG system with SGSC are developed in the multiple synchronous rotating reference frames. In order to counteract the adverse effects of the voltage harmonics upon the DFIG, the SGSC generates series compensation control voltages to keep the stator voltage sinusoidal and symmetrical, which allows the use of the conventional vector control strategy for the rotor-side converter (RSC, regardless of grid voltage harmonics. Meanwhile, two control targets for the parallel grid-side converter (PGSC are identified, including eliminating the oscillations in total active and reactive power entering the grid or suppressing the fifth- and seventh-order harmonic currents injected to the grid. Furthermore, the respective PI-R controller in the positive synchronous reference frame for the SGSC voltage control and PGSC current control have been developed to achieve precise and rapid regulation of the corresponding components. Finally, the proposed coordinated control strategy has been fully validated by the simulation results of a 2 MW DFIG-based wind turbine with SGSC under distorted grid voltage conditions.

  18. Stabilisation of acid generating waste rock with fly ash : immobilization of arsenic under alkaline conditions

    Energy Technology Data Exchange (ETDEWEB)

    Backstrom, M. [Orebro Univ. (Sweden). Man-Technology Environment Research Centre; Sartz, L. [Bergslagen, Kopparberg (Sweden)

    2010-07-01

    This study evaluated the potential for using fly ash as an alkaline material for increasing the pH and decreasing arsenic leaching from highly acidic mine waste. A wood ash sample known to contain high concentrations of both calcium and barium was tested with highly acidic mine waste samples that leached approximately 200 mg/L of arsenic at a liquid/solid ratio of 2. Samples were mixed with the fly ash. Control samples consisted of only mine waste, while the amended samples contained 10 g of mine waste and 10 g of wood ash. Ultra pure water was used as a leachant for both systems until the liquid-solid ratio that corresponded to 900 years of drainage for a waste pile that was 3 m high with an annual run-off of 300 mm. Results of the experimental study showed that the pH in the control increased from 1.7 to 2.7, while the pH in the amended system decreased from 12.6 to 11.5. Initial concentrations of arsenic decreased by almost 3 orders of magnitude in the amended systems. Co-precipitation with the iron, and the calcium arsenate precipitation process were identified as the principal arsenic immobilization mechanisms. The study demonstrated that under the right chemical conditions, alkaline amendments can be used to reduce arsenic leaching from mine wastes. 5 refs., 2 tabs., 1 fig.

  19. Optimization pretreatment condition of sweet sorghum bagasse for production of second generation bioethanol

    Science.gov (United States)

    Sudiyani, Yanni; Waluyo, Joko; Triwahyuni, Eka; Burhani, Dian; Muryanto, Primandaru, Prasetyo; Riandy, Andika Putra; Sumardi, Novia

    2017-01-01

    The bagasse residue of Sweet sorghum (Sorghum bicolor (L.) Moench) consist of cellulose 39.48%; hemicellulose 16.56% and lignin 24.77% that can be converted to ethanol. Pretreatment is of great importance to ethanol yield. In this study, pretreatment process was conducted in a 5-liter reactor using NaOH 10% at various temperature 110, 130, 150°C and reaction time 10, 20, 30 minutes and optimizing severity parameter (log R0 between 1.3 - 2.9). The statistical analysis using two way anova showed that third variations of temperature give different effects significant on lignin, hemicellulose and cellulose content at 95% the confidence level. The optimum pretreatment of bagasse sorghum were obtained with Log R0 value between 2.4-2.9. High severity value in pretreatment condition reduce lignin almost 84-86%, maximum reducing lignin content was 86% obtained at temperature 150°C for 20 minutes reaction time and cellulose increased almost two times the initial content.

  20. Statistical modelling and optimization of hydrolysis of urea to generate ammonia for flue gas conditioning

    Energy Technology Data Exchange (ETDEWEB)

    Mahalik, K. [Department of Chemical Engineering, Indian Institute of Technology (IIT), Kharagpur, P.O. Kharagpur Technology, West Bengal 721302 (India); Department of Chemical Engineering, Gandhi Institute of Engineering and Technology, Gunupur, Orissa (India); Sahu, J.N., E-mail: jay_sahu@yahoo.co.in [Department of Chemical Engineering, Indian Institute of Technology (IIT), Kharagpur, P.O. Kharagpur Technology, West Bengal 721302 (India); Department of Chemical Engineering, Faculty of Engineering, University of Malaya, Kuala Lumpur 50603 (Malaysia); Patwardhan, Anand V. [Department of Chemical Engineering, Institute of Chemical Technology (ICT), Mumbai 400019 (India); Meikap, B.C. [Department of Chemical Engineering, Indian Institute of Technology (IIT), Kharagpur, P.O. Kharagpur Technology, West Bengal 721302 (India); School of Chemical Engineering, University of KwaZulu-Natal, Howard College Campus, King George V. Avenue, Durban 4041 (South Africa)

    2010-10-15

    The present study is concerned with the technique of producing a relatively small quantity of ammonia which can be used safely in a coal-fired thermal power plant to improve the efficiency of electrostatic precipitator by removing the suspended particulate material mostly fly ash, from the flue gas. In this work hydrolysis of urea has been conducted in a batch reactor at atmospheric pressure to study the different reaction variables such as reaction temperature, initial concentration and stirring speed on the conversion by using design expert software. A 2{sup 3} full factorial central composite design (CCD) has been employed and a quadratic model equation has been developed. The study reveals that conversion increases exponentially with an increase in temperature, stirring speed and feed concentration. However the stirring speed has the greatest effect on the conversion with concentration and temperature exerting least and moderate effect respectively. The values of equilibrium conversion obtained through the developed models are found to agree well with their corresponding experimental counterparts with a satisfactory correlation coefficient of 93%. The developed quadratic model was optimized using quadratic programming to maximize conversion of urea within experimental range studied. The optimum production condition has been found to be at the temperature of 130 {sup o}C, feed concentration of 4.16 mol/l and stirring speed of 400 rpm and the corresponding conversion, 63.242%.

  1. Murine dendritic cells generated under serum-free conditions have a mature phenotype and efficiently induce primary immune responses.

    Science.gov (United States)

    Warncke, Max; Dodero, Anna; Dierbach, Heide; Follo, Marie; Veelken, Hendrik

    2006-03-20

    Vaccination with in vitro-generated dendritic cells (DC) that present tumor-associated antigens is a promising approach for immunotherapy of malignant tumors. For optimization of DC-based vaccination protocols, preclinical tumor models that mimic the clinical situation closely are highly desirable. Strong non-specific T cell activation was observed in experimental immunization of mice with syngeneic DC generated in standard FCS-supplemented culture medium. To avoid deviation of the immune response to FCS-derived antigens, a serum-free culture protocol for in vitro generation of murine DC from bone marrow progenitor cells was developed. In comparison to DC differentiated with FCS supplementation, DC generated under serum-free conditions (sfDC) have a more homogeneous phenotype with higher expression of IL-12 and the differentiation and activation markers CD11c, CD40, CD80, CD83, CD86, DEC-205, and MHC class II. Demonstration of strong uptake of protein and carbohydrate antigens and analysis of the in vivo migration behaviour of sfDC also indicated excellent APC function. Vaccination of mice with peptide-pulsed sfDC efficiently induced an antigen-specific T cell response as assessed by MHC tetramer staining, IFN-gamma ELISPOT and in vivo cytotoxicity assay. sfDC may therefore represent a valuable tool to improve active tumor immunotherapy in animal models.

  2. Particle dynamics in self-generated dunes over a range of hydraulic and sediment transport conditions using LES--DEM

    CERN Document Server

    Sun, Rui; Strom, Kyle

    2016-01-01

    Direct measurement of vertical and longitudinal sediment fluxes on migrating sandy bedforms are extremely difficult to perform in both the field and laboratory. In this study we use the LES--DEM (large eddy simulation--discrete element method) solver SediFoam to examine the individual particle motions and resulting fluxes in a domain of self-generated dunes. In SediFoam, the motions of, and collisions among, the sediment grains as well as their interactions with surrounding turbulent flows are resolved. The numerical simulations are performed over a range of transport settings, spanning bedform inception through washout conditions, to examine the individual particle dynamics. The space-time evolution of dune surfaces is demonstrated. The self-generated dunes are stable at relatively low Reynolds numbers, but then become increasingly unstable at higher Reynolds numbers; eventually washing out as the number of bypass grains and particles in suspension increase. Data from the simulation are used to examine the v...

  3. Mutational spectrum drives the rise of mutator bacteria.

    Directory of Open Access Journals (Sweden)

    Alejandro Couce

    Full Text Available Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.

  4. Generation of a gene-corrected isogenic control hiPSC line derived from a familial Alzheimer's disease patient carrying a L150P mutation in presenilin 1

    DEFF Research Database (Denmark)

    Poon, Anna Fong-Yee; Schmid, Benjamin; Pires, Carlota

    2016-01-01

    a familial AD patient carrying a L150P point mutation in PSEN1. Here we used CRISPR/Cas9 gene editing to correct for the single base pair mutation. This gene-corrected line, L150P-GC-hiPSC, serves as an isogenic control to the mutant line for future investigation of mechanisms and cellular phenotypes altered...

  5. Genetic Analysis for Some of Morphological Traits in Bread Wheat under Drought Stress Condition Using Generations Mean Analysis

    Directory of Open Access Journals (Sweden)

    Jamileh Abedi

    2015-06-01

    Full Text Available Perception of genes action controlling of quantitative traits is very important in genetic breeding methods the plant populations. to study and estimate the parameters of genetic and appointment the best genetically model for justification the genetic changing some of traits the bread wheat under drought stress condition, parents (P1 & P2 and F3, F4, F5 generations together the four control cultivars (Kharchia, Gaspard, Moghan and Mahuti were evaluated by generation mean analysis using a agoment design including six blocks. Generation mean analysis was performed for all traits with Mather and Jinks model using joint scaling test. Three parameter model [m d h] provided the best fit for all traits expect harvest index, main spike grain weight, number of grain per plant, Total spike weight of plant with significant at 5% and 1% levels . Though additive and dominance effect both had interfered in controlling often the traits but with attention to difference effects and variety component was determined that dominance is more impressive than additive effect for traits of number of tiller, main spike weight, grain yield and grain number of main spike. Therefore will benefit using of these traits in the collection and to improve these traits hybridization would be much efficient than the selection strategies. In this study additive Ч additive epistasis effect only observed for traits of Total spike weight of plant, number of grain per plant, main spike grain weight and harvest index and other traits hadn’t any epistasis effect that it was demonstration lack of existence the genes reciprocal effect in the inheritance studied traits. Therefore we can suggest that the selection strategies perform in terminal generations and additive Ч additive epistasis effect would be confirmed in selection under self-pollination condition.

  6. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    María Questa

    2016-03-01

    Full Text Available Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.

  7. Néstor-Guillermo progeria syndrome: a novel premature aging condition with early onset and chronic development caused by BANF1 mutations.

    Science.gov (United States)

    Cabanillas, Rubén; Cadiñanos, Juan; Villameytide, José A F; Pérez, Mercedes; Longo, Jesús; Richard, José M; Alvarez, Rebeca; Durán, Noelia S; Illán, Rafael; González, Daniel J; López-Otín, Carlos

    2011-11-01

    Progeria syndromes are rare disorders that involve premature aging. Mutations in BANF1 have been recently reported to cause a new hereditary progeroid syndrome that we now propose to call the Néstor-Guillermo progeria syndrome (NGPS). We describe herein the clinical features of the first two NGPS patients, who phenocopy features of classic progerias (i.e., Hutchinson-Gilford progeria syndrome or mandibuloacral dysplasia), such as aged appearance, growth retardation, decreased subcutaneous fat, thin limbs, and stiff joints. However, these NGPS patients have a distinctive phenotype. In their early adulthood (32 and 24 years of age), they have no signs of cardiovascular impairment, diabetes mellitus, or hypertriglyceridemia. In contrast, they suffer profound skeletal abnormalities that affect their quality of life. The observed differences are of utmost importance to patients and their families and palliation of osseous manifestations is a priority, given their relatively long lifespan. We define NGPS as a chronic progeria because of its slow clinical course and relatively long survival, despite its early onset. Understanding the differences between progeria syndromes might contribute to the development of treatment strategies for common skeletal conditions, as well as aging itself.

  8. Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation.

    Science.gov (United States)

    Sato, Wataru; Hitaoka, Seiji; Inoue, Kaoru; Imai, Mizue; Saio, Tomohide; Uchida, Takeshi; Shinzawa-Itoh, Kyoko; Yoshikawa, Shinya; Yoshizawa, Kazunari; Ishimori, Koichiro

    2016-07-15

    Based on the mutational effects on the steady-state kinetics of the electron transfer reaction and our NMR analysis of the interaction site (Sakamoto, K., Kamiya, M., Imai, M., Shinzawa-Itoh, K., Uchida, T., Kawano, K., Yoshikawa, S., and Ishimori, K. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 12271-12276), we determined the structure of the electron transfer complex between cytochrome c (Cyt c) and cytochrome c oxidase (CcO) under turnover conditions and energetically characterized the interactions essential for complex formation. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c-CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions that were almost cancelled out by large favorable (Columbic) electrostatic interactions but resulted in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, whereas the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.

  9. Fitness Effects of Spontaneous Mutations in Picoeukaryotic Marine Green Algae.

    Science.gov (United States)

    Krasovec, Marc; Eyre-Walker, Adam; Grimsley, Nigel; Salmeron, Christophe; Pecqueur, David; Piganeau, Gwenael; Sanchez-Ferandin, Sophie

    2016-07-07

    Estimates of the fitness effects of spontaneous mutations are important for understanding the adaptive potential of species. Here, we present the results of mutation accumulation experiments over 265-512 sequential generations in four species of marine unicellular green algae, Ostreococcus tauri RCC4221, Ostreococcus mediterraneus RCC2590, Micromonas pusilla RCC299, and Bathycoccus prasinos RCC1105. Cell division rates, taken as a proxy for fitness, systematically decline over the course of the experiment in O. tauri, but not in the three other species where the MA experiments were carried out over a smaller number of generations. However, evidence of mutation accumulation in 24 MA lines arises when they are exposed to stressful conditions, such as changes in osmolarity or exposure to herbicides. The selection coefficients, estimated from the number of cell divisions/day, varies significantly between the different environmental conditions tested in MA lines, providing evidence for advantageous and deleterious effects of spontaneous mutations. This suggests a common environmental dependence of the fitness effects of mutations and allows the minimum mutation/genome/generation rates to be inferred at 0.0037 in these species. Copyright © 2016 Krasovec et al.

  10. Successful treatment of Philadelphia chromosome-positive mixed phenotype acute leukemia by appropriate alternation of second-generation tyrosine kinase inhibitors according to BCR-ABL1 mutation status.

    Science.gov (United States)

    Kawajiri, Chika; Tanaka, Hiroaki; Hashimoto, Shinichiro; Takeda, Yusuke; Sakai, Shio; Takagi, Toshiyuki; Takeuchi, Masahiro; Ohwada, Chikako; Sakaida, Emiko; Shimizu, Naomi; Nakaseko, Chiaki

    2014-04-01

    Philadelphia chromosome-positive mixed phenotype acute leukemia (Ph(+)MPAL) is a rare type of acute leukemia having myeloid and lymphoid features. In the present study, we describe the successful treatment of a 71-year-old Japanese female patient with Ph(+)MPAL by the alternation of second-generation tyrosine kinase inhibitors according to BCR-ABL1 mutations. The patient survived in her third complete remission (CR) for over 4 years. In her first CR, the patient was treated with multiple-agent chemotherapy and underwent maintenance therapy with imatinib and monthly vincristine and prednisolone (VP). At the first relapse, an examination of the bone marrow revealed a transformation into acute lymphoblastic leukemia and an F317L mutation in BCR-ABL1 gene, which responded preferentially to nilotinib over dasatinib. She achieved second CR, and nilotinib with VP therapy was selected for maintenance treatment. At second relapse, BCR-ABL1 mutational analysis revealed Y253H mutation instead of F317L mutation, resulting in resistance to nilotinib. The patient achieved third CR with dasatinib and VP therapy, and maintained CR with this treatment. This suggests that appropriate alternation of TKIs may contribute to long-term survival in elderly patients with Ph(+)MPAL.

  11. Reactive oxygen species generation-scavenging and signaling during plant-arbuscular mycorrhizal and Piriformospora indica interaction under stress condition

    Directory of Open Access Journals (Sweden)

    Manoj Nath

    2016-10-01

    Full Text Available A defined balance between the generation and scavenging of reactive oxygen species (ROS is essential to utilize ROS as an adaptive defense response of plants under biotic and abiotic stress conditions. Moreover, ROS are not only a major determinant of stress response but also acts as signaling molecule that regulates various cellular processes including plant-microbe interaction. In particular, rhizosphere constitutes the biologically dynamic zone for plant–microbe interactions which forms a mutual link leading to reciprocal signaling in both the partners. Among plant–microbe interactions, symbiotic associations of arbuscular mycorrhizal fungi (AMF and arbuscular mycorrhizal-like fungus especially Piriformospora indica with plants are well known to improve plant growth by alleviating the stress-impacts and consequently enhance the plant fitness. AMF and P. indica colonization mainly enhances ROS-metabolism, maintains ROS-homeostasis, and thereby averts higher ROS-level accrued inhibition in plant cellular processes and plant growth and survival under stressful environments. This article summarizes the major outcomes of the recent reports on the ROS-generation and scavenging and signaling in biotic-abiotic stressed plants with AMF and P. indica colonization. Overall, a detailed exploration of ROS-signature kinetics during plant-AMF/P. indica interaction can help in designing innovative strategies for improving plant health and productivity under stress conditions.

  12. Reactive Oxygen Species Generation-Scavenging and Signaling during Plant-Arbuscular Mycorrhizal and Piriformospora indica Interaction under Stress Condition.

    Science.gov (United States)

    Nath, Manoj; Bhatt, Deepesh; Prasad, Ram; Gill, Sarvajeet S; Anjum, Naser A; Tuteja, Narendra

    2016-01-01

    A defined balance between the generation and scavenging of reactive oxygen species (ROS) is essential to utilize ROS as an adaptive defense response of plants under biotic and abiotic stress conditions. Moreover, ROS are not only a major determinant of stress response but also act as signaling molecule that regulates various cellular processes including plant-microbe interaction. In particular, rhizosphere constitutes the biologically dynamic zone for plant-microbe interactions which forms a mutual link leading to reciprocal signaling in both the partners. Among plant-microbe interactions, symbiotic associations of arbuscular mycorrhizal fungi (AMF) and arbuscular mycorrhizal-like fungus especially Piriformospora indica with plants are well known to improve plant growth by alleviating the stress-impacts and consequently enhance the plant fitness. AMF and P. indica colonization mainly enhances ROS-metabolism, maintains ROS-homeostasis, and thereby averts higher ROS-level accrued inhibition in plant cellular processes and plant growth and survival under stressful environments. This article summarizes the major outcomes of the recent reports on the ROS-generation, scavenging and signaling in biotic-abiotic stressed plants with AMF and P. indica colonization. Overall, a detailed exploration of ROS-signature kinetics during plant-AMF/P. indica interaction can help in designing innovative strategies for improving plant health and productivity under stress conditions.

  13. Operating conditions for the generation of stable anode spot plasma in front of a positively biased electrode

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yeong-Shin; Lee, Yuna; Dang, Jeong-Jeung [Department of Nuclear Engineering, Seoul National University, Seoul 151-744 (Korea, Republic of); Chung, Kyoung-Jae, E-mail: jkjlsh1@snu.ac.kr [Center for Advance Research in Fusion Reactor Engineering, Seoul National University, Seoul 151-744 (Korea, Republic of); Hwang, Y. S. [Department of Nuclear Engineering, Seoul National University, Seoul 151-744 (Korea, Republic of); Center for Advance Research in Fusion Reactor Engineering, Seoul National University, Seoul 151-744 (Korea, Republic of)

    2014-02-15

    Stability of an anode spot plasma, which is an additional high density plasma generated in front of a positively biased electrode immersed in ambient plasma, is a critical issue for its utilization to various types of ion sources. In this study, operating conditions for the generation of stable anode spot plasmas are experimentally investigated. Diagnostics of the bias current flowing into the positively biased electrode and the properties of ambient plasma reveal that unstable nature of the anode spot is deeply associated with the reduction of double layer potential between the anode spot plasma and the ambient plasma. It is found that stability of the anode spot plasma can be improved with increasing the ionization rate in ambient plasma so as to compensate the loss of electrons across the double layer or with enlarging the area of the biased electrode to prevent electron accumulation inside the anode spot. The results obtained from the present study give the guideline for operating conditions of anode spot plasmas as an ion source with high brightness.

  14. Efficient generation of long-distance conditional alleles using recombineering and a dual selection strategy in replicate plates

    Directory of Open Access Journals (Sweden)

    Liang Hong-Erh

    2009-07-01

    Full Text Available Abstract Background Conditional knockout mice are a useful tool to study the function of gene products in a tissue-specific or inducible manner. Classical approaches to generate targeting vectors for conditional alleles are often limited by the availability of suitable restriction sites. Furthermore, plasmid-based targeting vectors can only cover a few kB of DNA which precludes the generation of targeting vectors where the two loxP sites are placed far apart. These limitations have been overcome in the recent past by using homologous recombination of bacterial artificial chromosomes (BACs in Escherichia coli to produce large targeting vector containing two different loxP-flanked selection cassettes so that a single targeting event is sufficient to introduce loxP-sites a great distances into the mouse genome. However, the final targeted allele should be free of selection cassettes and screening for correct removal of selection cassettes can be a laborious task. Therefore, we developed a new strategy to rapidly identify ES cells containing the desired allele. Results Using BAC recombineering we generated a single targeting vector which contained two different selection cassettes that were flanked by loxP-loxP sites or by FRT-FRT/loxP sites so that they could be deleted sequentially by Cre- and FLPe-recombinases, respectively. Transfected ES cells were first selected in the presence of both antibiotics in vitro before correctly targeted clones were identified by Southern blot. After transfection of a Cre recombinase expression plasmid ES cell clones were selected on replicate plates to identify those clones which maintained the FRT-FRT/loxP flanked cassette and lost the loxP-loxP flanked cassette. Using this strategy facilitated the identification of ES cell clones containing the desired allele before blastocyst injection. Conclusion The strategy of ES cell cultures in replicate plates proved to be very efficient in identifying ES cells that had

  15. In vitro generation of monocyte-derived macrophages under serum-free conditions improves their tumor promoting functions.

    Directory of Open Access Journals (Sweden)

    Flora Rey-Giraud

    Full Text Available The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs, reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.

  16. Efficient post-processing of aqueous generator eluates facilitates {sup 68}Ga-labelling under anhydrous conditions

    Energy Technology Data Exchange (ETDEWEB)

    Zoller, F.; Riss, P.J.; Roesch, F. [University of Mainz (Germany). Institute of Nuclear Chemistry; Montforts, F.P. [University of Bremen (Germany). Institute of Organic Chemistry

    2010-07-01

    The present study reports a convenient method for {sup 68}Ga-labelling under anhydrous conditions using solid-phase derived gallium-68-acetylacetonate ([ {sup 68}Ga]Ga(acac){sub 3}) in a microwave-enhanced radiosynthesis. Commercial {sup 68}Ge/{sup 68}Ga-radionuclide generators utilizing TiO{sub 2} to adsorb {sup 68}Ge(IV) (Obninsk-generators) were used. The initial aqueous generator eluate was transferred online onto a cation exchange resin and {sup 68}Ga was absorbed quantitatively. From this resin, {sup 68}Ga was eluted with different acetone-based, non-aqueous solvent systems. More than 95% of the generator-eluted {sup 68}Ga was obtained from the cation exchange resin with 600 {mu}L of a 98% acetone/2% acetylacetone mixture providing n.c.a. [ {sup 68}Ga]Ga(acac){sub 3} as labelling agent. Water-insoluble macrocyclic polypyrrole derivatives were chosen as model compounds for a proof-of-principle labelling of lipophilic compounds with {sup 68}Ga. Labelling of two different porphyrin derivatives, meso -tetraphenyl-porphyrin (Tpp) and 3-(1-hydroxyheptyl)deuteroporphyrin dimethylester (HHDPD) was performed in chloroform in a focused microwave synthesis system in yields of up to 90% within 5 min using phenol as co-ligand. Moreover, new co-ligands were investigated to be more effective and significantly less toxic than phenol. Among the phenol alternatives, gentisic acid (2,5-dihydroxy benzoic acid, DHB, 5 mg) emerged as the most useful, non-toxic phenol substitute. It facilitates reducing the load of co-ligand by 95%, while providing an increased labeling yield of 97%. {sup 68}Ga-labelled porphyrins may facilitate the medical application for molecular imaging via positron emission tomography. (orig.)

  17. Targeted Next-Generation Sequencing of a 12.5 Mb Homozygous Region Reveals ANO10 Mutations in Patients with Autosomal-Recessive Cerebellar Ataxia

    NARCIS (Netherlands)

    Vermeer, Sascha; Hoischen, Alexander; Meijer, Rowdy P. P.; Gilissen, Christian; Neveling, Kornelia; Wieskamp, Nienke; de Brouwer, Arjan; Koenig, Michel; Anheim, Mathieu; Assoum, Mirna; Drouot, Nathalie; Todorovic, Slobodanka; Milic-Rasic, Vedrana; Lochmueller, Hanns; Stevanin, Giovanni; Goizet, Cyril; David, Albert; Durr, Alexandra; Brice, Alexis; Kremer, Berry; van de Warrenburg, Bart P. C.; Schijvenaars, Mascha M. V. A. P.; Heister, Angelien; Kwint, Michael; Arts, Peer; van der Wijst, Jenny; Veltman, Joris; Kamsteeg, Erik-Jan; Scheffer, Hans; Knoers, Nine

    2010-01-01

    Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provid

  18. Effect of Doubly Fed Induction GeneratorTidal Current Turbines on Stability of a Distribution Grid under Unbalanced Voltage Conditions

    Directory of Open Access Journals (Sweden)

    Dahai Zhang

    2017-02-01

    Full Text Available This paper analyses the effects of doubly fed induction generator (DFIG tidal current turbines on a distribution grid under unbalanced voltage conditions of the grid. A dynamic model of an electrical power system under the unbalanced network is described in the paper, aiming to compare the system performance when connected with and without DFIG at the same location in a distribution grid. Extensive simulations of investigating the effect of DFIG tidal current turbine on stability of the distribution grid are performed, taking into account factors such as the power rating, the connection distance of the turbine and the grid voltage dip. The dynamic responses of the distribution system are examined, especially its ability to ride through fault events under unbalanced grid voltage conditions. The research has shown that DFIG tidal current turbines can provide a good damping performance and that modern DFIG tidal current power plants, equipped with power electronics and low-voltage ride-through capability, can stay connected to weak electrical grids even under the unbalanced voltage conditions, whilst not reducing system stability.

  19. Mutation supply and the repeatability of selection for antibiotic resistance

    Science.gov (United States)

    van Dijk, Thomas; Hwang, Sungmin; Krug, Joachim; de Visser, J. Arjan G. M.; Zwart, Mark P.

    2017-10-01

    Whether evolution can be predicted is a key question in evolutionary biology. Here we set out to better understand the repeatability of evolution, which is a necessary condition for predictability. We explored experimentally the effect of mutation supply and the strength of selective pressure on the repeatability of selection from standing genetic variation. Different sizes of mutant libraries of antibiotic resistance gene TEM-1 β-lactamase in Escherichia coli, generated by error-prone PCR, were subjected to different antibiotic concentrations. We determined whether populations went extinct or survived, and sequenced the TEM gene of the surviving populations. The distribution of mutations per allele in our mutant libraries followed a Poisson distribution. Extinction patterns could be explained by a simple stochastic model that assumed the sampling of beneficial mutations was key for survival. In most surviving populations, alleles containing at least one known large-effect beneficial mutation were present. These genotype data also support a model which only invokes sampling effects to describe the occurrence of alleles containing large-effect driver mutations. Hence, evolution is largely predictable given cursory knowledge of mutational fitness effects, the mutation rate and population size. There were no clear trends in the repeatability of selected mutants when we considered all mutations present. However, when only known large-effect mutations were considered, the outcome of selection is less repeatable for large libraries, in contrast to expectations. We show experimentally that alleles carrying multiple mutations selected from large libraries confer higher resistance levels relative to alleles with only a known large-effect mutation, suggesting that the scarcity of high-resistance alleles carrying multiple mutations may contribute to the decrease in repeatability at large library sizes.

  20. Noise-mean relationship in mutated promoters.

    Science.gov (United States)

    Hornung, Gil; Bar-Ziv, Raz; Rosin, Dalia; Tokuriki, Nobuhiko; Tawfik, Dan S; Oren, Moshe; Barkai, Naama

    2012-12-01

    Gene expression depends on the frequency of transcription events (burst frequency) and on the number of mRNA molecules made per event (burst size). Both processes are encoded in promoter sequence, yet their dependence on mutations is poorly understood. Theory suggests that burst size and frequency can be distinguished by monitoring the stochastic variation (noise) in gene expression: Increasing burst size will increase mean expression without changing noise, while increasing burst frequency will increase mean expression and decrease noise. To reveal principles by which promoter sequence regulates burst size and frequency, we randomly mutated 22 yeast promoters chosen to span a range of expression and noise levels, generating libraries of hundreds of sequence variants. In each library, mean expression (m) and noise (coefficient of variation, η) varied together, defining a scaling curve: η(2) = b/m + η(ext)(2). This relation is expected if sequence mutations modulate burst frequency primarily. The estimated burst size (b) differed between promoters, being higher in promoter containing a TATA box and lacking a nucleosome-free region. The rare variants that significantly decreased b were explained by mutations in TATA, or by an insertion of an out-of-frame translation start site. The decrease in burst size due to mutations in TATA was promoter-dependent, but independent of other mutations. These TATA box mutations also modulated the responsiveness of gene expression to changing conditions. Our results suggest that burst size is a promoter-specific property that is relatively robust to sequence mutations but is strongly dependent on the interaction between the TATA box and promoter nucleosomes.

  1. Familial gigantism caused by an NSD1 mutation.

    NARCIS (Netherlands)

    Haelst, M.M. van; Hoogeboom, J.J.; Baujat, G.; Bruggenwirth, H.T.; Laar, I. van de; Coleman, K.; Rahman, N.; Niermeijer, M.F.; Drop, S.L.; Scambler, P.J.

    2005-01-01

    A three-generation family with autosomal dominant segregation of a novel NSD1 mutation (6605G --> A, resulting in Cys2202Tyr) is reported. Haploinsufficiency of NSD1 has been identified as the major cause of Sotos syndrome. The overgrowth condition (MIM 117550) is characterized by facial anomalie

  2. Experiments and Simulations on a Heat Exchanger of an Automotive Exhaust Thermoelectric Generation System Under Coupling Conditions

    Science.gov (United States)

    Liu, X.; Yu, C. G.; Chen, S.; Wang, Y. P.; Su, C. Q.

    2014-06-01

    The present experimental and computational study investigates an exhaust gas waste heat recovery system for vehicles, using thermoelectric modules and a heat exchanger to produce electric power. It proposes a new plane heat exchanger of a thermoelectric generation (TEG) system, producing electricity from a limited hot surface area. To investigate the new plane heat exchanger, we make a coupling condition of heat-flow and flow-solid coupling analysis on it to obtain the temperature, heat, and pressure field of the heat exchanger, and compared it with the old heat exchanger. These fields couple together to solve the multi-field coupling of the flow, solid, and heat, and then the simulation result is compared with the test bench experiment of TEG, providing a theoretical and experimental basis for the present exhaust gas waste heat recovery system.

  3. FEATURES SOCIALIZATION STUDENTS OF THE BOARDING SCHOOL IN THE FAR NORTH IN CONDITIONS OF INTRODUCTUIN OF THE GEF SECOND GENERATION

    Directory of Open Access Journals (Sweden)

    Zimin Aleksei Valerevich

    2013-02-01

    Full Text Available Purpose. Study the features of the socialization of students, of the efficiency of primary school textbooks, with the introduction of the federal state educational standards of the second generation-specific institution. Methodology. Questionnaire and the study of literature and documents. Results. As a result of the original research report the presence of three groups of students, requiring consideration of ethnic features when planning learning activities. The features of the formation of ethnic socialization boarding schools. The conclusions about the lack of effectiveness of the selection and application of teaching and learning materials to meet the specific conditions of the environment of students - students of the boarding school, to work towards improving the quality of training of the teaching staff of boarding schools. Practical implications. Methodical and personnel support of the educational process in the open secondary boarding schools in the Far North.

  4. Biofouling community composition across a range of environmental conditions and geographical locations suitable for floating marine renewable energy generation.

    Science.gov (United States)

    Macleod, Adrian K; Stanley, Michele S; Day, John G; Cook, Elizabeth J

    2016-01-01

    Knowledge of biofouling typical of marine structures is essential for engineers to define appropriate loading criteria in addition to informing other stakeholders about the ecological implications of creating novel artificial environments. There is a lack of information regarding biofouling community composition (including weight and density characteristics) on floating structures associated with future marine renewable energy generation technologies. A network of navigation buoys were identified across a range of geographical areas, environmental conditions (tidal flow speed, temperature and salinity), and deployment durations suitable for future developments. Despite the perceived importance of environmental and temporal factors, geographical location explained the greatest proportion of the observed variation in community composition, emphasising the importance of considering geography when assessing the impact of biofouling on device functioning and associated ecology. The principal taxa associated with variation in biofouling community composition were mussels (Mytilus edulis), which were also important when determining loading criteria.

  5. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma.

    Directory of Open Access Journals (Sweden)

    Alexandre Harlé

    Full Text Available Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies.Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM PCR, Real-time allele-specific amplification (RT-ASA PCR, Next generation sequencing (NGS, immunohistochemistry (IHC and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays.BRAF mutations were found in 28(47.5%, 29(49.2%, 31(52.5%, 29(49.2% and 27(45.8% samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2% samples were found bearing a c.1799T>A (p.Val600Glu mutation, three (9.4% with a c.1798_1799delinsAA (p.Val600Lys mutation and one with c.1789_1790delinsTC (p.Leu597Ser mutation. Two samples were found bearing complex mutations.HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays.

  6. An Optimal Integrated Control Scheme for Permanent Magnet Synchronous Generator-Based Wind Turbines under Asymmetrical Grid Fault Conditions

    Directory of Open Access Journals (Sweden)

    Dan Wang

    2016-04-01

    Full Text Available In recent years, the increasing penetration level of wind energy into power systems has brought new issues and challenges. One of the main concerns is the issue of dynamic response capability during outer disturbance conditions, especially the fault-tolerance capability during asymmetrical faults. In order to improve the fault-tolerance and dynamic response capability under asymmetrical grid fault conditions, an optimal integrated control scheme for the grid-side voltage-source converter (VSC of direct-driven permanent magnet synchronous generator (PMSG-based wind turbine systems is proposed in this paper. The optimal control strategy includes a main controller and an additional controller. In the main controller, a double-loop controller based on differential flatness-based theory is designed for grid-side VSC. Two parts are involved in the design process of the flatness-based controller: the reference trajectories generation of flatness output and the implementation of the controller. In the additional control aspect, an auxiliary second harmonic compensation control loop based on an improved calculation method for grid-side instantaneous transmission power is designed by the quasi proportional resonant (Quasi-PR control principle, which is able to simultaneously restrain the second harmonic components in active power and reactive power injected into the grid without the respective calculation for current control references. Moreover, to reduce the DC-link overvoltage during grid faults, the mathematical model of DC-link voltage is analyzed and a feedforward modified control factor is added to the traditional DC voltage control loop in grid-side VSC. The effectiveness of the optimal control scheme is verified in PSCAD/EMTDC simulation software.

  7. Expanding phenotype of p.Ala140Val mutation in MECP2 in a 4 generation family with X-linked intellectual disability and spasticity.

    Science.gov (United States)

    Lambert, Sophie; Maystadt, Isabelle; Boulanger, Sébastien; Vrielynck, Pascal; Destrée, Anne; Lederer, Damien; Moortgat, Stéphanie

    2016-10-01

    Mutations in MECP2 (MIM #312750), located on Xq28 and encoding a methyl CpG binding protein, are classically associated with Rett syndrome in female patients, with a lethal effect in hemizygous males. However, MECP2 mutations have already been reported in surviving males with severe neonatal-onset encephalopathy, or with X-linked intellectual disability associated with psychosis, pyramidal signs, parkinsonian features and macro-orchidism (PPM-X syndrome; MIM3 #300055). Here we report on the identification of the p.Ala140Val mutation in the MECP2 gene in 4 males and 3 females of a large Caucasian family affected with X-linked intellectual disability. Females present with mild cognitive impairment and speech difficulties. Males have moderate intellectual disability, impaired language development, friendly behavior, slowly progressive spastic paraparesis and dystonic movements of the hands. Two of them show microcephaly. The p.Ala140Val mutation is recurrent, as it was already described in 4 families with X-linked mental retardation and in three sporadic male patients with intellectual disability. We further delineate the phenotype associated with the p.Ala140Val mutation, illustrating a variable expressivity even within a given family, and we compare our patients with previous reported cases in the literature.

  8. In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy.

    Science.gov (United States)

    Cao, H; Vergé, V; Baron, C; Martinache, C; Leon, A; Scholl, S; Gorin, N C; Salamero, J; Assari, S; Bernard, J; Lopez, M

    2000-04-01

    DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.

  9. Muller's ratchet with compensatory mutations

    CERN Document Server

    Pfaffelhuber, Peter; Wakolbinger, Anton

    2011-01-01

    We consider an infinite dimensional system of stochastic differential equations which describes the evolution of type frequencies in a large population. Random reproduction is modeled by a Wright-Fisher noise whose inverse diffusion coefficient $N$ corresponds to the total population size. The type of an individual is the number $k$ of deleterious mutations it carries. We assume that fitness of individuals carrying $k$ mutations is decreased by $\\alpha k$ for some $\\alpha >0$. Along the individual lines of descent, (new) mutations accumulate at rate $\\lambda$ per generation, and each of these mutations has a small probability $\\gamma$ per generation to disappear. While the case $\\gamma =0 $ is known as (the Fleming-Viot version of) {\\em Muller's ratchet}, the case $\\gamma > 0$ is referred to as that of {\\em compensatory mutations} in the biological literature. In the former case ($\\gamma=0$), an ever increasing number of mutations is accumulated over time, while in the latter ($\\gamma > 0$) this is prevented ...

  10. Phenotypic variability in a seven-generation Swedish family segregating autosomal dominant hearing impairment due to a novel EYA4 frameshift mutation

    DEFF Research Database (Denmark)

    Frykholm, Carina; Klar, Joakim; Arnesson, Hanna;

    2015-01-01

    -sectional and longitudinal deterioration of pure tone average (PTA) once the process of hearing deterioration had started, and no gender, parent-of-origin or family branch differences on PTA could be found. Age at onset varied between the family branches. In summary, this is the ninth published genetically verified DFNA10...... family. The results imply that unidentified factors, genetic or environmental, other than the EYA4 mutation, are of importance for the age at onset of DFNA10, and that mutation early in the variable region of the EYA4 protein can occur in the absence of dilated cardiomyopathy....

  11. Escherichia coli frameshift mutation rate depends on the chromosomal context but not on the GATC content near the mutation site.

    Directory of Open Access Journals (Sweden)

    Mariana A Martina

    Full Text Available Different studies have suggested that mutation rate varies at different positions in the genome. In this work we analyzed if the chromosomal context and/or the presence of GATC sites can affect the frameshift mutation rate in the Escherichia coli genome. We show that in a mismatch repair deficient background, a condition where the mutation rate reflects the fidelity of the DNA polymerization process, the frameshift mutation rate could vary up to four times among different chromosomal contexts. Furthermore, the mismatch repair efficiency could vary up to eight times when compared at different chromosomal locations, indicating that detection and/or repair of frameshift events also depends on the chromosomal context. Also, GATC sequences have been proved to be essential for the correct functioning of the E. coli mismatch repair system. Using bacteriophage heteroduplexes molecules it has been shown that GATC influence the mismatch repair efficiency in a distance- and number-dependent manner, being almost nonfunctional when GATC sequences are located at 1 kb or more from the mutation site. Interestingly, we found that in E. coli genomic DNA the mismatch repair system can efficiently function even if the nearest GATC sequence is located more than 2 kb away from the mutation site. The results presented in this work show that even though frameshift mutations can be efficiently generated and/or repaired anywhere in the genome, these processes can be modulated by the chromosomal context that surrounds the mutation site.

  12. Escherichia coli frameshift mutation rate depends on the chromosomal context but not on the GATC content near the mutation site.

    Science.gov (United States)

    Martina, Mariana A; Correa, Elisa M E; Argaraña, Carlos E; Barra, José L

    2012-01-01

    Different studies have suggested that mutation rate varies at different positions in the genome. In this work we analyzed if the chromosomal context and/or the presence of GATC sites can affect the frameshift mutation rate in the Escherichia coli genome. We show that in a mismatch repair deficient background, a condition where the mutation rate reflects the fidelity of the DNA polymerization process, the frameshift mutation rate could vary up to four times among different chromosomal contexts. Furthermore, the mismatch repair efficiency could vary up to eight times when compared at different chromosomal locations, indicating that detection and/or repair of frameshift events also depends on the chromosomal context. Also, GATC sequences have been proved to be essential for the correct functioning of the E. coli mismatch repair system. Using bacteriophage heteroduplexes molecules it has been shown that GATC influence the mismatch repair efficiency in a distance- and number-dependent manner, being almost nonfunctional when GATC sequences are located at 1 kb or more from the mutation site. Interestingly, we found that in E. coli genomic DNA the mismatch repair system can efficiently function even if the nearest GATC sequence is located more than 2 kb away from the mutation site. The results presented in this work show that even though frameshift mutations can be efficiently generated and/or repaired anywhere in the genome, these processes can be modulated by the chromosomal context that surrounds the mutation site.

  13. Role of mutation in Pseudomonas aeruginosa biofilm development.

    Directory of Open Access Journals (Sweden)

    Tim C R Conibear

    Full Text Available The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor

  14. Generation and Validation of a Mouse Line with a Floxed SRC-3/AIB1 Allele for Conditional Knockout

    Directory of Open Access Journals (Sweden)

    Zhaoliang Liu, Lan Liao, Suoling Zhou, Jianming Xu

    2008-01-01

    Full Text Available The steroid receptor coactivator-3 (SRC-3, also known as AIB1, ACTR, p/CIP and NCOA3, is a transcriptional coactivator for nuclear receptors and certain other transcription factors. SRC-3 is widely expressed and plays important physiological functions and pathogenic roles in breast and prostate cancers. SRC-3 knockout (SRC-3-/- mice display genetic background-dependent embryonic lethality and multiple local and systemic abnormalities. Since both the partial lethality and the systemic effects caused by global SRC-3 knockout interfere with downstream investigation of tissue-specific function of SRC-3, we have generated floxed SRC-3 (SRC-3f/f mice with conditional alleles carrying loxP sites in introns 10 and 12 by a gene-targeting strategy. The two SRC-3f/f mouse lines (A and B are indistinguishable from wild type mice. To test if deletion of the floxed exons 11 and 12 for SRC-3 nuclear receptor interaction domains and disruption of its downstream sequence for transcriptional activation domains would inactivate SRC-3 function, SRC-3f/f mice were crossbred with EIIa-Cre mice to generate SRC-3d/d mice with germ line deletion of the floxed SRC-3 gene. Both lines of SRC-3d/d mice exhibited growth retardation and low IGF-I levels, which was similar to that observed in SRC-3-/- mice. The line A SRC-3d/d mice showed normal viability, while line B SRC-3d/d mice showed partial lethality similar to SRC-3-/- mice, probably due to variable distributions of genetic background during breeding. These results demonstrate that the floxed SRC-3 mouse lines have been successfully established. These mice will be useful for investigating the cell type- and developmental stage-specific functions of SRC-3.

  15. Generation of Human Alloantigen-Specific Regulatory T Cells Under Good Manufacturing Practice-Compliant Conditions for Cell Therapy.

    Science.gov (United States)

    Cheraï, Mustapha; Hamel, Yamina; Baillou, Claude; Touil, Soumia; Guillot-Delost, Maude; Charlotte, Frédéric; Kossir, Laila; Simonin, Ghislaine; Maury, Sébastien; Cohen, José L; Lemoine, François M

    2015-01-01

    Natural regulatory T cells (Tregs) may have a great therapeutic potential to induce tolerance in allogeneic cells and organ transplantations. In mice, we showed that alloantigen-specific Tregs (spe-Tregs) were more efficient than polyclonal Tregs (poly-Tregs) in controlling graft-versus-host disease (GVHD). Here we describe a clinical-grade compliant method for generating human spe-Tregs. Tregs were enriched from leukapheresis products with anti-CD25 immunomagnetic beads, primed twice by allogeneic mature monocyte-derived dendritic cells (mDCs), and cultured during 3 weeks in medium containing interleukin 2 (IL-2), IL-15, and rapamycin. After 3 weeks of culture, final cell products were expanded 8.3-fold from the initial CD25(+) purifications. Immunophenotypic analyses of final cells indicate that they were composed of 88 ± 2.6% of CD4(+) T cells, all expressing Treg-specific markers (FOXP3, Helios, GARP, LAP, and CD152). Spe-Tregs were highly suppressive in vitro and also in vivo using a xeno-GVHD model established in immunodeficient mice. The specificity of their suppressive activity was demonstrated on their ability to significantly suppress the proliferation of autologous effector T cells stimulated by the same mDCs compared to third-party mDCs. Our data provide evidence that functional alloantigen Tregs can be generated under clinical-grade compliant conditions. Taking into account that 130 × 10(6) CD25(+) cells can be obtained at large scale from standard leukapheresis, our cell process may give rise to a theoretical final number of 1 × 10(9) spe-Tregs. Thus, using our strategy, we can propose to prepare spe-Tregs for clinical trials designed to control HLA-mismatched GVHD or organ transplantation rejection.

  16. Direct electric current treatment under physiologic saline conditions kills Staphylococcus epidermidis biofilms via electrolytic generation of hypochlorous acid.

    Directory of Open Access Journals (Sweden)

    Elizabeth L Sandvik

    Full Text Available The purpose of this study was to investigate the mechanism by which a direct electrical current reduced the viability of Staphylococcus epidermidis biofilms in conjunction with ciprofloxacin at physiologic saline conditions meant to approximate those in an infected artificial joint. Biofilms grown in CDC biofilm reactors were exposed to current for 24 hours in 1/10(th strength tryptic soy broth containing 9 g/L total NaCl. Dose-dependent log reductions up to 6.7 log(10 CFU/cm(2 were observed with the application of direct current at all four levels (0.7 to 1.8 mA/cm(2 both in the presence and absence of ciprofloxacin. There were no significant differences in log reductions for wells with ciprofloxacin compared to those without at the same current levels. When current exposures were repeated without biofilm or organics in the medium, significant generation of free chlorine was measured. Free chlorine doses equivalent to the 24 hour endpoint concentration for each current level were shown to mimic killing achieved by current application. Current exposure (1.8 mA/cm(2 in medium lacking chloride and amended with sulfate, nitrate, or phosphate as alternative electrolytes produced diminished kills of 3, 2, and 0 log reduction, respectively. Direct current also killed Pseudomonas aeruginosa biofilms when NaCl was present. Together these results indicate that electrolysis reactions generating hypochlorous acid from chloride are likely a main contributor to the efficacy of direct current application. A physiologically relevant NaCl concentration is thus a critical parameter in experimental design if direct current is to be investigated for in vivo medical applications.

  17. Direct electric current treatment under physiologic saline conditions kills Staphylococcus epidermidis biofilms via electrolytic generation of hypochlorous acid.

    Science.gov (United States)

    Sandvik, Elizabeth L; McLeod, Bruce R; Parker, Albert E; Stewart, Philip S

    2013-01-01

    The purpose of this study was to investigate the mechanism by which a direct electrical current reduced the viability of Staphylococcus epidermidis biofilms in conjunction with ciprofloxacin at physiologic saline conditions meant to approximate those in an infected artificial joint. Biofilms grown in CDC biofilm reactors were exposed to current for 24 hours in 1/10(th) strength tryptic soy broth containing 9 g/L total NaCl. Dose-dependent log reductions up to 6.7 log(10) CFU/cm(2) were observed with the application of direct current at all four levels (0.7 to 1.8 mA/cm(2)) both in the presence and absence of ciprofloxacin. There were no significant differences in log reductions for wells with ciprofloxacin compared to those without at the same current levels. When current exposures were repeated without biofilm or organics in the medium, significant generation of free chlorine was measured. Free chlorine doses equivalent to the 24 hour endpoint concentration for each current level were shown to mimic killing achieved by current application. Current exposure (1.8 mA/cm(2)) in medium lacking chloride and amended with sulfate, nitrate, or phosphate as alternative electrolytes produced diminished kills of 3, 2, and 0 log reduction, respectively. Direct current also killed Pseudomonas aeruginosa biofilms when NaCl was present. Together these results indicate that electrolysis reactions generating hypochlorous acid from chloride are likely a main contributor to the efficacy of direct current application. A physiologically relevant NaCl concentration is thus a critical parameter in experimental design if direct current is to be investigated for in vivo medical applications.

  18. Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific homozygous mutation in CHMP2B

    DEFF Research Database (Denmark)

    Zhang, Yu; Schmid, Benjamin; Nielsen, Troels T

    2016-01-01

    Frontotemporal dementia (FTD) is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B) on chromosome 3 (FTD3), a component of the endosomal sorting complex required for transport III (ESCRT-III). ...

  19. Generation of a human induced pluripotent stem cell line via CRISPR-Cas9 mediated integration of a site-specific heterozygous mutation in CHMP2B

    DEFF Research Database (Denmark)

    Zhang, Yu; Schmid, Benjamin; Nielsen, Troels Tolstrup

    2016-01-01

    Frontotemporal dementia (FTD) is an early onset neurodegenerative disease. Mutations in several genes cause familial FTD and one of them is charged multivesicular body protein 2B (CHMP2B) on chromosome 3 (FTD3), a component of the endosomal sorting complex required for transport III (ESCRT-III). ...

  20. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1

    DEFF Research Database (Denmark)

    Li, Tong; Pires, Carlota; Nielsen, Troels T.

    2016-01-01

    Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1), causing Alzheimer's disease (AD). Induced pluripotent stem cell (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28...

  1. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a M146I mutation in PSEN1

    DEFF Research Database (Denmark)

    Li, Tong; Pires, Carlota; Nielsen, Troels Tolstrup

    2016-01-01

    Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1), responsible for causing Alzheimer's disease (AD). Induced pluripotent stem cells (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, ...

  2. NRAS germline variant G138R and multiple rare somatic mutations on APC in colorectal cancer patients in Taiwan by next generation sequencing.

    Science.gov (United States)

    Chang, Pi-Yueh; Chen, Jinn-Shiun; Chang, Nai-Chung; Chang, Shih-Cheng; Wang, Mei-Chia; Tsai, Shu-Hui; Wen, Ying-Hao; Tsai, Wen-Sy; Chan, Err-Cheng; Lu, Jang-Jih

    2016-06-21

    Colorectal cancer (CRC) arises from mutations in a subset of genes. We investigated the germline and somatic mutation spectrum of patients with CRC in Taiwan by using the AmpliSeq Cancer Hotspot Panel V2. Fifty paired freshly frozen stage 0-IV CRC tumors and adjacent normal tissue were collected. Blood DNA from 20 healthy donors were used for comparison of germline mutations. Variants were identified using an ion-torrent personal genomic machine and subsequently confirmed by Sanger sequencing or pyrosequencing. Five nonsynonymous germline variants on 4 cancer susceptible genes, CDH1, APC, MLH1, and NRAS, were observed in 6 patients with CRC (12%). Among them, oncogene NRAS G138R variant was identified as having a predicted damaging effect on protein function, which has never been reported by other laboratories. CDH1 T340A variants were presented in 3 patients. The germline variants in the cancer patients differed completely from those found in asymptomatic controls. Furthermore, a total of 56 COSMIC and 21 novel somatic variants distributed in 20 genes were detected in 44 (88%) of the CRC samples. High inter- and intra-tumor heterogeneity levels were observed. Nine rare variants located in the β-catenin binding region of the APC gene were discovered, 7 of which could cause amino acid frameshift and might have a pathogenic effect. In conclusion, panel-based mutation detection by using a high-throughput sequencing platform can elucidate race-dependent cancer genomes. This approach facilitates identifying individuals at high risk and aiding the recognition of novel mutations as targets for drug development.

  3. Direct generation of titanium dioxide nanoparticles dispersion under supercritical conditions for photocatalytic active thermoplastic surfaces for microbiological inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Zydziak, Nicolas, E-mail: nicolas.zydziak@kit.edu [Polymer Engineering Department, Fraunhofer Institute of Chemical Technology, Joseph-von-Fraunhofer-Str. 7, 76327 Pfinztal (Germany); Zanin, Maria-Helena Ambrosio [Laboratory of Chemical Processes and Particle Technology Bionanomanufacturing, Institute for Technological Research of the State of São Paulo – IPT, Av. Prof. Almeida Prado 532, Cidade Universitária, CEP 05508-901 São Paulo, SP (Brazil); Trick, Iris [Environmental Biotechnology and Bioprocess Engineering Department, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart (Germany); Hübner, Christof [Polymer Engineering Department, Fraunhofer Institute of Chemical Technology, Joseph-von-Fraunhofer-Str. 7, 76327 Pfinztal (Germany)

    2015-03-01

    Thermoplastic poly(propylene) (PP) and acrylonitrile-butadiene-styrene (ABS) surfaces were coated with silica based films via the sol–gel process, containing titanium dioxide (TiO{sub 2}) as photocatalyst. TiO{sub 2} was previously synthesized via sol–gel and treated under supercritical conditions in water dispersions. The characterization of the TiO{sub 2} dispersions was performed via disc centrifuge to determine the particle size and via Raman spectroscopy and X-Ray Diffraction (XRD) to characterize the crystallinity of TiO{sub 2}. The synthesized TiO{sub 2} dispersions and commercially available TiO{sub 2} particles were incorporated in silica based films which were synthesized under acidic or basic conditions, leading to dense or porous films respectively. The morphology of the films was characterized via Scanning Electron Microscopy (SEM). The incorporation of synthesized TiO{sub 2} in the coating led to photocatalytically more active thermoplastic surfaces than films formulated with commercially available TiO{sub 2} as determined via dye discoloration test. A microbiological test performed with Sarcina lutea confirmed this result and showed an inactivation factor of 6 (99.9999%) after 24 h UV irradiation, for synthesized TiO{sub 2} incorporated in acidic formulated silica layer on ABS surfaces. - Highlights: • We report about photocatalytic layers formulated on thermoplastic surfaces. • We synthesized silica layer and TiO{sub 2} via sol–gel and supercritical treatment. • Amorphous, crystalline and commercial dispersions were generated and characterized. • The morphology of dense and porous photocatalytic layers is observed via SEM. • Discoloration and microbiological tests correlate activity and surface morphology.

  4. The conditional nature of genetic interactions: the consequences of wild-type backgrounds on mutational interactions in a genome-wide modifier screen.

    Directory of Open Access Journals (Sweden)

    Sudarshan Chari

    Full Text Available The phenotypic outcome of a mutation cannot be simply mapped onto the underlying DNA variant. Instead, the phenotype is a function of the allele, the genetic background in which it occurs and the environment where the mutational effects are expressed. While the influence of genetic background on the expressivity of individual mutations is recognized, its consequences on the interactions between genes, or the genetic network they form, is largely unknown. The description of genetic networks is essential for much of biology; yet if, and how, the topologies of such networks are influenced by background is unknown. Furthermore, a comprehensive examination of the background dependent nature of genetic interactions may lead to identification of novel modifiers of biological processes. Previous work in Drosophila melanogaster demonstrated that wild-type genetic background influences the effects of an allele of scalloped (sd, with respect to both its principal consequence on wing development and its interactions with a mutation in optomotor blind. In this study we address whether the background dependence of mutational interactions is a general property of genetic systems by performing a genome wide dominant modifier screen of the sd(E3 allele in two wild-type genetic backgrounds using molecularly defined deletions. We demonstrate that ~74% of all modifiers of the sd(E3 phenotype are background-dependent due in part to differential sensitivity to genetic perturbation. These background dependent interactions include some with qualitative differences in the phenotypic outcome, as well as instances of sign epistasis. This suggests that genetic interactions are often contingent on genetic background, with flexibility in genetic networks due to segregating variation in populations. Such background dependent effects can substantially alter conclusions about how genes influence biological processes, the potential for genetic screens in alternative wild

  5. Effect of wire EDM conditions on generation of residual stresses in machining of aluminum 2014 T6 alloy

    Directory of Open Access Journals (Sweden)

    Pujari Srinivasa Rao

    2016-06-01

    Full Text Available Wire electrical discharge machining (EDM possesses many advantages over the conventional manufacturing process. Hence, this process was used for machining of all conductive materials; especially, nowadays this is the most common process for machining of aerospace aluminum alloys. This process produces complex shapes in aluminum alloys with extremely tight tolerances in a single setup. But, for good surface integrity and longer service life, the residual stresses generated on the components should be as low as possible and it depends on the setting of process parameters and the material to be machined. In wire EDM, much of the work was concentrated on Titanium alloys, Inconel alloys and various types of steels and partly on aluminum alloys. The present investigation was a parametric analysis of wire EDM parameters on residual stresses in the machining of aluminum alloy using Taguchi method. The results obtained had shown a wide range of residual stresses from 8.2 to 405.6 MPa. It also influenced the formation of various intermetallics such as AlCu and AlCu3. Microscopic examination revealed absence of surface cracks on aluminum surface at all the machining conditions. Here, an attempt was made to compare the results of aluminum alloy with the available machined data for other metals.

  6. Engineering of second generation HTS coated conductor architecture to enhance the normal zone propagation velocity in various operating conditions

    Science.gov (United States)

    Lacroix, C.; Sirois, F.; Fournier Lupien, J.-H.

    2017-06-01

    The effects of operating conditions, critical current and stabilizer geometry on the normal zone propagation velocity (NZPV) of second generation (2G) high-temperature superconductor (HTS) coated conductors (CCs) are investigated using finite element simulations. The NZPV of tapes with a low interfacial resistance between the HTS and stabilizer layers are first compared with tapes with a current flow diverter (CFD) architecture. Our results indicates that the CFD concept increases the NZPV for the whole range of operating temperatures investigated (10-77 K). In particular, for an operating temperature of 77 K and an operating current of 0.9I c, our numerical results indicate that the NZPV of a 2G HTS CC with a CFD architecture and a 2 μm thick stabilizer layer could reach a value of 50 m s-1. Furthermore, numerical simulations realized on the effect of the stabilizer geometry on the NZPV of 2G HTS CCs revealed that putting most of the stabilizer on the substrate side can enhance the NZPV by a factor of 7 or more, even for tape with thick stabilizer (20 μm or more). This is particularly promising for improving quench detection in applications requiring a thick stabilizer such as superconducting coils.

  7. A Fuzzy Logic Control Strategy for Doubly Fed Induction Generator for Improved Performance under Faulty Operating Conditions

    Directory of Open Access Journals (Sweden)

    G. Venu Madhav

    2014-12-01

    Full Text Available In this paper, decouple PI control for output active and reactive powers which is the common control technique for power converter of Doubly Fed Induction Generator (DFIG is presented. But there are some disadvantages with this control method like uncertainty about the exact model, behavior of some parameters or unpredictable wind speed and tuning of PI parameters. To overcome the mentioned disadvantages a fuzzy logic control of DFIG wind turbine is presented and is compared with PI controller. To validate the proposed scheme, simulation results are presented, these results showed that the performance of fuzzy control of DFIG is excellent and it improves power quality and stability of wind turbine compared to PI controller. The Fuzzy logic controller is applied to rotor side converter for active power control and voltage regulation of wind turbine. The entire work is carried out in MATLab/Simulink. Different faulty operating conditions are considered to prove the effective implementation of the proposed control scheme.

  8. Dataset in support of the generation of Niemann-Pick disease Type C1 patient-specific iPS cell lines carrying the novel NPC1 mutation c.1180T>C or the prevalent c.3182T>C mutation – Analysis of pluripotency and neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Franziska Peter

    2017-06-01

    Full Text Available Data presented in this article demonstrate the generation and characterization of two novel Niemann-Pick disease Type C1 (NPC1 patient-specific induced pluripotent stem cell (iPSC lines, related to the research article Trilck et al. (Diversity of Glycosphingolipid GM2 and Cholesterol Accumulation in NPC1 Patient-Specific iPSC-Derived Neurons; Brain Res.; 2017; 1657:52-61. doi: 10.1016/j.brainres.2016.11.031. For reprogramming fibroblasts, carrying the novel homozygous mutation c.1180T>C and the prevalent homozygous mutation c.3182T>C, were used. Reprogramming into patient-specific iPSCs was induced by retroviral transduction of the transcription factors Sox2, Klf4, Oct4 and c-Myc, and confirmed according to their pluripotency. The iPSCs were subsequently differentiated into neural progenitor cells, which were terminally differentiated into functional neurons and glial cells. The generation of these cell lines provides further valuable tools to investigate pathogenic mechanism of NPC1 in human neuronal cells carrying different NPC1 mutations.

  9. Dataset in support of the generation of Niemann-Pick disease Type C1 patient-specific iPS cell lines carrying the novel NPC1 mutation c.1180T>C or the prevalent c.3182T>C mutation - Analysis of pluripotency and neuronal differentiation.

    Science.gov (United States)

    Peter, Franziska; Trilck, Michaela; Rabenstein, Michael; Rolfs, Arndt; Frech, Moritz J

    2017-06-01

    Data presented in this article demonstrate the generation and characterization of two novel Niemann-Pick disease Type C1 (NPC1) patient-specific induced pluripotent stem cell (iPSC) lines, related to the research article Trilck et al. (Diversity of Glycosphingolipid GM2 and Cholesterol Accumulation in NPC1 Patient-Specific iPSC-Derived Neurons; Brain Res.; 2017; 1657:52-61. doi: 10.1016/j.brainres.2016.11.031). For reprogramming fibroblasts, carrying the novel homozygous mutation c.1180T>C and the prevalent homozygous mutation c.3182T>C, were used. Reprogramming into patient-specific iPSCs was induced by retroviral transduction of the transcription factors Sox2, Klf4, Oct4 and c-Myc, and confirmed according to their pluripotency. The iPSCs were subsequently differentiated into neural progenitor cells, which were terminally differentiated into functional neurons and glial cells. The generation of these cell lines provides further valuable tools to investigate pathogenic mechanism of NPC1 in human neuronal cells carrying different NPC1 mutations.

  10. Generation and evaluation of typical meteorological year datasets for greenhouse and external conditions on the Mediterranean coast

    Science.gov (United States)

    Fernández, M. D.; López, J. C.; Baeza, E.; Céspedes, A.; Meca, D. E.; Bailey, B.

    2015-08-01

    A typical meteorological year (TMY) represents the typical meteorological conditions over many years but still contains the short term fluctuations which are absent from long-term averaged data. Meteorological data were measured at the Experimental Station of Cajamar `Las Palmerillas' (Cajamar Foundation) in Almeria, Spain, over 19 years at the meteorological station and in a reference greenhouse which is typical of those used in the region. The two sets of measurements were subjected to quality control analysis and then used to create TMY datasets using three different methodologies proposed in the literature. Three TMY datasets were generated for the external conditions and two for the greenhouse. They were assessed by using each as input to seven horticultural models and comparing the model results with those obtained by experiment in practical trials. In addition, the models were used with the meteorological data recorded during the trials. A scoring system was used to identify the best performing TMY in each application and then rank them in overall performance. The best methodology was that of Argiriou for both greenhouse and external conditions. The average relative errors between the seasonal values estimated using the 19-year dataset and those using the Argiriou greenhouse TMY were 2.2 % (reference evapotranspiration), -0.45 % (pepper crop transpiration), 3.4 % (pepper crop nitrogen uptake) and 0.8 % (green bean yield). The values obtained using the Argiriou external TMY were 1.8 % (greenhouse reference evapotranspiration), 0.6 % (external reference evapotranspiration), 4.7 % (greenhouse heat requirement) and 0.9 % (loquat harvest date). Using the models with the 19 individual years in the historical dataset showed that the year to year weather variability gave results which differed from the average values by ± 15 %. By comparison with results from other greenhouses it was shown that the greenhouse TMY is applicable to greenhouses which have a solar

  11. Modeling and optimization of energy generation and storage systems for thermal conditioning of buildings targeting conceptual building design

    Energy Technology Data Exchange (ETDEWEB)

    Grahovac, Milica

    2012-11-29

    The thermal conditioning systems are responsible for almost half of the energy consump-tion by commercial buildings. In many European countries and in the USA, buildings account for around 40% of primary energy consumption and it is therefore vital to explore further ways to reduce the HVAC (Heating, Ventilation and Air Conditioning) system energy consumption. This thesis investigates the relationship between the energy genera-tion and storage systems for thermal conditioning of buildings (shorter: primary HVAC systems) and the conceptual building design. Certain building design decisions irreversibly influence a building's energy performance and, conversely, many generation and storage components impose restrictions on building design and, by their nature, cannot be introduced at a later design stage. The objective is, firstly, to develop a method to quantify this influence, in terms of primary HVAC system dimensions, its cost, emissions and energy consumption and, secondly, to enable the use of the developed method by architects during the conceptual design. In order to account for the non-stationary effects of the intermittent renewable energy sources (RES), thermal storage and for the component part load efficiencies, a time domain system simulation is required. An abstract system simulation method is proposed based on seven pre-configured primary HVAC system models, including components such as boil-ers, chillers and cooling towers, thermal storage, solar thermal collectors, and photovoltaic modules. A control strategy is developed for each of the models and their annual quasi-stationary simulation is performed. The performance profiles obtained are then used to calculate the energy consumption, carbon emissions and costs. The annuity method has been employed to calculate the cost. Optimization is used to automatically size the HVAC systems, based on their simulation performance. Its purpose is to identify the system component dimensions that provide

  12. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying a M146I mutation in PSEN1.

    Science.gov (United States)

    Li, Tong; Pires, Carlota; Nielsen, Troels T; Waldemar, Gunhild; Hjermind, Lena E; Nielsen, Jørgen E; Dinnyes, Andras; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K

    2016-03-01

    Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1), responsible for causing Alzheimer's disease (AD). Induced pluripotent stem cells (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. M146I-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported M146I-iPSCs line may be a useful resource for in vitro modeling of familial AD. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  13. Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1.

    Science.gov (United States)

    Li, Tong; Pires, Carlota; Nielsen, Troels T; Waldemar, Gunhild; Hjermind, Lena E; Nielsen, Jørgen E; Dinnyes, Andras; Hyttel, Poul; Freude, Kristine K

    2016-03-01

    Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1), causing Alzheimer's disease (AD). Induced pluripotent stem cell (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. A79V-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported A79V-iPSCs line may be a useful resource for in vitro modeling of familial AD. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  14. Generation of induced pluripotent stem cells (iPSCs from an Alzheimer's disease patient carrying a M146I mutation in PSEN1

    Directory of Open Access Journals (Sweden)

    Tong Li

    2016-03-01

    Full Text Available Skin fibroblasts were obtained from a 46-year-old symptomatic man carrying a M146I mutation in the presenilin 1 gene (PSEN1, responsible for causing Alzheimer's disease (AD. Induced pluripotent stem cells (iPSCs were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. M146I-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported M146I-iPSCs line may be a useful resource for in vitro modeling of familial AD.

  15. Generation of induced pluripotent stem cells (iPSCs from an Alzheimer's disease patient carrying an A79V mutation in PSEN1

    Directory of Open Access Journals (Sweden)

    Tong Li

    2016-03-01

    Full Text Available Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1, causing Alzheimer's disease (AD. Induced pluripotent stem cell (iPSCs were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28 and shTP53 genes. A79V-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported A79V-iPSCs line may be a useful resource for in vitro modeling of familial AD.

  16. Activating somatic mutations in diffuse large B-cell lymphomas: lessons from next generation sequencing and key elements in the precision medicine era.

    Science.gov (United States)

    Bohers, Elodie; Mareschal, Sylvain; Bertrand, Philippe; Viailly, Pierre Julien; Dubois, Sydney; Maingonnat, Catherine; Ruminy, Philippe; Tilly, Hervé; Jardin, Fabrice

    2015-05-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30-40% of newly diagnosed non-Hodgkin lymphomas. Historically, DLBCL has been thought to involve recurrent translocations of the immunoglobulin heavy (IGH) locus and the deregulation of rearranged oncogenes. Whole exome sequencing (WES) of more than 200 DLBCLs has completely redefined the genetic landscape of the disease by identifying recurrent single nucleotide variants and providing new therapeutic opportunities in DLBCL molecular subtypes. Some of these somatic mutations target genes that play a crucial role in B-cell function (B cell receptor [BCR] signaling, nuclear factor κB [NF-κB] pathway, Toll-like receptor [TLR] signaling and phosphatidylinositol 3-kinase [PI3K] pathway), immunity, cell cycle/apoptosis or chromatin modification. In this review, following an overview of the somatic mutations reported in DLBCL, we focus on activating and clustered mutations targeting genes including MYD88, CD79A/B, EZH2 and CARD11 and discuss their clinical and therapeutic relevance in the precision medicine era.

  17. Generation of a Tph2 Conditional Knockout Mouse Line for Time- and Tissue-Specific Depletion of Brain Serotonin.

    Directory of Open Access Journals (Sweden)

    Barbara Pelosi

    Full Text Available Serotonin has been gaining increasing attention during the last two decades due to the dual function of this monoamine as key regulator during critical developmental events and as neurotransmitter. Importantly, unbalanced serotonergic levels during critical temporal phases might contribute to the onset of neuropsychiatric disorders, such as schizophrenia and autism. Despite increasing evidences from both animal models and human genetic studies have underpinned the importance of serotonin homeostasis maintenance during central nervous system development and adulthood, the precise role of this molecule in time-specific activities is only beginning to be elucidated. Serotonin synthesis is a 2-step process, the first step of which is mediated by the rate-limiting activity of Tph enzymes, belonging to the family of aromatic amino acid hydroxylases and existing in two isoforms, Tph1 and Tph2, responsible for the production of peripheral and brain serotonin, respectively. In the present study, we generated and validated a conditional knockout mouse line, Tph2flox/flox, in which brain serotonin can be effectively ablated with time specificity. We demonstrated that the Cre-mediated excision of the third exon of Tph2 gene results in the production of a Tph2null allele in which we observed the near-complete loss of brain serotonin, as well as the growth defects and perinatal lethality observed in serotonin conventional knockouts. We also revealed that in mice harbouring the Tph2null allele, but not in wild-types, two distinct Tph2 mRNA isoforms are present, namely Tph2Δ3 and Tph2Δ3Δ4, with the latter showing an in-frame deletion of amino acids 84-178 and coding a protein that could potentially retain non-negligible enzymatic activity. As we could not detect Tph1 expression in the raphe, we made the hypothesis that the Tph2Δ3Δ4 isoform can be at the origin of the residual, sub-threshold amount of serotonin detected in the brain of Tph2null/null mice

  18. Next-generation EGFR/HER tyrosine kinase inhibitors for the treatment of patients with non-small-cell lung cancer harboring EGFR mutations: a review of the evidence

    Directory of Open Access Journals (Sweden)

    Wang X

    2016-09-01

    Full Text Available Xiaochun Wang,1,2 David Goldstein,3 Philip J Crowe,1,2 Jia-Lin Yang1,2 1Department of Surgery, 2Sarcoma and Nanooncology Group, Adult Cancer Program, Lowy Cancer Research Centre, 3Department of Medical Oncology, Prince of Wales Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia Abstract: Tyrosine kinase inhibitors (TKIs against human epidermal growth factor receptor (EGFR/HER family have been introduced into the clinic to treat cancers, particularly non-small-cell lung cancer (NSCLC. There have been three generations of the EGFR/HER-TKIs. First-generation EGFR/HER-TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR TK domain, show a significant breakthrough treatment in selected NSCLC patients with activating EGFR mutations (actEGFRm EGFRL858R and EGFRDel19, in terms of safety, efficacy, and quality of life. However, all those responders inevitably develop acquired resistance within 12 months, because of the EGFRT790M mutation, which prevents TKI binding to ATP-pocket of EGFR by steric hindrance. The second-generation EGFR/HER-TKIs were developed to prolong and maintain more potent response as well as overcome the resistance to the first-generation EGFR/HER-TKIs. They are different from the first-generation EGFR/HER-TKIs by covalently binding to the ATP-binding site, irreversibly blocking enzymatic activation, and targeting EGFR/HER family members, including EGFR, HER2, and HER4. Preclinically, these compounds inhibit the enzymatic activation for actEGFRm, EGFRT790M, and wtEGFR. The second-generation EGFR/HER-TKIs improve overall survival in cancer patients with actEGFRm in a modest way. However, they are not clinically active in overcoming EGFRT790M resistance, mainly because of dose-limiting toxicity due to simultaneous inhibition against wtEGFR. The third-generation EGFR/HER-TKIs selectively and irreversibly target EGFRT790M and actEGFRm while sparing wtEGFR. They yield

  19. Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation.

    Science.gov (United States)

    Swenson, P A; Norton, I L

    1986-05-01

    A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occurring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA+, lexA+ and recBC+ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30 degrees C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30 degrees C but not at 37 degrees C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37 degrees C and shows a temperature conditional UV-induced respiration shutoff response at 30 degrees C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37 degrees This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.

  20. Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors

    DEFF Research Database (Denmark)

    Bahrami, Shervin; Pedersen, Finn Skou; Duch, Mogens R.

    2003-01-01

    envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random......The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV...... mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells...

  1. SOD isoforms play no role in lifespan in ad lib or dietary restricted conditions, but mutational inactivation of SOD-1 reduces life extension by cold.

    Science.gov (United States)

    Yen, Kelvin; Patel, Harshil B; Lublin, Alex L; Mobbs, Charles V

    2009-03-01

    The free radical theory of aging is one of the most prominent theories of aging and senescence, but has yet to be definitively proven. If free radicals are the cause of senescence, then the cellular anti-oxidant system should play a large role in lifespan determination. Because superoxide dismutase (SOD) plays a central role in detoxifying superoxide radicals, we have examined the effects of mutational inactivation of each isoform of sod on normal lifespan and lifespan extension by dietary restriction (DR) or cold-/hypothermic-induced longevity (CHIL). We find no significant decrease in lifespan for control worms or worms undergoing DR when sod isoforms are knocked-out even though sod mutational inactivation produces hypersensitivity to paraquat. In contrast, sod-1 inactivation significantly reduces lifespan extension by CHIL, suggesting that CHIL requires a specific genetic program beyond simple reduction in metabolic rate. Furthermore, CHIL paradoxically increases lifespan while reducing resistance to oxidative stress, further disassociating oxidative stress resistance and lifespan.

  2. Calreticulin Mutations in Myeloproliferative Neoplasms

    Directory of Open Access Journals (Sweden)

    Noa Lavi

    2014-10-01

    Full Text Available With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph− myeloproliferative neoplasms (MPNs in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET and primary myelofibrosis (PMF. At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations and recurrent 5-bp insertions (type 2 mutations in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph− MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

  3. Optimization of Mutation Pressure in Relation to Properties of Protein-Coding Sequences in Bacterial Genomes.

    Science.gov (United States)

    Błażej, Paweł; Miasojedow, Błażej; Grabińska, Małgorzata; Mackiewicz, Paweł

    2015-01-01

    Most mutations are deleterious and require energetically costly repairs. Therefore, it seems that any minimization of mutation rate is beneficial. On the other hand, mutations generate genetic diversity indispensable for evolution and adaptation of organisms to changing environmental conditions. Thus, it is expected that a spontaneous mutational pressure should be an optimal compromise between these two extremes. In order to study the optimization of the pressure, we compared mutational transition probability matrices from bacterial genomes with artificial matrices fulfilling the same general features as the real ones, e.g., the stationary distribution and the speed of convergence to the stationarity. The artificial matrices were optimized on real protein-coding sequences based on Evolutionary Strategies approach to minimize or maximize the probability of non-synonymous substitutions and costs of amino acid replacements depending on their physicochemical properties. The results show that the empirical matrices have a tendency to minimize the effects of mutations rather than maximize their costs on the amino acid level. They were also similar to the optimized artificial matrices in the nucleotide substitution pattern, especially the high transitions/transversions ratio. We observed no substantial differences between the effects of mutational matrices on protein-coding sequences in genomes under study in respect of differently replicated DNA strands, mutational cost types and properties of the referenced artificial matrices. The findings indicate that the empirical mutational matrices are rather adapted to minimize mutational costs in the studied organisms in comparison to other matrices with similar mathematical constraints.

  4. Optimization of Mutation Pressure in Relation to Properties of Protein-Coding Sequences in Bacterial Genomes.

    Directory of Open Access Journals (Sweden)

    Paweł Błażej

    Full Text Available Most mutations are deleterious and require energetically costly repairs. Therefore, it seems that any minimization of mutation rate is beneficial. On the other hand, mutations generate genetic diversity indispensable for evolution and adaptation of organisms to changing environmental conditions. Thus, it is expected that a spontaneous mutational pressure should be an optimal compromise between these two extremes. In order to study the optimization of the pressure, we compared mutational transition probability matrices from bacterial genomes with artificial matrices fulfilling the same general features as the real ones, e.g., the stationary distribution and the speed of convergence to the stationarity. The artificial matrices were optimized on real protein-coding sequences based on Evolutionary Strategies approach to minimize or maximize the probability of non-synonymous substitutions and costs of amino acid replacements depending on their physicochemical properties. The results show that the empirical matrices have a tendency to minimize the effects of mutations rather than maximize their costs on the amino acid level. They were also similar to the optimized artificial matrices in the nucleotide substitution pattern, especially the high transitions/transversions ratio. We observed no substantial differences between the effects of mutational matrices on protein-coding sequences in genomes under study in respect of differently replicated DNA strands, mutational cost types and properties of the referenced artificial matrices. The findings indicate that the empirical mutational matrices are rather adapted to minimize mutational costs in the studied organisms in comparison to other matrices with similar mathematical constraints.

  5. Estimation on gas generation and corrosion rates of carbon steel, stainless steel and zircaloy in alkaline solutions under low oxygen condition

    Energy Technology Data Exchange (ETDEWEB)

    Mihara, Morihiro; Honda, Akira [Japan Nuclear Cycle Development Inst., Tokai Works, Tokai, Ibaraki (Japan); Nishimura, Tsutomu; Wada, Ryutaro [Kobe Steel Ltd., Engineering Company, Energy and Nuclear System Center, Osaka (Japan)

    2002-06-01

    Hydrogen gas generated by corrosion of metals in TRU waste repository may degrade the function of the engineered barrier system for nuclide migration. Therefore, estimation of gas generation rates of metals under the repository condition is important. In this study, we obtained gas generation rates of carbon steel, stainless steel and zircaloy in alkaline solutions under low oxygen conditions and evaluated the corrosion rates based on these data in order to compare with the published data. The magnitude of corrosion rates of carbon steel, stainless steel and zircaloy were 10{sup -1} {mu}m/y, 10{sup -2} {mu}m/y and 10{sup -3} {mu}m/y, respectively. These values agreed with the published corrosion rates from gas generation rates by others. (author)

  6. Under What Conditions Are Embedded versus Generative Cognitive Strategy Activators Effective? Two Prescriptive Models for Designing Instruction.

    Science.gov (United States)

    Darwazeh, Afnan N.

    In an embedded cognitive-strategy system, learners are forced to use a given cognitive strategy prepared by the instructor, designer, or researcher. In a generative cognitive-strategy system, learners are directed to generate a certain cognitive strategy activator by themselves. This is a review of experimental and theoretical studies conducted on…

  7. Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

    Science.gov (United States)

    Jaing, Crystal J.; McLoughlin, Kevin S.; Thissen, James B.; Zemla, Adam; Vergez, Lisa M.; Bourguet, Feliza; Mabery, Shalini; Fofanov, Viacheslav Y.; Koshinsky, Heather; Jackson, Paul J.

    2016-01-01

    Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms. PMID:27668749

  8. Evolutionary loss of the rdar morphotype in Salmonella as a result of high mutation rates during laboratory passage.

    Science.gov (United States)

    Davidson, Carla J; White, Aaron P; Surette, Michael G

    2008-03-01

    Rapid evolution of microbes under laboratory conditions can lead to domestication of environmental or clinical strains. In this work, we show that domestication due to laboratory passage in rich medium is extremely rapid. Passaging of wild-type Salmonella in rich medium led to diversification of genotypes contributing to the loss of a spatial phenotype, called the rdar morphotype, within days. Gene expression analysis of the rdar regulatory network demonstrated that mutations were primarily within rpoS, indicating that the selection pressure for scavenging during stationary phase had the secondary effect of impairing this highly conserved phenotype. If stationary phase was omitted from the experiment, radiation of genotypes and loss of the rdar morphotype was also demonstrated, but due to mutations within the cellulose biosynthesis pathway and also in an unknown upstream regulator. Thus regardless of the selection pressure, rapid regulatory changes can be observed on laboratory timescales. The speed of accumulation of rpoS mutations during daily passaging could not be explained by measured fitness and mutation rates. A model of mutation accumulation suggests that to generate the observed accumulation of sigma 38 mutations, this locus must experience a mutation rate of approximately 10(-4) mutations/gene/generation. Sequencing and gene expression of population isolates indicated that there were a wide variety of sigma 38 phenotypes within each population. This suggests that the rpoS locus is highly mutable by an unknown pathway, and that these mutations accumulate rapidly under common laboratory conditions.

  9. Simulation of an air conditioning absorption refrigeration system in a co-generation process combining a proton exchange membrane fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Pilatowsky, I.; Gamboa, S.A.; Rivera, W. [Centro de Investigacion en Energia - UNAM, Temixco, Morelos (Mexico); Romero, R.J. [Centro de Investigacion en Ingenieria y Ciencias Aplicadas - UAEM, Cuernavaca, Morelos (Mexico); Isaza, C.A. [Universidad Pontificia Bolivariana, Medellin (Colombia). Instituto de Energia y Termodinamica; Sebastian, P.J. [Centro de Investigacion en Energia - UNAM, Temixco, Morelos (Mexico); Cuerpo Academico de Energia y Sustentabilidad-UP Chiapas, Tuxtla Gutierrez, Chiapas (Mexico); Moreira, J. [Cuerpo Academico de Energia y Sustentabilidad-UP Chiapas, Tuxtla Gutierrez, Chiapas (Mexico)

    2007-10-15

    In this work, a computer simulation program was developed to determine the optimum operating conditions of an air conditioning system during the co-generation process. A 1 kW PEMFC was considered in this study with a chemical/electrical theoretical efficiency of 40% and a thermal efficiency of 30% applying an electrical load of 100%. A refrigeration-absorption cycle (RAC) operating with monomethylamine-water solutions (MMA-WS), with low vapor generation temperatures (up to 80 C) is proposed in this work. The computer simulation was based on the refrigeration production capacity at the maximum power capacity of the PEMFC. Heat losses between the fuel cell and the absorption air conditioning system at standard operating conditions were considered to be negligible. The results showed the feasibility of using PEMFC for cooling, increasing the total efficiency of the fuel cell system. (author)

  10. Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.

    Science.gov (United States)

    Moyer, Tyler C; Holland, Andrew J

    2015-01-01

    The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5-6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research.

  11. Automatic generation of boundary conditions using Demons non-rigid image registration for use in 3D modality-independent elastography

    Science.gov (United States)

    Pheiffer, Thomas S.; Ou, Jao J.; Miga, Michael I.

    2010-02-01

    Modality-independent elastography (MIE) is a method of elastography that reconstructs the elastic properties of tissue using images acquired under different loading conditions and a biomechanical model. Boundary conditions are a critical input to the algorithm, and are often determined by time-consuming point correspondence methods requiring manual user input. Unfortunately, generation of accurate boundary conditions for the biomechanical model is often difficult due to the challenge of accurately matching points between the source and target surfaces and consequently necessitates the use of large numbers of fiducial markers. This study presents a novel method of automatically generating boundary conditions by non-rigidly registering two image sets with a Demons diffusion-based registration algorithm. The use of this method was successfully performed in silico using magnetic resonance and X-ray computed tomography image data with known boundary conditions. These preliminary results have produced boundary conditions with accuracy of up to 80% compared to the known conditions. Finally, these boundary conditions were utilized within a 3D MIE reconstruction to determine an elasticity contrast ratio between tumor and normal tissue. Preliminary results show a reasonable characterization of the material properties on this first attempt and a significant improvement in the automation level and viability of the method.

  12. Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

    NARCIS (Netherlands)

    de Leng, Wendy W J; Gadellaa-Van Hooijdonk, Christa G.; Barendregt-Smouter, Françoise A S; Koudijs, Marco J.; Nijman, Ies; Hinrichs, John W.J.; Cuppen, Edwin; van Lieshout, Stef; Loberg, Robert D.; De Jonge, Maja; Voest, Emile E; De Weger, Roel A.; Steeghs, Neeltje; Langenberg, Marlies H G; Sleijfer, Stefan; Willems, Stefan M.; Lolkema, Martijn P.

    2016-01-01

    BACKGROUND: Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed

  13. Enhanced Control of a DFIG-Based Wind-Power Generation System With Series Grid-Side Converter Under Unbalanced Grid Voltage Conditions

    DEFF Research Database (Denmark)

    Yao, Jun; Li, Hui; Chen, Zhe

    2013-01-01

    This paper presents an enhanced control method for a doubly fed induction generator (DFIG)-based wind-power generation system with series grid-side converter (SGSC) under unbalanced grid voltage conditions. The behaviors of the DFIG system with SGSC during network unbalance are described. By inje......This paper presents an enhanced control method for a doubly fed induction generator (DFIG)-based wind-power generation system with series grid-side converter (SGSC) under unbalanced grid voltage conditions. The behaviors of the DFIG system with SGSC during network unbalance are described....... By injecting a series control voltage generated from the SGSC to balance the stator voltage, the adverse effects of voltage unbalance upon the DFIG, such as stator and rotor current unbalances, electromagnetic torque, and power pulsations, can be removed, and then the conventional vector control strategy...... for the rotor-side converter remains in full force under unbalanced conditions. Meanwhile, three control targets for the parallel grid-side converter (PGSC) are identified, including eliminating the oscillations in the total active power or reactive power, or eliminating negative-sequence current injected...

  14. THE TEMPERATURE FIELD OF HEAT-GENERATING FLUID IN A SQUARE AREA WITH A NON-UNIFORM FIRST KIND BOUNDARY CONDITIONS

    Directory of Open Access Journals (Sweden)

    V. I. Ryazhskikh

    2012-01-01

    Full Text Available Based on the of the integral Fourier transforms the problem of stationary temperature fields distribution of heat-generating fluid in a square area is analytically solved under non-uniform first kind boundary conditions for the conductive heat transfer mode.

  15. Numerical investigation on thermal conditions for a tee junction of LMFBR coolant pipes. 5. Investigations on flow conditions for arched-vortex generation

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, Satoshi [Customer System Co. Ltd., Tokai, Ibaraki (Japan); Muramatsu, Toshiharu [Japan Nuclear Cycle Development Inst., Oarai, Ibaraki (Japan). Oarai Engineering Center

    2001-04-01

    A fundamental experiment, the aim of which is to quantify turbulent mixing characteristics, was carried out at Hiroshima University in the framework of a cooperative study with Japan Nuclear Cycle Development Institute (JNC). Turbulent mixing analyses for the fundamental experiment were carried out using a direct numerical simulation code DINUS-3 at JNC. >From the analyses, the following results have been obtained: (1) It was confirmed that the numerical analysis is applicable to the evaluation of the three flow patterns observed in the experiment: an adhesion flow, a deflection flow and an impinging flow. (2) In the deflection flow analysis, arched-vortex characteristics with lower frequency fluctuations agreed well with the experimental results. (3) Frequency of arched-vortex transportation depended on the Reynolds number. The frequency of arched-vortex was increased with increasing the Reynolds number under the condition of the constant velocity ratio between coolant pipes. (author)

  16. Peopling an Unaccustomed Earth with a New Generation: Jhumpa Lahiri’s Supreme Fictional Journey into Human Conditions

    Directory of Open Access Journals (Sweden)

    Neela Bhattacharya SAXENA

    2012-06-01

    Full Text Available Using a theoretical framework derived from my ongoing engagement with what I havecalled a ‘Gynocentric matrix’ of Indic sensibility, along with James Hillman’s polytheisticpsychology and Wallace Stevens’ notion of a Supreme Fiction, this paper offers a readingof Jhumpa Lahiri’s (b. 1967 short stories beyond postcolonial criticism. Stemming froma depth consciousness where life, living and death, joy, indifference and sorrow, generation, de/re-generation, and transformation are intricately intertwined, Lahiri’s fictionalmultiverse is peopled by a new generation of characters who speak to the soul of the reader; in the process, she sculpts a reality that does not tolerate any homogenizing impulse in the name of an abstract unity.

  17. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases.

    Science.gov (United States)

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A; Jenkins, Andrew; Traynelis, Stephen F

    2015-07-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases.

  18. A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

    NARCIS (Netherlands)

    van der Geize, R.; de Jong, W.; Hessels, G. I.; Grommen, A. W. F.; Jacobs, A. A. C.; Dijkhuizen, L.

    2008-01-01

    A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular

  19. The R21C Mutation in Cardiac Troponin I Imposes Differences in Contractile Force Generation between the Left and Right Ventricles of Knock-In Mice

    Directory of Open Access Journals (Sweden)

    Jingsheng Liang

    2015-01-01

    Full Text Available We investigated the effect of the hypertrophic cardiomyopathy-linked R21C (arginine to cysteine mutation in human cardiac troponin I (cTnI on the contractile properties and myofilament protein phosphorylation in papillary muscle preparations from left (LV and right (RV ventricles of homozygous R21C+/+ knock-in mice. The maximal steady-state force was significantly reduced in skinned papillary muscle strips from the LV compared to RV, with the latter displaying the level of force observed in LV or RV from wild-type (WT mice. There were no differences in the Ca2+ sensitivity between the RV and LV of R21C+/+ mice; however, the Ca2+ sensitivity of force was higher in RV-R21C+/+ compared with RV-WT and lower in LV- R21C+/+ compared with LV-WT. We also observed partial loss of Ca2+ regulation at low [Ca2+]. In addition, R21C+/+-KI hearts showed no Ser23/24-cTnI phosphorylation compared to LV or RV of WT mice. However, phosphorylation of the myosin regulatory light chain (RLC was significantly higher in the RV versus LV of R21C+/+ mice and versus LV and RV of WT mice. The difference in RLC phosphorylation between the ventricles of R21C+/+ mice likely contributes to observed differences in contractile force and the lower tension monitored in the LV of HCM mice.

  20. Effects of polycyclic aromatic compounds in fine particulate matter generated from household coal combustion on response to EGFR mutations in vitro.

    Science.gov (United States)

    Ho, Kin-Fai; Chang, Chih-Cheng; Tian, Linwei; Chan, Chi-Sing; Musa Bandowe, Benjamin A; Lui, Ka-Hei; Lee, Kang-Yun; Chuang, Kai-Jen; Liu, Chien-Ying; Ning, Zhi; Chuang, Hsiao-Chi

    2016-11-01

    Induction of PM2.5-associated lung cancer in response to EGFR-tyrosine kinase inhibitors (EGFR-TKI) remains unclear. Polycyclic aromatic hydrocarbons (PAHs) and their polar derivatives (oxygenated PAHs: OPAHs and azaarenes: AZAs) were characterized in fine particulates (PM2.5) emitted from indoor coal combustion. Samples were collected in Xuanwei (Yunnan Province), a region in China with a high rate of lung cancer. Human lung adenocarcinoma cells A549 (with wild-type EGFR) and HCC827 (with EGFR mutation) were exposed to the PM2.5, followed by treatment with EGFR-TKI. Two samples showed significant and dose-dependent reduction in the cell viability in A549. EGFR-TKI further demonstrated significantly decreased in cell viability in A549 after exposure to the coal emissions. Chrysene and triphenylene, dibenzo[a,h]anthracene, benzo[ghi]perylene, azaarenes and oxygenated polycyclic aromatic hydrocarbons (carbonyl-OPAHs) were all associated with EGFR-TKI-dependent reduced cell viability after 72-h exposure to the PM2.5. The findings suggest the coal emissions could influence the response of EGFR-TKI in lung cancer cells in Xuanwei.

  1. Enzymatic generation of whey protein hydrolysates under pH-controlled and non pH-controlled conditions: Impact on physicochemical and bioactive properties.

    Science.gov (United States)

    Le Maux, Solène; Nongonierma, Alice B; Barre, Chloé; FitzGerald, Richard J

    2016-05-15

    Enzymatic hydrolysis of whey protein (WP) was carried out under pH-controlled and non pH-controlled conditions using papain and a microbial-derived alternative (papain-like activity). The impact of such conditions on physicochemical and bioactive properties was assessed. WP hydrolysates (WPH) generated with the same enzyme displayed similar degree of hydrolysis. However, their reverse-phase liquid chromatograph mass spectrometry peptide profiles differed. A significantly higher oxygen radical absorbance capacity (ORAC) value was obtained for WP hydrolysed with papain at constant pH of 7.0 compared to the associated WPH generated without pH regulation. In contrast, there was no significant effect of pH regulation on dipeptidyl peptidase IV (DPP-IV) properties. WP hydrolysed with papain-like activity under pH regulation at 7.0 displayed higher ORAC activity and DPP-IV inhibitory properties compared to the associated WPH generated without pH regulation. This study has demonstrated that pH conditions during WPH generation may impact on peptide release and therefore on WPH bioactive properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Targeted next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material

    NARCIS (Netherlands)

    W.W.J. de Leng (Wendy); C.G.M. Gadellaa-Van Hooijdonk (C. G M); F.A.S. Barendregt-Smouter (Françoise A.S.); M.J. Koudijs (Marco J.); I. Nijman (Ies); J.W.J. Hinrichs (John W.J.); E. Cuppen (Edwin); S. van Lieshout (Stef); R.D. Loberg (Robert D.); M.J.A. de Jonge (Maja); E.E. Voest (Emile); R.A. de Weger (Roel); N. Steeghs (Neeltje); M.H. Langenberg (Marlies); S. Sleijfer (Stefan); S.M. Willems (Stefan Martin); M.P. Lolkema (Martijn)

    2016-01-01

    textabstractBackground Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study ther

  3. Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease patient iPSC line carrying a A79V mutation in PSEN1

    DEFF Research Database (Denmark)

    Pires, Carlota; Schmid, Benjamin; Petræus, Carina;

    2016-01-01

    Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease causing neural cell degeneration and brain atrophy and is considered to be the most common form of dementia. We previously generated an induced pluripotent stem cell (iPSC) line from an AD patient carrying an A79V...

  4. Limited phenotypic variation of hypocalcified amelogenesis imperfecta in a Danish five-generation family with a novel FAM83H nonsense mutation

    DEFF Research Database (Denmark)

    Haubek, Dorte; Gjørup, Hans; Jensen, Lillian Gryesten

    2011-01-01

    BACKGROUND.  Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI) is a disease with severe dental manifestations. OBJECTIVES.  The aims were by means of a genome-wide linkage scan to search for the gene underlying the ADHCAI phenotype in a Danish five-generation family and to study...

  5. Factors that affect the molecular nature of germ-line mutations recovered in the mouse specific-locus test

    Energy Technology Data Exchange (ETDEWEB)

    Russell, L.B. (Oak Ridge National Lab., TN (United States))

    1991-01-01

    The morphological specific locus test (SLT), which allows the scoring of 2,000 loci/hr/person, has been in use for four decades for measuring mammalian germ-line mutation rates under various conditions of exposure. More recently, the SLT's capabilities for the qualitative characterization of mutations have been exploited. The large sets of mutations centered on specific loci that have been accumulated over the years, including sets of nested deletions, have provided prime material for fine-structure genetic analyses. Subsequent molecular entry to these regions has led to intensive physical/functional mapping of megabase segments of the genome. In turn, these investigations have generated genetic and molecular tools for analyzing individual mutations as to extent and nature of the genomic lesion. These and related quantitative findings now make it possible to optimize conditions for the use of mutagens in providing desired types of mutations as tools.

  6. Carbocations generated under stable conditions by ionization of matrix-Isolated radicals: the allyl and benzyl cations

    OpenAIRE

    Mišić, Vladimir; Piech, Krzysztof; Bally, Thomas

    2013-01-01

    Carbocations are crucial intermediates in many chemical reactions; hence, considerable effort has gone into investigating their structures and properties, for example, in superacids, in salts, or in the gas phase. However, studies of the vibrational structure of carbocations are not abundant, because their infrared spectra are difficult to obtain in superacids or salts (where furthermore the cations may be perturbed by counterions), and the generation of gas-phase carbocations in discharges u...

  7. Application of Neural Networks to Predict UH-60L Electrical Generator Condition using (IMD-HUMS) Data

    Science.gov (United States)

    2006-12-01

    the two-year period where the IMD-HUMS were installed. Each IMD-HUMS acquisition concerning the shaft, spur gear , and bearing of generators results... gear train, the gear mesh is the dominating source of mechanical vibration. This vibration primarily stems from the non-uniformity in the transmission ... torque fluctuations and deflections of the gearbox can also be 32 sources of vibration in gears . Clearly, any damage that occurs to the gear

  8. Conditional preparation of a quantum state in the continuous variable regime generation of a sub-Poissonian state from twin beams

    CERN Document Server

    Laurat, J; Treps, N; Maitre, A; Fabre, C; Laurat, Julien; Coudreau, Thomas; Treps, Nicolas; Maitre, Agnes; Fabre, Claude

    2003-01-01

    We report the first experimental demonstration of conditional preparation of a non classical state of light in the continuous variable regime. Starting from a non degenerate OPO which generates above threshold quantum intensity correlated signal and idler "twin beams", we keep the recorded values of the signal intensity only when the idler falls inside a band of values narrower than its standard deviation. By this very simple technique, we generate a sub-Poissonian state 4.4dB below shot noise from twin beams exhibiting 7.5dB of noise reduction in the intensity difference.

  9. Setting boundary conditions on the Khokhlov-Zabolotskaya equation for modeling ultrasound fields generated by strongly focused transducers

    Science.gov (United States)

    Rosnitskiy, P. B.; Yuldashev, P. V.; Vysokanov, B. A.; Khokhlova, V. A.

    2016-03-01

    An equivalent source model is developed for setting boundary conditions on the parabolic diffraction equation in order to simulate ultrasound fields radiated by strongly focused medical transducers. The equivalent source is defined in a plane; corresponding boundary conditions for pressure amplitude, aperture, and focal distance are chosen so that the axial solution to the parabolic model in the focal region of the beam matches the solution to the full diffraction model (Rayleigh integral) for a spherically curved uniformly vibrating source. It is shown that the proposed approach to transferring the boundary condition from a spherical surface to a plane makes it possible to match the solutions over an interval of several diffraction maxima around the focus even for focused sources with F-numbers less than unity. This method can be used to accurately simulate nonlinear effects in the fields of strongly focused therapeutic transducers using the parabolic Khokhlov-Zabolotskaya equation.

  10. PPARγ mutations, lipodystrophy and diabetes.

    Science.gov (United States)

    Astapova, Olga; Leff, Todd

    2014-11-01

    The focus of this review is the lipodystrophy syndrome caused by mutation in the PPARγ nuclear receptor - partial familial lipodystrophy FPLD3. To provide a broader context for how these mutations act to generate the clinical features of partial lipodystrophy we will review the basic biology of PPARγ and also survey the set PPARγ genetic variants that do not cause lipodystrophy, but are nonetheless associated with clinically related syndromes, specifically type 2 diabetes.

  11. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.

    Science.gov (United States)

    Quadros, Rolen M; Miura, Hiromi; Harms, Donald W; Akatsuka, Hisako; Sato, Takehito; Aida, Tomomi; Redder, Ronald; Richardson, Guy P; Inagaki, Yutaka; Sakai, Daisuke; Buckley, Shannon M; Seshacharyulu, Parthasarathy; Batra, Surinder K; Behlke, Mark A; Zeiner, Sarah A; Jacobi, Ashley M; Izu, Yayoi; Thoreson, Wallace B; Urness, Lisa D; Mansour, Suzanne L; Ohtsuka, Masato; Gurumurthy, Channabasavaiah B

    2017-05-17

    Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring. Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

  12. A low-copy-number plasmid for retrieval of toxic genes from BACs and generation of conditional targeting constructs.

    Science.gov (United States)

    Na, Giyoun; Wolfe, Andrew; Ko, Chemyong; Youn, Hyesook; Lee, Young-Min; Byun, Sung June; Jeon, Iksoo; Koo, Yongbum

    2013-06-01

    Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.

  13. Dominance effects of deleterious and beneficial mutations in a single gene of the RNA virus ϕ6.

    Directory of Open Access Journals (Sweden)

    Sarah B Joseph

    Full Text Available Most of our knowledge of dominance stems from studies of deleterious mutations. From these studies we know that most deleterious mutations are recessive, and that this recessivity arises from a hyperbolic relationship between protein function (i.e., protein concentration or activity and fitness. Here we investigate whether this knowledge can be used to make predictions about the dominance of beneficial and deleterious mutations in a single gene. We employed a model system--the bacteriophage φ6--that allowed us to generate a collection of mutations in haploid conditions so that it was not biased toward either dominant beneficial or recessive deleterious mutations. Screening for the ability to infect a bacterial host that does not permit infection by the wildtype φ6, we generated a collection of mutations in P3, a gene involved in attachment to the host and in phage particle assembly. The resulting collection contained mutations with both deleterious and beneficial effects on fitness. The deleterious mutations in our collection had additive effects on fitness and the beneficial mutations were recessive. Neither of these observations were predicted from previous studies of dominance. This pattern is not consistent with the hyperbolic (diminishing returns relationship between protein function and fitness that is characteristic of enzymatic genes, but could have resulted from a curve of increasing returns.

  14. Do Conditional Cash Transfers for Schooling Generate Lasting Benefits? A Five-Year Followup of PROGRESA/Oportunidades

    Science.gov (United States)

    Behrman, Jere R.; Parker, Susan W.; Todd, Petra E.

    2011-01-01

    Conditional cash transfer (CCT) programs link public transfers to human capital investment in hopes of alleviating current poverty and reducing its intergenerational transmission. However, little is known about their long-term impacts. This paper evaluates longer-run impacts on schooling and work of the best-known CCT program, Mexico's…

  15. A Study on Effects of Initial Conditions to the Fuel Integrity Analysis of Steam Generator Tube Rupture Accident

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Min Seok; Kim, Ung Soo; Park, Min Soo; Huh, Jae Young; Lee, Gyu Cheon [KEPCO, Daejeon (Korea, Republic of)

    2016-05-15

    During SGTR accident, RCS pressure continuously decreases while the core power, core flow rate and core average temperature almost do not change until reactor trip occurs. As a result, the departure from nucleate boiling ratio (DNBR) also continuously decreases, thus eroding the thermal margin to DNB. To identify no damage of the fuel cladding due to the onset of film boiling, it must be analyzed whether the heat flux is maintained below the critical heat flux or not. For this purpose, it is used to define the specified acceptable fuel design limit (SAFDL) of DNBR and evaluate that the DNBR stays above the SAFDL. Evaluating the DNBR in SGTR accident, several initial conditions are varied as that would challenge to the safety limit, or SAFDL. However, the effects of some initial conditions are complicate and difficult to be intuitively identified. The initial conditions mainly affect the minimum DNBR during SGTR accident are the initial core power, the initial RCS flow rate, the initial core inlet temperature and the initial pressurizer pressure. The conservative initial conditions for the core power and the RCS flow rate can be determined easily by qualitative evaluation. The others such as the initial core inlet temperature and the initial pressurizer pressure are complicate and difficult to be determined. From this study, it is identified that the maximum initial pressurizer pressure and the minimum initial core inlet temperature on SGTR accident most postpones the reactor trip resulting in smaller RCS inventory at the reactor trip time point. And the smaller RCS inventory at the time point of the reactor trip tends to make the DNBR undershoot larger which is a dominant factor to determine the minimum DNBR under a LOOP condition.

  16. Software Mutational Robustness: Bridging The Gap Between Mutation Testing and Evolutionary Biology

    CERN Document Server

    Schulte, Eric; Fast, Ethan; Forrest, Stephanie; Weimer, Westley

    2012-01-01

    In the mutation testing paradigm, test suite quality is measured by its ability to detect variant programs generated through application of random changes to an original program. In evolutionary biology however, neutral mutations that leave fitness unchanged are considered to be beneficial---improving the system's robustness and ability to discover evolutionary improvements. In this paper, we generate a population of variant programs from an original program by applying lightweight random mutations. We adopt biological terminology and refer to undetected variants as neutral, and the percentage of all variants that are neutral as mutational robustness. Although they are related to equivalent mutants in mutation testing, which are viewed as problematic, we show positive properties of neutral variants which are easily generated and can be used to protect software against unknown defects. Even when mutations are restricted to statements executed by the test suit, we find that mutational robustness is high: 36.75%...

  17. Automatic Control Systems (ACS for Generation and Sale of Electric Power Under Conditions of Industry-Sector Liberalization

    Directory of Open Access Journals (Sweden)

    Yu. Petrusha

    2013-01-01

    Full Text Available Possible risks pertaining to transition of electric-power industry to market relations have been considered in the paper. The paper presents an integrated ACS for generation and sale of electric power as an improvement of methodology for organizational and technical management. The given system is based on integration of operating Automatic Dispatch Control System (ADCS and developing Automatic Electricity Meter Reading System (AEMRS. The paper proposes to form an inter-branch sector of ACS PLC (Automatic Control System for Prolongation of Life Cycle users which is oriented on provision of development strategy.

  18. [Potential difference across the membrane of subcellular particles. V. Generation of potential differences by mitochondria and submitochondrial particles under anaerobic conditions].

    Science.gov (United States)

    Liberman, E A; Vladimirova, M A; Tsofina, L M

    1977-01-01

    It is shown by the mehtod of penetrating ions that Site O and I of the respiratory chain of submitochondrial particles are able to generate a membrane potential of the normal value under anaerobic conditons. When succinate is an electron donor and ferricyanide-an acceptor (Site II), the oxygen addition sharply increases the membrane potential at pH above 7.5 and does not change or even decreases it in reaction conditions more acid than pH 6.5. The generation of the membrane potential at low pH and in the absence of oxygen is predicted by the chemielectric hypothesis and cannot be explained by the chemiosmotic one. Mitochondria usually generate the membrane potential without O2 at pH 7.5 in the presence of ferricyanide when the substrate concentration exceeds 5 mM.

  19. A quality comparison of protein crystals grown under containerless conditions generated by diamagnetic levitation, silicone oil and agarose gel.

    Science.gov (United States)

    Cao, Hui-Ling; Sun, Li-Hua; Li, Jian; Tang, Lin; Lu, Hui-Meng; Guo, Yun-Zhu; He, Jin; Liu, Yong-Ming; Xie, Xu-Zhuo; Shen, He-Fang; Zhang, Chen-Yan; Guo, Wei-Hong; Huang, Lin-Jun; Shang, Peng; He, Jian-Hua; Yin, Da-Chuan

    2013-10-01

    High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

  20. Computation of a leakage in a stream generator heating tube with realistic initial and boundary conditions; Berechnung eines Dampferzeugerheizrohrlecks mit realistischen Anfangs- und Randbedingungen

    Energy Technology Data Exchange (ETDEWEB)

    Sarkadi, Peter; Schaffrath, Andreas [TUeV NORD SysTec GmbH und Co. KG, Hamburg (Germany)

    2009-07-01

    In the frame of a PWR reactor safety analysis the TUEV Nord Sys Tec GmbH has analyzed the plant behavior in case of a steam generator tube leakage using the thermal hydraulic code ATHLET and realistic initial and boundary conditions. The aim of the analysis was to show that the response of the emergency cooling criteria including the activation of safety injection pumps can be avoided. The Activation of the safety injection pumps could jeopardize the activity retention.

  1. Rapid knockout and reporter mouse line generation and breeding colony establishment using EUCOMM conditional-ready embryonic stem cells: A case study

    Directory of Open Access Journals (Sweden)

    James L. J. Coleman

    2015-06-01

    Full Text Available As little as a decade ago, generation of a single knockout mouse line was an expensive and time-consuming undertaking available to relatively few researchers. The International Knockout Mouse Consortium, established in 2007, has revolutionized the use of such models by creating an open-access repository of ES cells that, through sequential breeding with first FlpE and then Cre recombinase transgenic mice, facilitates germline global or conditional deletion of almost every gene in the mouse genome. In this Case Study, we describe our experience using the repository to create mouse lines for a variety of experimental purposes. Specifically, we discuss the process of obtaining germline transmission of two EUCOMM ‘knockout-first’ gene targeted constructs and the advantages and pitfalls of using this system. We then outline our breeding strategy and the outcomes of our efforts to generate global and conditional knockouts and reporter mice for the genes of interest. Line maintenance, removal of recombinase transgenes and cryopreservation are also considered. Our approach led to the generation of heterozygous knockout mice within 6 months of commencing breeding to the founder mice. By describing our experiences with the EUCOMM ES cells and subsequent breeding steps, we hope to assist other researchers with the application of this valuable approach to generating versatile knockout mouse lines.

  2. Atmospheric Dispersion Modeling of 137Cs generated from Nuclear Spent Fuel under Hypothetic Accidental Condition in the BNPP Area

    Science.gov (United States)

    Lee, Jongkuk; Lee, Kwan-Hee; Yook, Daesik; Kim, Sung Il; Lee, Byung Soo

    2016-04-01

    This study presents the results of atmosphere dispersion modeling using CALPUFF code that are based on computational simulation to evaluate the environmental characteristics of the Barakah nuclear power plant (BNPP) in west area of UAE. According to meteorological data analysis (2012~2013), the winds from the north(7.68%) and west(9.05%) including NNW(41.63%), NW(28.55%), and WNW(6.31%) winds accounted for more than 90% of the wind directions. East(0.2%) and south(0.6%) direction wind, including ESE(0.31%), SE(0.38%), and SSE(0.38%) were rarely distributed during the simulation period. Seasonal effects were not showed. However, a discrepancy in the tendency between daytime and night-time was observed. Approximately 87% of the wind speed was distributed below 5.4m/s (17%, 47% and 23% between the speeds of 0.5-1.8m/s 1.8-3.3m/s and 3.3-5.4m/s, respectively) during the annual period. Seasonal wind speed distribution results presented very similar pattern of annual distribution. Wind speed distribution of day and night, on the other hand, had a discrepancy with annual modeling results than seasonal distribution in some sections. The results for high wind speed (more than 10.8m/s) showed that this wind blew from the west. This high wind speed is known locally as the 'Shamal', which occurs rarely, lasting one or two days with the strongest winds experienced in association with gust fronts and thunderstorms. Six variations of cesium-137 (137Cs) dispersion test were simulated under hypothetic severe accidental condition. The 137Cs dispersion was strongly influenced by the direction and speed of the main wind. From the test cases, east-south area of the BNPP site was mainly influenced by 137Cs dispersion. A virtual receptor was set and calculated for observation of the 137Cs movement and accumulation. Surface roughness tests were performed for the analysis of topographic conditions. According to the surface condition, there are various surface roughness length. Four types

  3. Sex and deleterious mutations.

    Science.gov (United States)

    Gordo, Isabel; Campos, Paulo R A

    2008-05-01

    The evolutionary advantage of sexual reproduction has been considered as one of the most pressing questions in evolutionary biology. While a pluralistic view of the evolution of sex and recombination has been suggested by some, here we take a simpler view and try to quantify the conditions under which sex can evolve given a set of minimal assumptions. Since real populations are finite and also subject to recurrent deleterious mutations, this minimal model should apply generally to all populations. We show that the maximum advantage of recombination occurs for an intermediate value of the deleterious effect of mutations. Furthermore we show that the conditions under which the biggest advantage of sex is achieved are those that produce the fastest fitness decline in the corresponding asexual population and are therefore the conditions for which Muller's ratchet has the strongest effect. We also show that the selective advantage of a modifier of the recombination rate depends on its strength. The quantification of the range of selective effects that favors recombination then leads us to suggest that, if in stressful environments the effect of deleterious mutations is enhanced, a connection between sex and stress could be expected, as it is found in several species.