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Sample records for reverse mutation assay

  1. Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames Test

    Directory of Open Access Journals (Sweden)

    Eliana Aparecida Varanda

    2012-05-01

    Full Text Available The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone, the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

  2. The sequence spectrum of frameshift reversions obtained with a novel adaptive mutation assay in Saccharomyces cerevisiae

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    Erich Heidenreich

    2016-12-01

    Full Text Available Research on the mechanisms of adaptive mutagenesis in resting, i.e. non-replicating cells relies on appropriate mutation assays. Here we provide a novel procedure for the detection of frameshift-reverting mutations in yeast. Proliferation of non-reverted cells in this assay is suppressed by the lack of a fermentable carbon source. The test allele was constructed in a way that the reversions mimic microsatellite instability, a condition often found in cancer cells. We show the cell numbers during these starvation conditions and provide a DNA sequence spectrum of a representative set of revertants. The data in this article support the publication "Glucose starvation as a selective tool for the study of adaptive mutations in Saccharomyces cerevisiae" (Heidenreich and Steinboeck, 2016 [1].

  3. Absence of mutagenic effects of a particular Symphytum officinale L. liquid extract in the bacterial reverse mutation assay.

    Science.gov (United States)

    Benedek, Birgit; Ziegler, Andreas; Ottersbach, Peter

    2010-03-01

    Comfrey (Symphytum officinale L.) root is traditionally used for the topical treatment of contusions, strains and sprains. Besides allantoin and rosmarinic acid, which are discussed as pharmacologically active principles, the drug contains pyrrolizidine alkaloids (PAs) known for their hepatotoxic, carcinogenic and mutagenic properties. The topical herbal medicinal products Kytta-Salbe f and Kytta-Plasma f contain a PA-free liquid extract from comfrey root as active substance. The aim of this study was to demonstrate the absence of genotoxic effects of this special extract in the bacterial reverse mutation assay (Ames test). Briefly, comfrey root liquid extract was investigated for its ability to induce gene mutations in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation using the mammalian microsomal fraction S9 mix. Reference mutagens were used to check the validity of the experiments. Comfrey root fluid extract showed no biologically relevant increases in revertant colony numbers of any of the five tester strains, neither in the presence nor in the absence of metabolic activation. In conclusion, the comfrey root fluid extract contained in Kytta-Salbe f and Kytta-Plasma f was not mutagenic in the bacterial reverse mutation assay. (c) 2009 John Wiley & Sons, Ltd.

  4. Mutagenic and antimutagenic activities of Artemisia absinthium volatile oil by the bacterial reverse mutation assay in Salmonella typhimurium strains TA98 and TA100

    Directory of Open Access Journals (Sweden)

    Mahboubeh Taherkhani

    2014-09-01

    Full Text Available Objective: To investigate the mutagenic and antimutagenic activities of Artemisia absinthium L. (A. absinthium essential oil by the bacterial reverse mutation assay in Salmonella typhimurium (S. typhimurium strains. Methods: Water-distilled essential oil of A. absinthium collected from Ardabil, NorthWestern Iran, was investigated for mutagenic and antimutagenic activities. In present study, the mutagenic and antimutagenic activities of A. absinthium oil were investigated by the bacterial revere mutation assay in S. typhimurium TA98 and TA100 strains with and without S9 (microsomal mutagenesis assay. Results: The comparative mutagenicity effect was seen in 1.5 mg/plate by the bacterial reverse mutation assay in S. typhimurium TA98 strains, without S9 and the excellent antimutagenicity effect was seen in 1.5 mg/plate against S. typhimurium TA100, without S9. Conclusions: The mutagenicity and antimutagenicity effects of the volatile oil of A. absinthium were seen without the presence of metabolic activation.

  5. 40 CFR 798.5265 - The salmonella typhimurium reverse mutation assay.

    Science.gov (United States)

    2010-07-01

    ... mean number of revertant colonies per plate and standard deviation shall be presented for test chemical... revertant colonies per plate, standard deviation. (vi) Dose-response relationship, if applicable. (g... Salmonella/ mammalian-microsome mutagenicity test,” Mutation Research 31:347-364 (1975). (2) de Serres, F.J...

  6. [Safety Evaluation of Rare Sugar Syrup: Single-dose Oral Toxicity in Rats, Reverse Mutation Assay, Chromosome Aberration Assay, and Acute Non-Effect Level for Diarrhea of a Single Dose in Humans].

    Science.gov (United States)

    Yamada, Takako; Iida, Tetsuo; Takamine, Satoshi; Hayashi, Noriko; Okuma, Kazuhiro

    2015-01-01

    The safety of rare sugar syrup obtained from high-fructose corn syrup under slightly alkaline conditions was studied. Mutagenicity of rare sugar syrup was assessed by a reverse mutation assay using Salmonella typhimurium and Escherichia coli, and an in vitro chromosomal aberration assay using Chinese hamster lung cell line (CHL/IU). No mutagenicity of rare sugar syrup was detected under these experimental conditions. Oral administration of single dose (15,000 mg/kg) of rare sugar syrup to rats caused no abnormalities, suggesting no adverse effect of rare sugar syrup. In humans, the acute non-effect level of rare sugar syrup for causing diarrhea was estimated as 0.9 g/kg body weight as dry solid base in both males and females.

  7. Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

    Science.gov (United States)

    Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

    2014-07-01

    A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

  8. Telomerase reverse transcriptase promoter mutations in bladder cancer

    DEFF Research Database (Denmark)

    Allory, Yves; Beukers, Willemien; Sagrera, Ana

    2014-01-01

    for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription...... surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease...... frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC...

  9. SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

    Science.gov (United States)

    A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

  10. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1984-01-01

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

  11. Prospects for cellular mutational assays in human populations

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  12. Assessment of the safety of hydrogenated resistant maltodextrin: reverse mutation assay, acute and 90-day subchronic repeated oral toxicity in rats, and acute no-effect level for diarrhea in humans.

    Science.gov (United States)

    Yoshikawa, Yuko; Kishimoto, Yuka; Tagami, Hiroyuki; Kanahori, Sumiko

    2013-01-01

    A series of safety assessments were performed on hydrogenated resistant maltodextrin prepared by converting the reducing terminal glucose of resistant maltodextrin into sorbitol. The reverse mutation assay did not show mutagenicity. Acute and 90-day subchronic oral toxicity studies in rats showed no death was observed in any groups, including the group receiving the highest single dose of 10 g/kg body weight or the highest dose of 5 g/kg body weight per day for 90 days. Mucous or watery stools were observed in the hydrogenated resistant maltodextrin treatment group on the acute study, which were transient and were associated with the osmotic pressure caused by intake of the high concentrations. Subchronic study showed dose-dependent increases in the weights of cecum alone, cecal contents alone, and cecum with cecal contents as well as hypertrophy of the cecal mucosal epithelium, which are considered to be common physiological responses after intake of indigestible carbohydrates. These results indicated that the no observed adverse effect level (NOAEL) of hydrogenated resistant maltodextrin was 10 g/kg body weight or more on the acute oral toxicity study and 5.0 g/kg body weight/day or more on the 90-day subchronic repeated oral toxicity study in rats. Further study performed in healthy adult humans showed that the acute no-effect level of hydrogenated resistant maltodextrin for diarrhea was 0.8 g/kg body weight for men and more than 1.0 g/kg body weight for women. The results of the current safety assessment studies suggest that hydrogenated resistant maltodextrin is safe for human consumption.

  13. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...

  14. Prospects for cellular mutational assays in human populations

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1985-01-01

    Practical, sensitive, effective, human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis. When available, such assays should allow us to fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. We will be able to validate the role of somatic mutations in carcinogenesis, to identify environmental factors that affect human germ cells, to integrate the effects of complex mixtures and the environment in the human subject, and to identify people who are hypersusceptible to genetic injury. Human cellular mutational assays, particularly when combined with cytogenetic and heritable mutational tests, promise to play pivotal roles in estimating the risk from low-dose radiation and chemical exposures. These combined methods avoid extrapolations of dose and from species to species, and may be sensitive enough and credible enough to permit politically, socially and scientifically acceptable risk management. 16 references

  15. A source of artifact in the lacZ reversion assay in Escherichia coli.

    Science.gov (United States)

    Hoffmann, George R; Gray, Carol L; Lange, Paulina B; Marando, Christie I

    2015-06-01

    The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its

  16. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  17. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    International Nuclear Information System (INIS)

    Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

    2006-01-01

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

  18. Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

    Science.gov (United States)

    Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

    2015-10-01

    Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

  19. Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

    Science.gov (United States)

    Li, R; Kast, J

    2017-01-01

    Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

  20. Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

    Science.gov (United States)

    Azeem, Syeda Maryam; Muwonge, Alecia N; Thakkar, Nehaben; Lam, Kristina W; Frey, Kathleen M

    2018-01-01

    Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development. Published by Elsevier Inc.

  1. The induction of somatic mutations by high-LET radiation observed using the Drosophila assay system

    International Nuclear Information System (INIS)

    Yoshikawa, Isao; Takatsuji, Toshihiro; Nagano, Masaaki; Hoshi, Masaharu; Takada, Jun; Endo, Satoru

    1999-01-01

    To evaluate the mutagenic potential of high-LET radiation, an analysis was made on the production of somatic mutations by 252 Cf fission neutron s and heavy particle ions accelerated by a synchrotron. A Drosophila strain that allows simultaneous detection of two types of mutations in an identical fly was constructed. One was a wing-hair mutation and the other was an eye-color mosaic spot mutation. Measurements were made using a combined assay system of both mutation assays. Larvae were exposed to radiation at the age of post-ovipositional day-3. The efficiency of 252 Cf neutrons for inducing wing-hair mosaic spots was very high, the relative biological effectiveness (RBE) = 8.5, but the efficiency for eye-color mosaic spot was nearly equal (RBE = 1.2) to that of 137 Cs γ-rays. The RBE of carbon ions for inducing wing-hair mosaic spots increased as an increase in LET values. The RBE for the induction of eye-color mutants did not change with LET. These relationships suggest that more complex types of DNA damages such as non-rejoinable strand break or clustered double strand break, which increase with LET may be responsible for the induction of wing-hair mutation, while simpler forms of molecular damage may induce a reversion in the white-ivory allele. (M.N.)

  2. Progress in hprt mutation assay and its application in radiation biology

    International Nuclear Information System (INIS)

    He Jing; Li Qiang

    2008-01-01

    hprt gene is an X-linked locus that has been well studied and widely used as a bio-marker in mutation detection, hprt mutation assay is a gene mutation test system in mammalian cells in vitro which has been used as a biological dosimeter. In this paper, the biological characteristics of hprt gene, hprt mutation detection methodology and the application of hprt mutation assay in radiation biology are comprehensively reviewed. (authors)

  3. Standardized comparison of the relative impacts of HIV-1 reverse transcriptase (RT) mutations on nucleoside RT inhibitor susceptibility.

    Science.gov (United States)

    Melikian, George L; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V; Zolopa, Andrew; Robbins, Gregory K; Kagan, Ron; Israelski, Dennis; Shafer, Robert W

    2012-05-01

    Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.

  4. The induction of somatic mutations by high-LET radiations using the drosophila assay system

    International Nuclear Information System (INIS)

    Yoshikawa, Isao; Takatsuji, Toshihiro

    2004-01-01

    Two types of somatic mutation in Drosophila melanogaster were examined to evaluate the relative biological effectiveness (RBE) of 252 Cf neutrons and heavy ions (carbon ions and neon ions) accelerated with a synchrotron for inducing mutations as a function of linear energy transfer (LET). One is the loss of heterozygosity for wing-hair mutations and the other the reversion of the mutant white-ivory. The measurements were made using a combined mutation assay system; so that induced mutant wing-hair clones as well as revertant eye-color clones could be detected simultaneously in the same fly. Larvae were irradiated at the age of 3 days post-oviposition. The efficiency of 252 Cf neutrons for inducing wing-hair mosaic spots is very high, RBE=8.5, but that for eye-color mosaic spot is almost equal (RBE=1.2) to that of 137 Cs γ-rays. RBE-LET relationships were obtained for the induction of wing-hair and eye-color mosaic spots. The RBE of carbon and neon ions for producing wing-hair mosaic spots increased with increasing LET values. The RBE for the induction of eye-color mutants did not change with LET. These relationships suggest that more complex types of DNA damage such as non-rejoinable strand breaks or clustered double strand breaks that increase with LET may be responsible for inducing the wing-hair mutation, while simpler forms of molecular damage may induce reversion in the white-ivory allele. (author)

  5. 40 CFR 79.68 - Salmonella typhimurium reverse mutation assay.

    Science.gov (United States)

    2010-07-01

    ...) AIR PROGRAMS (CONTINUED) REGISTRATION OF FUELS AND FUEL ADDITIVES Testing Requirements for... of E. coli: partial purification and some properties,” Journal of Biological Chemistry. 218:97-106...

  6. Preclinical test: bacterial reverse mutation test for {sup 18}F-fluorocholine produced in CDTN

    Energy Technology Data Exchange (ETDEWEB)

    Mendes, Bruno M.; Bispo, Ana Carolina A.; Campos, Danielle C.; Silva, Juliana B., E-mail: bmm@cdtn.br, E-mail: acab@cdtn.br, E-mail: dcc@cdtn.br, E-mail: silvajb@cdtn.b [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical due to its effective detection of many type of malignant neoplasm. The research related to {sup 18}F-fluorocholine synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register {sup 18}FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the {sup 18}FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to 'cold' fluorocholine molecule (19FCH). Subsequently, the decayed product of {sup 18}FCH synthesis was evaluated. The first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically significant increases in revertant colonies rate for any strains tested after their exposure to decayed {sup 18}FCH or {sup 19}FCH. The number of revertant colonies in positive controls was significantly higher than that observed in significant increases in revertant colonies rate for any strains tested after their exposure to decayed {sup 18}FCH or {sup 19}FCH. The number of revertant colonies in positive controls was significantly higher than that observed in negative controls. Based on results of this assay, {sup 18}FCH and {sup 19}FCH, at tested doses, were found to be non-mutagenic in bacterial reverse mutation test. (author)

  7. Preclinical test: bacterial reverse mutation test for 18F-fluorocholine produced in CDTN

    International Nuclear Information System (INIS)

    Mendes, Bruno M.; Bispo, Ana Carolina A.; Campos, Danielle C.; Silva, Juliana B.

    2013-01-01

    The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical due to its effective detection of many type of malignant neoplasm. The research related to 18 F-fluorocholine synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register 18 FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the 18 FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to 'cold' fluorocholine molecule (19FCH). Subsequently, the decayed product of 18 FCH synthesis was evaluated. The first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18 FCH or 19 FCH. The number of revertant colonies in positive controls was significantly higher than that observed in significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18 FCH or 19 FCH. The number of revertant colonies in positive controls was significantly higher than that observed in negative controls. Based on results of this assay, 18 FCH and 19 FCH, at tested doses, were found to be non-mutagenic in bacterial reverse mutation test. (author)

  8. Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuscoe, J.C.; O' Neill, J.P.; Machanoff, R.; Hsie, A.W.

    1982-01-01

    An assay is described for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10/sup 6/ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10..mu..M) is then added directly to the growing culture and AS-resistant (AS/sup r/) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT/sup -/ clones. The AS/sup r/ phenotype is characterized both physiologically and biochemically. All AS/sup r/ clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT.

  9. From bad to good: Fitness reversals and the ascent of deleterious mutations.

    Directory of Open Access Journals (Sweden)

    Matthew C Cowperthwaite

    2006-10-01

    Full Text Available Deleterious mutations are considered a major impediment to adaptation, and there are straightforward expectations for the rate at which they accumulate as a function of population size and mutation rate. In a simulation model of an evolving population of asexually replicating RNA molecules, initially deleterious mutations accumulated at rates nearly equal to that of initially beneficial mutations, without impeding evolutionary progress. As the mutation rate was increased within a moderate range, deleterious mutation accumulation and mean fitness improvement both increased. The fixation rates were higher than predicted by many population-genetic models. This seemingly paradoxical result was resolved in part by the observation that, during the time to fixation, the selection coefficient (s of initially deleterious mutations reversed to confer a selective advantage. Significantly, more than half of the fixations of initially deleterious mutations involved fitness reversals. These fitness reversals had a substantial effect on the total fitness of the genome and thus contributed to its success in the population. Despite the relative importance of fitness reversals, however, the probabilities of fixation for both initially beneficial and initially deleterious mutations were exceedingly small (on the order of 10(-5 of all mutations.

  10. In vitro cross-resistance profile of nucleoside reverse transcriptase inhibitor (NRTI) BMS-986001 against known NRTI resistance mutations.

    Science.gov (United States)

    Li, Zhufang; Terry, Brian; Olds, William; Protack, Tricia; Deminie, Carol; Minassian, Beatrice; Nowicka-Sans, Beata; Sun, Yongnian; Dicker, Ira; Hwang, Carey; Lataillade, Max; Hanna, George J; Krystal, Mark

    2013-11-01

    BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

  11. A 3-plex methylation assay combined with the FGFR3 mutation assay sensitively detects recurrent bladder cancer in voided urine

    DEFF Research Database (Denmark)

    Kandimalla, Raju; Masius, Roy; Beukers, Willemien

    2013-01-01

    is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard. Experimental Design: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study......Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non–muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study......, and nonmalignant urines (n = 130). Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57...

  12. The glycophorin A assay for somatic cell mutations in humans

    International Nuclear Information System (INIS)

    Langlois, R.G.; Bigbee, W.L.; Jensen, R.H.

    1989-01-01

    In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs

  13. Reversible optic neuropathy with OPA1 exon 5b mutation

    DEFF Research Database (Denmark)

    Cornille, K.; Milea, D.; Amati-Bonneau, P.

    2008-01-01

    A new c.740G>A (R247H) mutation in OPA1 alternate spliced exon 5b was found in a patient presenting with bilateral optic neuropathy followed by partial, spontaneous visual recovery. R247H fibroblasts from the patient and his unaffected father presented unusual highly tubular mitochondrial network......, significant increased susceptibility to apoptosis, oxidative phosphorylation uncoupling, and altered OPA1 protein profile, supporting the pathogenicity of this mutation. These results suggest that the clinical spectrum of the OPA1-associated optic neuropathies may be larger than previously described...

  14. Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.

    Science.gov (United States)

    Li, Ru; Huang, Jiqing; Kast, Juergen

    2015-05-01

    Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.

  15. Reversal or protection by light of the ethidium bromide induced petite mutation in yeast

    International Nuclear Information System (INIS)

    Hixon, S.C.; Burnham, A.D.; Irons, R.L.

    1979-01-01

    An intermediate in the ethidium bromide (EB) induced petite mutation pathway may be destabilized by daylight light to cause a reversion to the normal grande phenotype. Starved cells preincubated in the dark for up to 6 h with 100μg/ml EB could be reverted to grandes after one hour of light exposure, whereas similarly treated cells maintained in the dark expresse the petite mutation in more than 80 percent of the population. In addition, the production of petite mutants by EB in buffer could be prevented if cell suspensions were exposed to light immediately upon the addition of EB. Photoreversal of the EB-derived petite mutation in growing cells as less efficient presumably because the availability of an energy source caused a continuation of mutation events beyond the light revertible step to a non-reversible fixation of the mutation. Cells treated with EB in growth and reversal of the mutation. This may be due to the cold inhibition of an enzyme which comes into play beyond the light sensitive step in the mutation pathway. (orig.) [de

  16. Reversible Congenital Hypogonadotropic Hypogonadism in Patients with CHD7, FGFR1 or GNRHR Mutations

    Science.gov (United States)

    Laitinen, Eeva-Maria; Tommiska, Johanna; Sane, Timo; Vaaralahti, Kirsi; Toppari, Jorma; Raivio, Taneli

    2012-01-01

    Background Congenital hypogonadotropic hypogonadism (HH) is a rare cause for delayed or absent puberty. These patients may recover from HH spontaneously in adulthood. To date, it is not possible to predict who will undergo HH reversal later in life. Herein we investigated whether Finnish patients with reversal of congenital hypogonadotropic hypogonadism (HH) have common phenotypic or genotypic features. Methods and Findings Thirty-two male HH patients with anosmia/hyposmia (Kallmann Syndrome, KS; n = 26) or normal sense of smell (nHH; n = 6) were enrolled (age range, 18–61 yrs). The patients were clinically examined, and reversal of HH was assessed after treatment withdrawal. KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, GNRHR, GNRH1, KISS1R, KISS1, TAC3, TACR3, and LHβ were screened for mutations. Six HH patients (2 KS, 4 nHH) were verified to have reversal of HH. In the majority of cases, reversal occurred early in adulthood (median age, 23 yrs; range, 21–39 yrs). All had spontaneous testicular growth while on testosterone replacement therapy (TRT). One nHH subject was restarted on TRT due to a decline in serum T. Two reversal variants had a same GNRHR mutation (R262Q), which was accompanied by another GNRHR mutation (R139H or del309F). In addition, both of the KS patients had a mutation in CHD7 (p.Q51X) or FGFR1 (c.91+2T>A). Conclusions Considerable proportion of patients with HH (8% of KS probands) may recover in early adulthood. Spontaneous testicular enlargement during TRT was highly suggestive for reversal of HH. Those with the GNRHR mutation R262Q accompanied by another GNRHR mutation may be prone to reversal, although even patients with a truncating mutation in CHD7 or a splice-site mutation in FGFR1 can recover. We recommend that all adolescents and young adults with congenital HH should be informed on the possibility of reversal. PMID:22724017

  17. Reversible congenital hypogonadotropic hypogonadism in patients with CHD7, FGFR1 or GNRHR mutations.

    Directory of Open Access Journals (Sweden)

    Eeva-Maria Laitinen

    Full Text Available BACKGROUND: Congenital hypogonadotropic hypogonadism (HH is a rare cause for delayed or absent puberty. These patients may recover from HH spontaneously in adulthood. To date, it is not possible to predict who will undergo HH reversal later in life. Herein we investigated whether Finnish patients with reversal of congenital hypogonadotropic hypogonadism (HH have common phenotypic or genotypic features. METHODS AND FINDINGS: Thirty-two male HH patients with anosmia/hyposmia (Kallmann Syndrome, KS; n = 26 or normal sense of smell (nHH; n = 6 were enrolled (age range, 18-61 yrs. The patients were clinically examined, and reversal of HH was assessed after treatment withdrawal. KAL1, FGFR1, FGF8, PROK2, PROKR2, CHD7, WDR11, GNRHR, GNRH1, KISS1R, KISS1, TAC3, TACR3, and LHβ were screened for mutations. Six HH patients (2 KS, 4 nHH were verified to have reversal of HH. In the majority of cases, reversal occurred early in adulthood (median age, 23 yrs; range, 21-39 yrs. All had spontaneous testicular growth while on testosterone replacement therapy (TRT. One nHH subject was restarted on TRT due to a decline in serum T. Two reversal variants had a same GNRHR mutation (R262Q, which was accompanied by another GNRHR mutation (R139H or del309F. In addition, both of the KS patients had a mutation in CHD7 (p.Q51X or FGFR1 (c.91+2T>A. CONCLUSIONS: Considerable proportion of patients with HH (8% of KS probands may recover in early adulthood. Spontaneous testicular enlargement during TRT was highly suggestive for reversal of HH. Those with the GNRHR mutation R262Q accompanied by another GNRHR mutation may be prone to reversal, although even patients with a truncating mutation in CHD7 or a splice-site mutation in FGFR1 can recover. We recommend that all adolescents and young adults with congenital HH should be informed on the possibility of reversal.

  18. Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.

    Science.gov (United States)

    Gu, Lijun; Kawana-Tachikawa, Ai; Shiino, Teiichiro; Nakamura, Hitomi; Koga, Michiko; Kikuchi, Tadashi; Adachi, Eisuke; Koibuchi, Tomohiko; Ishida, Takaomi; Gao, George F; Matsushita, Masaki; Sugiura, Wataru; Iwamoto, Aikichi; Hosoya, Noriaki

    2014-01-01

    Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research. We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method. Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%). We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.

  19. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

    2016-04-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  20. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    International Nuclear Information System (INIS)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee

    2016-01-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125 I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  1. Telomerase reverse transcriptase promoter mutations in glandular lesions of the urinary bladder.

    Science.gov (United States)

    Vail, Eric; Zheng, Xiaoyong; Zhou, Ming; Yang, Ximing; Fallon, John T; Epstein, Jonathan I; Zhong, Minghao

    2015-10-01

    Glandular lesions of the urinary bladder include a broad spectrum of entities ranging from completely benign to primary and secondary malignancies. The accurate diagnosis of these lesions is both important and challenging. Recently, studies suggest that telomerase reverse transcriptase (TERT) promoter mutations could be a biomarker for urothelial carcinoma (UC). We hypothesized that these mutations can distinguish UC with glandular differentiation from nephrogenic adenoma, primary adenocarcinoma of the urinary bladder (PAUB), or secondary malignancies. Twenty-five cases of benign glandular lesions (including nephrogenic adenoma); 29 cases of UC with glandular differentiation; 10 cases of PAUB; and 10 cases each of metastatic colon cancer, prostatic carcinoma, and carcinoma from Mullerian origin were collected. Slides were reviewed and selected to make sure the lesion was at least 10% to 20% of all tissue. Macrodissection was performed in some of cases, and genomic DNA was extracted from the tissue. Telomerase reverse transcriptase promoter mutations were determined by standard polymerase chain reaction sequencing. Twenty-one cases (72%) of UC with glandular differentiation were positive for TERT promoter mutations. However, none of the remaining cases (total 65 cases of benign lesions, PAUB, and metastatic carcinomas) was positive for TERT promoter mutation. Telomerase reverse transcriptase promoter mutations were highly associated with UC including UC with glandular differentiation but not other glandular lesions of bladder. Therefore, in conjunction with morphologic features, Immunohistochemistry stain profile, and clinical information, TERT promoter mutations could distinguish UC with glandular differentiation from other bladder glandular lesions. In addition, lack of TERT promoter mutations in primary adenocarcinoma of bladder suggests that this entity may have different origin or carcinogenesis from those of UC. Published by Elsevier Inc.

  2. Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen

    NARCIS (Netherlands)

    Cento, Valeria; van Hemert, Formijn; Neumann-Fraune, Maria; Mirabelli, Carmen; Di Maio, Velia-Chiara; Salpini, Romina; Bertoli, Ada; Micheli, Valeria; Gubertini, Guido; Romano, Sara; Visca, Michela; de Sanctis, Giuseppe-Maria; Berkhout, Ben; Marino, Nicoletta; Mazzotta, Francesco; Cappiello, Giuseppina; Spanò, Alberto; Sarrecchia, Cesare; Ceccherini-Silberstein, Francesca; Andreoni, Massimo; Angelico, Mario; Verheyen, Jens; Perno, Carlo Federico; Svicher, Valentina

    2013-01-01

    The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. 356 full-length HBV-RT sequences

  3. Photoreactivation reverses ultraviolet radition induced premutagenic lesions leading to frameshift mutations in Escherichia coli

    International Nuclear Information System (INIS)

    Yamamoto, Kazuo

    1985-01-01

    The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvr A6 derivative of Escherichia coli K12. Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps. The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsueqently exposed to visible light. The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp + was 3.6 and 3.4, respectively. UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation. The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp + was 6.5 and 6.3, respectively. The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7-2.5. Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent. We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvr A6 background. (orig.)

  4. Zidovudine (AZT monotherapy selects for the A360V mutation in the connection domain of HIV-1 reverse transcriptase.

    Directory of Open Access Journals (Sweden)

    Jessica H Brehm

    Full Text Available We previously demonstrated in vitro that zidovudine (AZT selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the C-terminal domains of HIV-1 RT.Full-length RT sequences in plasma obtained pre- and post-therapy were compared in 23 participants who received AZT monotherapy from the AIDS Clinical Trials Group study 175. Five of the 23 participants reached a primary study endpoint. Mutations significantly associated with AZT monotherapy included K70R (p = 0.003 and T215Y (p = 0.013 in the polymerase domain of HIV-1 RT, and A360V (p = 0.041 in the connection domain of HIV-1 RT. HIV-1 drug susceptibility assays demonstrated that A360V, either alone or in combination with thymidine analog mutations, decreased AZT susceptibility in recombinant viruses containing participant-derived full-length RT sequences or site-directed mutant RT. Biochemical studies revealed that A360V enhances the AZT-monophosphate excision activity of purified RT by significantly decreasing the frequency of secondary RNase H cleavage events that reduce the RNA/DNA duplex length and promote template/primer dissociation.The A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance.

  5. New prognostic factor telomerase reverse transcriptase promotor mutation presents without MR imaging biomarkers in primary glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Ersoy, Tunc F.; Simon, Matthias [University Hospital Bonn, Department of Neurosurgery and Stereotaxy, Bonn (Germany); Ev. Krankenhaus Bielefeld, Department of Neurosurgery, Bielefeld (Germany); Keil, Vera C.; Hadizadeh, Dariusch R.; Schild, Hans H. [University Hospital Bonn, Department of Radiology, Bonn (Germany); Gielen, Gerrit H.; Waha, Andreas [University Hospital Bonn, Institute of Neuropathology, Bonn (Germany); Fimmers, Rolf [IMBIE, University Hospital Bonn, Bonn (Germany); Heidenreich, Barbara; Kumar, Rajiv [DFKZ, Department of Molecular Genetic Epidemiology, Heidelberg (Germany)

    2017-12-15

    Magnetic resonance (MR) imaging biomarkers can assist in the non-invasive assessment of the genetic status in glioblastomas (GBMs). Telomerase reverse transcriptase (TERT) promoter mutations are associated with a negative prognosis. This study was performed to identify MR imaging biomarkers to forecast the TERT mutation status. Pre-operative MRIs of 64/67 genetically confirmed primary GBM patients (51/67 TERT-mutated with rs2853669 polymorphism) were analyzed according to Visually AcceSAble Rembrandt Images (VASARI) (https: //wiki.cancerimagingarchive.net/display/Public/VASARI+Research+Project) imaging criteria by three radiological raters. TERT mutation and O{sup 6}-methylguanine-DNA methyltransferase (MGMT) hypermethylation data were obtained through direct and pyrosequencing as described in a previous study. Clinical data were derived from a prospectively maintained electronic database. Associations of potential imaging biomarkers and genetic status were assessed by Fisher and Mann-Whitney U tests and stepwise linear regression. No imaging biomarkers could be identified to predict TERT mutational status (alone or in conjunction with TERT promoter polymorphism rs2853669 AA-allele). TERT promoter mutations were more common in patients with tumor-associated seizures as first symptom (26/30 vs. 25/37, p = 0.07); these showed significantly smaller tumors [13.1 (9.0-19.0) vs. 24.0 (16.6-37.5) all cm{sup 3}; p = 0.007] and prolonged median overall survival [17.0 (11.5-28.0) vs. 9.0 (4.0-12.0) all months; p = 0.02]. TERT-mutated GBMs were underrepresented in the extended angularis region (p = 0.03), whereas MGMT-methylated GBMs were overrepresented in the corpus callosum (p = 0.03) and underrepresented temporomesially (p = 0.01). Imaging biomarkers for prediction of TERT mutation status remain weak and cannot be derived from the VASARI protocol. Tumor-associated seizures are less common in TERT mutated glioblastomas. (orig.)

  6. Prediction of mutational tolerance in HIV-1 protease and reverse transcriptase using flexible backbone protein design.

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    Elisabeth Humphris-Narayanan

    Full Text Available Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model.

  7. Ex vivo reversal of effects of rivaroxaban evaluated using thromboelastometry and thrombin generation assay

    Science.gov (United States)

    Schenk, B.; Würtinger, P.; Streif, W.; Sturm, W.; Fries, D.; Bachler, M.

    2016-01-01

    Background In major bleeding events, the new direct oral anticoagulants pose a great challenge for physicians. The aim of the study was to test for ex vivo reversal of the direct oral anticoagulant rivaroxaban with various non-specific reversal agents: prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and fibrinogen concentrate (FI). Methods Blood was obtained from healthy volunteers and from patients treated with rivaroxaban. Blood samples from healthy volunteers were spiked with rivaroxaban to test the correlation between rivaroxaban concentration and coagulation tests. Patient blood samples were spiked with various concentrations of the above-mentioned agents and analysed using thromboelastometry and thrombin generation. Results When added in vitro, rivaroxaban was significantly (P<0.05) correlated with ROTEM® thromboelastometry EXTEM (extrinsic coagulation pathway) clotting time (CT), time to maximal velocity (MaxV−t), and with all measured thrombin generation parameters. In vivo, CT, MaxV−t, lag time, and peak thrombin generation (Cmax) were significantly correlated with rivaroxaban concentrations. Regarding reversal of rivaroxaban, all tested agents significantly (P<0.05) reduced EXTEM CT, but to different extents: rFVIIa by 68%, aPCC by 47%, PCC by 17%, and FI by 9%. Only rFVIIa reversed EXTEM CT to baseline values. Both PCC (+102%) and aPCC (+232%) altered overall thrombin generation (area under the curve) and increased Cmax (+461% for PCC, +87.5% for aPCC). Conclusions Thromboelastometry and thrombin generation assays do not favour the same reversal agents for rivaroxaban anticoagulation. Controlled clinical trials are urgently needed to establish doses and clinical efficacy of potential reversal agents. Clinical trial registration EudracCT trial no. 213-00474-30. PMID:27623677

  8. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    Science.gov (United States)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  9. Reversed phase parallel artificial membrane permeation assay for log P measurement

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    Zihao Song

    2016-03-01

    Full Text Available A reversed phase parallel artificial membrane permeation assay (RP-PAMPA was newly invented for log P measurement. An oil/water/oil sandwich was constructed using a conventional PAMPA instrument. 1 % agarose was used to improve the physical stability of the water phase. A linear correlation between log P and the apparent permeability was observed in the -0.24 < log P < 2.85 region (R2 = 0.98. RP-PAMPA was also applied to pKa measurement.

  10. Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test

    International Nuclear Information System (INIS)

    Chen Zhijian; Lou Jianlin; Chen Shijie; Zheng Wei; Wu Wei; Jin Lifen; Deng Hongping; He Jiliang

    2006-01-01

    To evaluate the genotoxic effects of lead (Pb) exposure, 25 workers in a workplace producing storage battery were monitored for three genetic end-points using micronucleus (MN) assay, comet assay and TCR gene mutation test. Twenty-five controls were matched with workers according to age, gender and smoking. The air Pb concentration in the workplace was 1.26 mg/m 3 . All subjects were measured for Pb concentration of blood by atom absorption spectrophotometry. The mean Pb concentration of blood in workers (0.32 mg/l) was significantly higher than that in controls (0.02 mg/l). The results of MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in workers were 9.04 ± 1.51 per mille and 7.76 ± 1.23 per mille , respectively, which were significantly higher than those (2.36 ± 0.42 per mille and 1.92 ± 0.31 per mille ) in controls (P -4 and 1.74 ± 0.17 x 10 -4 , respectively, there was no significant difference between workers and controls (P > 0.05). The results of our study indicated that the genetic damage was detectable in 25 workers occupationally exposed to lead

  11. The KRAS Strip Assay for detection of KRAS mutation in Egyptian patients with colorectal cancer (CRC): A pilot study

    International Nuclear Information System (INIS)

    Abd El Kader, Y.; Safwat, E.; Kassem, H.A.; Kassem, N.M.; Emera, G.

    2013-01-01

    Background: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefltinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. Purpose: Detection of KRAS mutation in Egyptian colorectal cancer (CRC) patients by the KRAS Strip Assay. Methods: Examination of 20 colorectal cancer (CRC) patients is done to detect KRAS mutations by KRAS Strip Assay. For the Strip Assay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. Results: Among 20 patients, KRAS mutations were identified in 80% of patients by the KRAS Strip Assay. Conclusions: Our preliminary results suggest that KRAS Strip Assay is an alternative to protocols currently in use for KRAS mutation detection

  12. Development of a human somatic mutation detection method--GPA assay

    International Nuclear Information System (INIS)

    Mao Jianping; Dong Yan; Liu Bin; Lin Ruxian; Sun Zhixian

    2000-01-01

    Objective: To study the damage to human body caused by environmental radiation, and supervise the somatic mutations. Methods: Three monoclonal antibodies specific to M-type(3G4), N-type(6A8), and MN-type (3C5) of glycophorin A, respectively, were prepared. Fluorescence or biotin conjugated antibodies were bound specifically to formalin and/or dimethyl suber-imidate fixed erythrocytes. M, MN and N type cells were divided by cytometry to demonstrate the erythrocyte mutation characteristics (MN→MO, MM, NO, NN) and give out the variant frequency. Results: 1Wa, 1Wb and 2Wa methods of GPA assay were developed. Erythrocytes of MN type individuals could be separated to normal and single locus variant groups by 1W methods and they could be sorted as normal (MN), single gene deletion mutants (MO, NO), homozygous mutants (MM, NN) cell groups by 2Wa method. Conclusion: The assay is applicable to evaluating the frequency of variant erythrocytes from human somatic mutation

  13. Development of elvitegravir resistance and linkage of integrase inhibitor mutations with protease and reverse transcriptase resistance mutations.

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    Mark A Winters

    Full Text Available Failure of antiretroviral regimens containing elvitegravir (EVG and raltegravir (RAL can result in the appearance of integrase inhibitor (INI drug-resistance mutations (DRMs. While several INI DRMs have been identified, the evolution of EVG DRMs and the linkage of these DRMs with protease inhibitor (PI and reverse transcriptase inhibitor (RTI DRMs have not been studied at the clonal level. We examined the development of INI DRMs in 10 patients failing EVG-containing regimens over time, and the linkage of INI DRMs with PI and RTI DRMs in these patients plus 6 RAL-treated patients. A one-step RT-nested PCR protocol was used to generate a 2.7 kB amplicon that included the PR, RT, and IN coding region, and standard cloning and sequencing techniques were used to determine DRMs in 1,277 clones (mean 21 clones per time point. Results showed all patients had multiple PI, NRTI, and/or NNRTI DRMs at baseline, but no primary INI DRM. EVG-treated patients developed from 2 to 6 strains with different primary INI DRMs as early as 2 weeks after initiation of treatment, predominantly as single mutations. The prevalence of these strains fluctuated and new strains, and/or strains with new combinations of INI DRMs, developed over time. Final failure samples (weeks 14 to 48 typically showed a dominant strain with multiple mutations or N155H alone. Single N155H or multiple mutations were also observed in RAL-treated patients at virologic failure. All patient strains showed evidence of INI DRM co-located with single or multiple PI and/or RTI DRMs on the same viral strand. Our study shows that EVG treatment can select for a number of distinct INI-resistant strains whose prevalence fluctuates over time. Continued appearance of new INI DRMs after initial INI failure suggests a potent, highly dynamic selection of INI resistant strains that is unaffected by co-location with PI and RTI DRMs.

  14. Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

    Science.gov (United States)

    Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat

    2018-01-01

    The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

  15. New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

    NARCIS (Netherlands)

    Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

    2000-01-01

    A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

  16. Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

    Science.gov (United States)

    Paoletta, Martina Soledad; López Arias, Ludmila; de la Fournière, Sofía; Guillemi, Eliana Carolina; Luciani, Carlos; Sarmiento, Néstor Fabián; Mosqueda, Juan; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

    2018-02-01

    Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine.

    Science.gov (United States)

    Akimoto, Masaru; Hokazono, Eisaku; Ota, Eri; Tateishi, Takiko; Kayamori, Yuzo

    2016-01-01

    Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results. We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components. Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations. The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein. © The Author(s) 2015.

  18. Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

    Science.gov (United States)

    Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

    2010-04-01

    Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

  19. Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

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    Marilanda Ferreira Bellini

    2008-01-01

    Full Text Available Functional food investigations have demonstrated the presence of substances that could be beneficial to human health when consumed. However, the toxic effects of some substances contained in foods have been determined. Reported medicinal and nutritive properties have led to the extensive commercialization of the basidiomycete fungi Agaricus blazei Murrill (sensu Heinemann, also known as Agaricus brasiliensis Wasser et al., Agaricus subrufescens Peck or the Brazilian medical mushroom (BMM. Different methanolic extract fractions (ME of this mushroom were submitted to the cytokinesis-block micronucleus (CBMN clastogenic assay and the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT assay for gene mutation, both using Chinese hamster ovary cells clone K1 (CHO-K1. The results suggest that all the fractions tested possess cytotoxic and mutagenic potential but no clastogenic effects. Further information is needed on the biochemical components of the A. blazei methanol fractions to identify any substances with cytotoxic and/or mutagenicity potential. These findings indicate that A. blazei methanolic extract should not be used due to their genotoxicity and care should be taken in the use of A. blazei by the general population until further biochemical characterization of this fungi is completed.

  20. Increased Chromosomal Radiosensitivity in Women Carrying BRCA1/BRCA2 Mutations Assessed With the G2 Assay

    International Nuclear Information System (INIS)

    Ernestos, Beroukas; Nikolaos, Pandis; Koulis, Giannoukakos; Eleni, Rizou; Konstantinos, Beroukas; Alexandra, Giatromanolaki; Michael, Koukourakis

    2010-01-01

    Purpose: Several in vitro studies suggest that BRCA1 and BRCA2 mutation carriers present increased sensitivity to ionizing radiation. Different assays for the assessment of deoxyribonucleic acid double-strand break repair capacity have been used, but results are rather inconsistent. Given the concerns about the possible risks of breast screening with mammography in mutation carrier women and the potentially damaging effects of radiotherapy, the purpose of this study was to further investigate the radiosensitivity of this population. Methods and Materials: The G2 chromosomal radiosensitivity assay was used to assess chromosomal breaks in lymphocyte cultures after exposure to 1 Gy. A group of familiar breast cancer patients carrying a mutation in the BRCA1 or BRCA2 gene (n = 15) and a group of healthy mutation carriers (n = 5) were investigated and compared with a reference group of healthy women carrying no mutation (n = 21). Results: BRCA1 and BRCA2 mutation carriers had a significantly higher number of mean chromatid breaks per cell (p = 0.006) and a higher maximum number of breaks (p = 0.0001) as compared with their matched controls. Both healthy carriers and carriers with a cancer history were more radiosensitive than controls (p = 0.002 and p = 0.025, respectively). Age was not associated with increased radiosensitivity (p = 0.868). Conclusions: Our results indicate that BRCA1 and BRCA2 mutation carriers show enhanced radiosensitivity, presumably because of the involvement of the BRCA genes in deoxyribonucleic acid repair and cell cycle control mechanisms.

  1. Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

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    S. Domenice

    2004-01-01

    Full Text Available In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N, was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.

  2. Characterization of a method for quantitating food consumption for mutation assays in Drosophila

    International Nuclear Information System (INIS)

    Thompson, E.D.; Reeder, B.A.; Bruce, R.D.

    1991-01-01

    Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period. Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity

  3. Simple PCR assays improve the sensitivity of HIV-1 subtype B drug resistance testing and allow linking of resistance mutations.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Johnson

    Full Text Available BACKGROUND: The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing. METHODOLOGY: We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing. SIGNIFICANCE: Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

  4. Association of telomerase reverse transcriptase promoter mutations with clinicopathological features and prognosis of thyroid cancer: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Su X

    2016-11-01

    Full Text Available Xingyun Su,1 Xiaoxia Jiang,1 Weibin Wang,1 Haiyong Wang,1 Xin Xu,2 Aihui Lin,1 Xiaodong Teng,3 Huiling Wu,4 Lisong Teng1 1Department of Surgical Oncology, 2Department of Medical Oncology, 3Department of Pathology, 4Department of Plastic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China Abstract: The clinicopathological and prognostic significance of telomerase reverse transcriptase (TERT promoter mutations have been widely investigated in thyroid cancer; however, the results are still discrepant. Systematic searches were performed in PubMed, Web of Science, Scopus, Ovid, and the Cochran Library databases for relevant articles prior to April 2016. Mutation rates were synthesized by R statistical software. The odds ratio or standardized mean difference with 95% confidence interval was pooled by Stata. A total of 22 studies with 4,907 cases were included in this meta-analysis. TERT promoter mutations tended to present in aggressive histological types including poorly differentiated thyroid cancer (33.37%, anaplastic thyroid cancer (38.69%, and tall-cell variant papillary thyroid cancer (30.23%. These promoter mutations were likely to exist in older patients and males and were well associated with larger tumor size, extrathyroidal extension, vascular invasion, lymph node metastasis, distant metastasis, advanced tumor stage, disease recurrence/persistence, and mortality. In addition, TERT promoter mutations (especially C228T tended to coexist with BRAFV600E mutation, which indicated more aggressive tumor behavior. Therefore, TERT promoter mutations may be promising biomarkers for early diagnosis, risk stratification, prognostic prediction, and management of thyroid cancer. Keywords: TERT promoter mutations, thyroid cancer, clinicopathological features, prognosis, BRAFV600E mutation

  5. Real-time reverse transcription-polymerase chain reaction assays for identification of wild poliovirus 1 & 3.

    Science.gov (United States)

    Sharma, Deepa K; Nalavade, Uma P; Deshpande, Jagadish M

    2015-10-01

    The poliovirus serotype identification and intratypic differentiation by real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is suitable for serotype mixtures but not for intratypic mixtures of wild and vaccine poliovirus strains. This study was undertaken to develop wild poliovirus 1 and 3 (WPV1 and WPV3) specific rRT-PCR assays for use. Specific primers and probes for rRT-PCR were designed based on VP1 sequences of WPV1 and WPV3 isolated in India since 2000. The specificity of the rRT-PCR assays was evaluated using WPV1 and WPV3 of different genetic lineages, non-polio enteroviruses (NPEVs) and mixtures of wild/wild and wild/Sabin vaccine strains. The sensitivity of the assays was determined by testing serial 10-fold dilutions of wild poliovirus 1 and 3 stock suspensions of known titre. No cross-reactivity with Sabin strains, intertypic wild poliovirus isolates or 27 types of NPEVs across all the four Enterovirus species was found for both the wild poliovirus 1 and 3 rRT-PCR assays. All WPV1 and WPV3 strains isolated since 2000 were successfully amplified. The rRT-PCR assays detected 10 4.40 CCID 50 /ml of WPV1 and 10 4.00 CCID 50 /ml of WPV3, respectively either as single isolate or mixture with Sabin vaccine strains or intertypic wild poliovirus. rRT-PCR assays for WPV1 and WPV3 have been validated to detect all the genetic variations of the WPV1 and WPV3 isolated in India for the last decade. When used in combination with the current rRT-PCR assay testing was complete for confirmation of the presence of wild poliovirus in intratypic mixtures.

  6. Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

    Science.gov (United States)

    Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

    2014-01-01

    A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

  7. Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

    Directory of Open Access Journals (Sweden)

    Hiraku Takei

    Full Text Available A gain-of-function mutation in the myeloproliferative leukemia virus (MPL gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs. The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5% of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

  8. Nature of unstable insertional mutations and reversions at the cut locus of Drosophila melanogaster: Molecular mechanism for transpositional memory

    International Nuclear Information System (INIS)

    Mizrokhi, L.Yu.; Georgieva, S.G.; Obolenkova, L.A.; Priimyagi, A.F.; Gerasimova, T.I.; Il'in, Yu.V.

    1988-01-01

    A segment of the cut locus containing an mdg4 insertion as a result of ct MR and ct MRp10 mutations was cloned. Clones were obtained for the phenotypically different ct MR2 and ct MRpN10 mutants and for stable and unstable revertants. All mutations studied are associated with mdg4 insertion at an identical nucleotide sequence of the cut locus, the same site at which mdg4 is inserted at the ct 6 allele. The ct MRpN line differs from ct MR2 in that the mobile element jockey (3 kbp) is inserted in mdg4. Jockey is represented by about 1,000 copies per genome; it is homogeneous and lacks long terminal repeats (LTRs). In stable ct + reversions, mdg4 is completely excised. In unstable ct + reversions, in which there is a high degree of reverse directed transposition of mdg4 to the cut locus, an LTR of mdg4 is preserved at the site of the mutation. It is a sequence along which new copies of mdg4 or jockey-containing mdg4 are inserted into the genome. The authors discuss a molecular mechanism for transpositional memory involving homologous recombination of the remnant LTR and circular extrachromosomal copies of mdg4

  9. Melt analysis of mismatch amplification mutation assays (Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Single nucleotide polymorphisms (SNPs are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA, is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg. Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of

  10. Assaying the Mutagenic Potential of ELF Radiation through Reverse Mutagenesis via the Ames Test

    National Research Council Canada - National Science Library

    Moga, Paul

    1996-01-01

    ...) on certain strains of Salmonella typhimurium. The strain of S-typhimurium used in the Ames test has a mutation on one gene of the histidine operon which prevents it from growing and replicating without the presence of histidine in the media...

  11. A ''reverse'' solid-phase radio-immuno-assay for IgM-antibodies to hepatitis A virus

    International Nuclear Information System (INIS)

    Meurman, O.H.; Matter, L.; Krishna, R.V.; Krech, U.H.

    1981-01-01

    A ''reverse'' solid-phase radio-immuno-assay for IgM antibodies to hepatitis A virus (HAV) was developed. Anti-human IgM immunoglobulins were bound on the wells of polyvinylchloride microtiter plates. Serum specimens were incubated in the anti-human IgM coated wells and bound IgM antibodies were then assayed for antigen specificity by subsequent incubations with HAV antigen and 125 I-labelled human anti-HAV IgG. The test showed a high sensitivity and specificity for anti-HAV IgM antibodies. No false-positive reactions were observed either in the sera from patients with hepatobiliary disorders other than HAV infection or in the sera containing both rheumatoid factor and anti-HAV IgG antibodies. In acute HAV infections specific IgM antibodies were present already in the first specimens taken within a few days after the onset of jaundice. The persistence of the IgM antibodies was from 4 to 6 months. IgM antibody titers up to 1,000,000 were observed in the acute phase of HAV infection. In routine diagnostic work the titration of the sera was not necessary, since a reliable qualitative result was obtained by testing the sera in a single dilution of 1:100. A similar ''reverse'' immuno-assay principle may be adaptable for the diagnostic determination of IgM antibodies to different viral and microbial antigens. (author)

  12. Investigation of mutations in the SRY, SOX9, and DAX1 genes in sex reversal patients from the Sichuan region of China.

    Science.gov (United States)

    Chen, L; Ding, X P; Wei, X; Li, L X

    2014-03-12

    We investigated the molecular genetic mechanism of sex reversal by exploring the relationship between mutations in the sex-determining genes SRY, SOX9, and DAX1 with genetic sex reversal disease. Mutations in the three key genes were detected by polymerase chain reaction (PCR) and sequencing after karyotype analysis. The mutations detected were then aligned with a random sample of 100 normal sequences and the NCBI sequence database in order to confirm any new mutations. Furthermore, the copy number of SOX9 was measured by fluorescence quantitative PCR. Seven of the 10 male sex reversal patients (46, XX) contained an excess copy of the SRY gene, while one of the eight female sex reversal patients (46, XY) was lacking the SRY gene. Additionally, a new mutation (T-A, Asp24Lys) was detected in one female sex reversal patient (46, XY). No other mutation was detected in the analysis of SOX9 and DAX1, with the exception of an insertion mutation (c.35377791insG) found in the testicular-specific enhancer (TESCO) sequences in an SRY-positive female sex reversal patient (46, XY). Eight of the 18 sex reversal cases (44.4%) showed obvious connections with SRY gene translocations, mutations, or deletions, which was significantly higher than that reported previously (33.3%), indicating a need to further expand the range of sample collection. Overall, these results indicated that the main mechanism of sex reversal are not associated with mutations in the coding regions of SOX9 and DAX1 or copy number variations of SOX9, which is consistent with results of previous studies.

  13. Detection of Rickettsia in Rhipicephalus sanguineus Ticks and Ctenocephalides felis Fleas from Southeastern Tunisia by Reverse Line Blot Assay

    Science.gov (United States)

    Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene

    2014-01-01

    Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia. PMID:24226919

  14. A HRM assay for identification of low level BRAF V600E and V600K mutations using the CADMA principle in FFPE specimens.

    Science.gov (United States)

    Huebner, Claudia; Weber, Remeny; Lloydd, Richard

    2017-12-01

    Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment. Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

  15. Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus.

    Science.gov (United States)

    Qin, Shaomin; Underwood, Darren; Driver, Luke; Kistler, Carol; Diallo, Ibrahim; Kirkland, Peter D

    2018-06-01

    We evaluated a fluorogenic probe-based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21-4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

  16. Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; van Lenthe, H.; van Gennip, A. H.

    1999-01-01

    A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-beta-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by

  17. Trends of drug-resistance-associated mutations in the reverse transcriptase gene of HIV type 1 isolates from North India.

    Science.gov (United States)

    Azam, Mohd; Malik, Abida; Rizvi, Meher; Rai, Arvind

    2014-04-01

    A major cause of failure of antiretroviral therapy (ART) is the presence of drug-resistance-associated mutations in the polymerase gene of HIV-1. The paucity of data regarding potential drug resistance to reverse transcriptase inhibitors (RTIs) prompted us to carry out this study. This information will shed light on the extent of drug resistance already present in HIV strains and will give future directions in patient treatment and in drug design. Drug resistance genotyping of a partial reverse transcriptase gene was done in 103 HIV-1-infected patients, including the ART-naive and ART-experienced population. The drug resistance pattern was analyzed using the Stanford HIV-DR database, the IAS-USA mutation list and the REGA algorithm-v8.0. Subtyping was done using the REGA HIV-1 subtyping tool-v2.01. The majority of our sequences (96 %) were found to be subtype C, and four (3.8 %) were subtype A1. Significant prevalence of DR mutations (28 %) was observed in the RT gene. Major amino acid substitutions were seen at positions 41, 90, 98, 103, 106, 108, 138, 181, 184, 190, 215, and 219, which confer high/intermediate levels of resistance to most RTIs, independently or together. Our results show that there is an urgent need to tailor ART drug regimens to the individual to achieve optimum therapeutic outcome in North India.

  18. Comparison of reverse transcriptase PCR, reverse transcriptase loop-mediated isothermal amplification, and culture-based assays for Salmonella detection from pork processing environments.

    Science.gov (United States)

    Techathuvanan, Chayapa; Draughon, Frances Ann; D'Souza, Doris Helen

    2011-02-01

    Novel rapid Salmonella detection assays without the need for sophisticated equipment or labor remain in high demand. Real-time reverse transcriptase PCR (RT-PCR) assays, though rapid and sensitive, require expensive thermocyclers, while a novel RT loop-mediated isothermal amplification (RT-LAMP) method requires only a simple water bath. Our objective was to compare the detection sensitivity of Salmonella Typhimurium from the pork processing environment by RT-LAMP, RT-PCR, and culture-based assays. Carcass and surface swabs and carcass rinses were obtained from a local processing plant. Autoclaved carcass rinses (500 ml) were spiked with Salmonella Typhimurium and filtered. Filters were placed in stomacher bags containing tetrathionate broth (TTB) and analyzed with or without 10-h enrichment at 37 °C. Natural swabs were stomached with buffered peptone water, and natural carcass rinses were filtered, preenriched, and further enriched in TTB. Serially-diluted enriched samples were enumerated by spread plating on xylose lysine Tergitol 4 agar. RNA was extracted from 5 ml of enriched TTB with TRIzol. RT-LAMP assay using previously described invA primers was conducted at 62 °C for 90 min in a water bath with visual detection and by gel electrophoresis. SYBR Green I-based-real-time RT-PCR was carried out with invA primers followed by melt temperature analysis. The results of RT-LAMP detection for spiked carcass rinses were comparable to those of RT-PCR and cultural plating, with detection limits of 1 log CFU/ml, although they were obtained significantly faster, within 24 h including preenrichment and enrichment. RT-LAMP showed 4 of 12 rinse samples positive, while RT-PCR showed 1 of 12 rinse samples positive. For swabs, 6 of 27 samples positive by RT-LAMP and 5 of 27 by RT-PCR were obtained. This 1-day RT-LAMP assay shows promise for routine Salmonella screening by the pork industry. Copyright ©, International Association for Food Protection

  19. Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay

    International Nuclear Information System (INIS)

    Bigbee, W.L.; Jensen, R.H.; Veidebaum, T.

    1997-01-01

    The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method. 25 refs., 2 figs., 3 tabs

  20. Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids.

    NARCIS (Netherlands)

    Meletiadis, J.; Melchers, W.J.G.; Meis, J.F.G.M.; Hurk, P.J.J.C. van den; Jannes, G.; Verweij, P.E.

    2003-01-01

    An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical

  1. CHARGE syndrome: a recurrent hotspot of mutations in CHD7 IVS25 analyzed by bioinformatic tools and minigene assays.

    Science.gov (United States)

    Legendre, Marine; Rodriguez-Ballesteros, Montserrat; Rossi, Massimiliano; Abadie, Véronique; Amiel, Jeanne; Revencu, Nicole; Blanchet, Patricia; Brioude, Frédéric; Delrue, Marie-Ange; Doubaj, Yassamine; Sefiani, Abdelaziz; Francannet, Christine; Holder-Espinasse, Muriel; Jouk, Pierre-Simon; Julia, Sophie; Melki, Judith; Mur, Sébastien; Naudion, Sophie; Fabre-Teste, Jennifer; Busa, Tiffany; Stamm, Stephen; Lyonnet, Stanislas; Attie-Bitach, Tania; Kitzis, Alain; Gilbert-Dussardier, Brigitte; Bilan, Frédéric

    2018-02-01

    CHARGE syndrome is a rare genetic disorder mainly due to de novo and private truncating mutations of CHD7 gene. Here we report an intriguing hot spot of intronic mutations (c.5405-7G > A, c.5405-13G > A, c.5405-17G > A and c.5405-18C > A) located in CHD7 IVS25. Combining computational in silico analysis, experimental branch-point determination and in vitro minigene assays, our study explains this mutation hot spot by a particular genomic context, including the weakness of the IVS25 natural acceptor-site and an unconventional lariat sequence localized outside the common 40 bp upstream the acceptor splice site. For each of the mutations reported here, bioinformatic tools indicated a newly created 3' splice site, of which the existence was confirmed using pSpliceExpress, an easy-to-use and reliable splicing reporter tool. Our study emphasizes the idea that combining these two complementary approaches could increase the efficiency of routine molecular diagnosis.

  2. A Fast, Sensitive and Accurate High Resolution Melting (HRM Technology-Based Assay to Screen for Common K-ras Mutations

    Directory of Open Access Journals (Sweden)

    D. Kramer

    2009-01-01

    Full Text Available Background: Increasing evidence points to a negative correlation between K-ras mutations and patient’s response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed.

  3. A Panel of High Resolution Melting (HRM Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

    Directory of Open Access Journals (Sweden)

    D. A. M. Heideman

    2009-01-01

    Full Text Available Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.

  4. Gene-mutation assays in lambda-lacZ transgenic mice : comparison of lacZ with endogenous genes in splenocytes and small intestinal epithelium

    NARCIS (Netherlands)

    Delft, J.H.M. van; Bergmans, A.; Dam, F.J. van; Tates, A.D.; Howard, L.; Winton, D.J.; Baan, R.A.

    1998-01-01

    Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used λlacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen

  5. A reverse transcriptase-PCR assay for detecting filarial infective larvae in mosquitoes.

    Directory of Open Access Journals (Sweden)

    Sandra J Laney

    2008-06-01

    Full Text Available Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3 capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript.Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes.This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971 that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens may also be useful for detecting infective stages of other vector-borne parasites.

  6. Autosomal recessive mutations in THOC6 cause intellectual disability: syndrome delineation requiring forward and reverse phenotyping.

    Science.gov (United States)

    Amos, J S; Huang, L; Thevenon, J; Kariminedjad, A; Beaulieu, C L; Masurel-Paulet, A; Najmabadi, H; Fattahi, Z; Beheshtian, M; Tonekaboni, S H; Tang, S; Helbig, K L; Alcaraz, W; Rivière, J-B; Faivre, L; Innes, A M; Lebel, R R; Boycott, K M

    2017-01-01

    THOC6 is a part of the THO complex, which is involved in coordinating mRNA processing with export. The THO complex interacts with additional components to form the larger TREX complex (transcription export complex). Previously, a homozygous missense mutation in THOC6 in the Hutterite population was reported in association with syndromic intellectual disability. Using exome sequencing, we identified three unrelated patients with bi-allelic mutations in THOC6 associated with intellectual disability and additional clinical features. Two of the patients were compound heterozygous for a stop and a missense mutation, and the third was homozygous for a missense mutation; the missense mutations were predicted to be pathogenic by in silico analysis and modeling. Clinical features of the three newly identified patients and those previously reported are reviewed; intellectual disability is moderate to severe, and malformations are variable including renal and heart defects, cleft palate, microcephaly, and corpus callosum dysgenesis. Facial features are variable and include tall forehead, short upslanting palpebral fissures +/- deep set eyes, and a long nose with overhanging columella. These subtle facial features render the diagnosis difficult to make in isolation with certainty. Our results expand the mutational and clinical spectrum of this rare disease, confirm that THOC6 is an intellectual disability causing gene, while providing insight into the importance of the THO complex in neurodevelopment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

    Directory of Open Access Journals (Sweden)

    Paula González-Alonso

    2015-08-01

    Full Text Available Mutations in Human Epidermal Growth Factor Receptors (HER are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS, alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC, ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  8. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, R.J.; Bobrow, M.; Roberts, R.G. [St. Thomas`s Hospitals, London (United Kingdom)

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  9. Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.

    Directory of Open Access Journals (Sweden)

    Windy A Boyd

    2007-12-01

    Full Text Available Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative.Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants, and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50 of 2 microM.The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or

  10. Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

    Science.gov (United States)

    Kho, S L; Chua, K H; George, E; Tan, J A M A

    2013-07-15

    Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

  11. IFN-γ release assay conversions and reversions. Challenges with serial testing in U.S. health care workers.

    Science.gov (United States)

    Joshi, Manish; Monson, Thomas P; Joshi, Anita; Woods, Gail L

    2014-03-01

    IFN-γ release assays (IGRAs) including the QuantiFERON-TB gold in-tube test (QFT-GIT) are increasingly used in place of the tuberculin skin test (TST) in surveillance programs for Mycobacterium tuberculosis infection in the United States. However, data on conversions, reversions, and predictive value of QFT in such programs for health care workers (HCWs) are limited. The purpose of this study is to assess long-term reproducibility and conversion and reversion rates of QFT-GIT among HCWs who underwent serial testing at a tertiary care center in the United States. Retrospective chart review of HCWs at the Central Arkansas Veterans Healthcare System (CAVHS) who underwent serial testing with QFT-GIT as a part of their employee screening between November 1, 2008 and January 31, 2011. A total of 2,303 HCWs had at least 2 QFT-GITs 1 year apart. The initial QFT-GIT was positive for 69 and 2 were indeterminate. Of these 69 HCWs, 31 (45%) reverted on repeat testing, and 25 of 31 (80.6%) HCWs who reverted had a negative look-back TST. Of the 2,232 HCWs with an initial negative QFT-GIT, 71 (3.2%) converted on repeat testing. A third QFT-GIT assay was performed in 41 of the 71 converters and 90% (37 of 41) reverted back to negative. Only two HCWs had TST and QFT-GIT conversion. Poor IGRA reproducibility and a low predictive value of QFT-GIT conversions indicate that QFT-GIT with current interpretation criteria should not be used for serial screening of U.S. HCWs. Negative TSTs have higher reproducibility than QFT-GIT for serial testing of HCWs in low tuberculosis incidence settings.

  12. Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

    Science.gov (United States)

    Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

    2018-04-06

    Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

  13. A novel paper-based assay for the simultaneous determination of Rh typing and forward and reverse ABO blood groups.

    Science.gov (United States)

    Noiphung, Julaluk; Talalak, Kwanrutai; Hongwarittorrn, Irin; Pupinyo, Naricha; Thirabowonkitphithan, Pannawich; Laiwattanapaisal, Wanida

    2015-05-15

    We propose a new, paper-based analytical device (PAD) for blood typing that allows for the simultaneous determination of ABO and Rh blood groups on the same device. The device was successfully fabricated by using a combination of wax printing and wax dipping methods. A 1:2 blood dilution was used for forward grouping, whereas whole blood could be used for reverse grouping. A 30% cell suspension of A-cells or B-cells was used for haemagglutination on the reverse grouping side. The total assay time was 10 min. The ratio between the distance of red blood cell movement and plasma separation is the criterion for agglutination and indicates the presence of the corresponding antigen or antibody. The proposed PAD has excellent reproducibility in that the same blood groups, namely A, AB, and O, were reported by using different PADs that were fabricated on the same day (n=10). The accuracy for detecting blood group A (n=12), B (n=13), AB (n=9), O (n=14), and Rh (n=48) typing were 92%, 85%, 89%, 93%, and 96%, respectively, in comparison with the conventional slide test method. The haematocrit of the sample affects the accuracy of the results, and appropriate dilution is suggested before typing. In conclusion, this study proposes a novel method that is straightforward, time-saving, and inexpensive for the simultaneous determination of ABO and Rh blood groups, which is promising for use in developing countries. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. [Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations].

    Science.gov (United States)

    Liu, Yan-chen; Huang, Ai-long; Hu, Yuan; Hu, Jie-li; Lai, Guo-qi; Zhang, Wen-lu

    2011-12-01

    To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test. Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

  15. A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

    Science.gov (United States)

    Erickson, A; Fisher, M; Furukawa-Stoffer, T; Ambagala, A; Hodko, D; Pasick, J; King, D P; Nfon, C; Ortega Polo, R; Lung, O

    2018-04-01

    Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional

  16. High-resolution melting (HRM) assay for the detection of recurrent BRCA1/BRCA2 germline mutations in Tunisian breast/ovarian cancer families.

    Science.gov (United States)

    Riahi, Aouatef; Kharrat, Maher; Lariani, Imen; Chaabouni-Bouhamed, Habiba

    2014-12-01

    Germline deleterious mutations in the BRCA1/BRCA2 genes are associated with an increased risk for the development of breast and ovarian cancer. Given the large size of these genes the detection of such mutations represents a considerable technical challenge. Therefore, the development of cost-effective and rapid methods to identify these mutations became a necessity. High resolution melting analysis (HRM) is a rapid and efficient technique extensively employed as high-throughput mutation scanning method. The purpose of our study was to assess the specificity and sensitivity of HRM for BRCA1 and BRCA2 genes scanning. As a first step we estimate the ability of HRM for detection mutations in a set of 21 heterozygous samples harboring 8 different known BRCA1/BRCA2 variations, all samples had been preliminarily investigated by direct sequencing, and then we performed a blinded analysis by HRM in a set of 68 further sporadic samples of unknown genotype. All tested heterozygous BRCA1/BRCA2 variants were easily identified. However the HRM assay revealed further alteration that we initially had not searched (one unclassified variant). Furthermore, sequencing confirmed all the HRM detected mutations in the set of unknown samples, including homozygous changes, indicating that in this cohort, with the optimized assays, the mutations detections sensitivity and specificity were 100 %. HRM is a simple, rapid and efficient scanning method for known and unknown BRCA1/BRCA2 germline mutations. Consequently the method will allow for the economical screening of recurrent mutations in Tunisian population.

  17. Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

    Science.gov (United States)

    Frawley, Thomas; O'Brien, Cathal P; Conneally, Eibhlin; Vandenberghe, Elisabeth; Percy, Melanie; Langabeer, Stephen E; Haslam, Karl

    2018-02-01

    The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.

  18. An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification.

    Science.gov (United States)

    Lin, Zhenyu; Yang, Weiqiang; Zhang, Guiyun; Liu, Qida; Qiu, Bin; Cai, Zongwei; Chen, Guonan

    2011-08-28

    A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.

  19. Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

    2017-04-10

    HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.

  20. Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

    Science.gov (United States)

    Singh, Om P; Dykes, Cherry L; Lather, Manila; Agrawal, Om P; Adak, Tridibes

    2011-03-14

    Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

  1. Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.

    Science.gov (United States)

    Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan; Qi, Shunxiang; Ma, Xuejun

    2014-06-01

    Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71

  2. Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

    Science.gov (United States)

    Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

    2009-10-01

    Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

  3. Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.

    LENUS (Irish Health Repository)

    Menton, John F

    2007-01-01

    BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII\\/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII\\/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

  4. Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

    Science.gov (United States)

    Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

    2006-01-01

    A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

  5. Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene.

    Science.gov (United States)

    Mariani, Sara; Bertero, Luca; Osella-Abate, Simona; Di Bello, Cristiana; Francia di Celle, Paola; Coppola, Vittoria; Sapino, Anna; Cassoni, Paola; Marchiò, Caterina

    2017-07-25

    Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies. We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs. By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12). mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

  6. The strains recommended for use in the bacterial reverse mutation test (OECD guideline 471) can be certified as non-genetically modified organisms.

    Science.gov (United States)

    Sugiyama, Kei-Ichi; Yamada, Masami; Awogi, Takumi; Hakura, Atsushi

    2016-01-01

    The bacterial reverse mutation test, commonly called Ames test, is used worldwide. In Japan, the genetically modified organisms (GMOs) are regulated under the Cartagena Domestic Law, and organisms obtained by self-cloning and/or natural occurrence would be exempted from the law case by case. The strains of Salmonella typhimurium and Escherichia coli recommended for use in the bacterial reverse mutation test (OECD guideline 471), have been considered as non-GMOs because they can be constructed by self-cloning or naturally occurring bacterial strains, or do not disturb the biological diversity. The present article explains the reasons why these tester strains should be classified as non-GMOs.

  7. Peptide bond-forming reagents HOAt and HATU are not mutagenic in the bacterial reverse mutation test.

    Science.gov (United States)

    Nicolette, John; Neft, Robin E; Vanosdol, Jessica; Murray, Joel

    2016-04-01

    The peptide bond-forming reagents 1-hydroxy-7-azabenzotriazole (HOAt, CAS 39968-33-7) and O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, CAS 148893-10-1) either have structural alerts, unclassified features or are considered out of domain when evaluated for potential mutagenicity with in silico programs DEREK and CaseUltra. Since they are commonly used reagents in pharmaceutical drug syntheses, they may become drug substance or drug product impurities and would need to be either controlled to appropriately safe levels or tested for mutagenicity. Both reagents were tested in the bacterial reverse mutation (Ames) test at Covance, under GLP conditions, following the OECD test guideline and ICH S2(R1) recommendations and found to be negative. Our data show that HOAt and HATU-common pharmaceutical synthesis reagents-are not mutagenic, and can be treated as ordinary drug impurities. © 2016 Wiley Periodicals, Inc.

  8. A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

    Directory of Open Access Journals (Sweden)

    Meng Shuang

    2010-06-01

    Full Text Available Abstract Background The hepatitis C virus (HCV genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM, at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP/COBAS TaqMan (CTM assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

  9. Reversion of a live porcine reproductive and respiratory virus vaccine investigated by parallel mutations

    DEFF Research Database (Denmark)

    Nielsen, Henriette S.; Oleksiewicz, Martin B; Forsberg, R

    2001-01-01

    A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates was sequen......A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates...... in the vaccine virus sequence during cell-culture adaptation. Evaluation of the remaining mutations in the ORF1 sequence revealed stronger selective pressure for amino acid conservation during spread in pigs than during vaccine production. Furthermore, it was found that the selective pressure did not change...

  10. Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations

    DEFF Research Database (Denmark)

    Nielsen, Henriette S.; Oleksiewicz, M.B.; Forsberg, R.

    2001-01-01

    A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates was sequen......A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates...... in the vaccine virus sequence during cell-culture adaptation. Evaluation of the remaining mutations in the ORF1 sequence revealed stronger selective pressure for amino acid conservation during spread in pigs than during vaccine production. Furthermore, it was found that the selective pressure did not change...

  11. Comparing BMD-derived genotoxic potency estimations across variants of the transgenic rodent gene mutation assay.

    Science.gov (United States)

    Wills, John W; Johnson, George E; Battaion, Hannah L; Slob, Wout; White, Paul A

    2017-12-01

    There is growing interest in quantitative analysis of in vivo genetic toxicity dose-response data, and use of point-of-departure (PoD) metrics such as the benchmark dose (BMD) for human health risk assessment (HHRA). Currently, multiple transgenic rodent (TGR) assay variants, employing different rodent strains and reporter transgenes, are used for the assessment of chemically-induced genotoxic effects in vivo. However, regulatory issues arise when different PoD values (e.g., lower BMD confidence intervals or BMDLs) are obtained for the same compound across different TGR assay variants. This study therefore employed the BMD approach to examine the ability of different TGR variants to yield comparable genotoxic potency estimates. Review of over 2000 dose-response datasets identified suitably-matched dose-response data for three compounds (ethyl methanesulfonate or EMS, N-ethyl-N-nitrosourea or ENU, and dimethylnitrosamine or DMN) across four commonly-used murine TGR variants (Muta™Mouse lacZ, Muta™Mouse cII, gpt delta and BigBlue® lacI). Dose-response analyses provided no conclusive evidence that TGR variant choice significantly influences the derived genotoxic potency estimate. This conclusion was reliant upon taking into account the importance of comparing BMD confidence intervals as opposed to directly comparing PoD values (e.g., comparing BMDLs). Comparisons with earlier works suggested that with respect to potency determination, tissue choice is potentially more important than choice of TGR assay variant. Scoring multiple tissues selected on the basis of supporting toxicokinetic information is therefore recommended. Finally, we used typical within-group variances to estimate preliminary endpoint-specific benchmark response (BMR) values across several TGR variants/tissues. We discuss why such values are required for routine use of genetic toxicity PoDs for HHRA. Environ. Mol. Mutagen. 58:632-643, 2017. © 2017 Her Majesty the Queen in Right of Canada

  12. Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

    Science.gov (United States)

    A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

  13. Development of a reverse transcriptase loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Leishmania parasites in clinical samples

    NARCIS (Netherlands)

    Adams, Emily R.; Schoone, Gerard J.; Ageed, Al Farazdag; El Safi, Sayda; Schallig, Henk D. F. H.

    2010-01-01

    Here we describe a generic, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) assay, for the identification of Leishmania species from clinical samples. LAMP is an isothermal reaction recently developed as a point-of-care diagnostic tool. Primers were designed in the conserved

  14. Profile of Mutations in the Reverse Transcriptase and Overlapping Surface Genes of Hepatitis B Virus (HBV) in Treatment-Naïve Indonesian HBV Carriers.

    Science.gov (United States)

    Yamani, Laura Navika; Yano, Yoshihiko; Utsumi, Takako; Wasityastuti, Widya; Rinonce, Hanggoro Tri; Widasari, Dewiyani Indah; Juniastuti; Lusida, Maria Inge; Soetjipto; Hayashi, Yoshitake

    2017-11-22

    Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome are an important factor in low therapeutic effectiveness. Nonetheless, the prevalence of these mutations in HBV strains isolated previously in Indonesia has not been systematically examined. Therefore, in this study, we investigated the profile of mutations in the RT region and the associations of these mutations with amino acid changes in the surface protein in the virus of treatment-naïve Indonesian HBV carriers. Overall, 96 sequences of the full-length Indonesian HBV genomes (genotype B, n = 54; genotype C, n = 42) were retrieved from the National Center for Biotechnology Information. Naturally occurring primary and/or compensatory drug resistance mutations were found in 6/54 (11.1%) genotype B strains and in 1/42 (2.4%) genotype C strains. The potential mutations underlying resistance to a nucleos(t)ide analog and/or pretreatment mutations were more frequent in both genotypes but more frequent in genotype C strains than in genotype B strains. The A-B interdomain region in the RT gene was more frequently mutated in genotype C than in genotype B (3.51 ± 2.53 vs. 1.08 ± 1.52, P < 0.001). Knowledge about the mutational profiles of the RT gene and changes in the surface protein may help clinicians to select the most appropriate antiviral drug and vaccination or HBV immunoglobulin regimen for management of HBV infection in Indonesia.

  15. A Standard Addition Method to Assay the Concentration of Biologically Interesting Polyphenols in Grape Berries by Reversed-Phase HPLC

    Directory of Open Access Journals (Sweden)

    Serkos A. Haroutounian

    2007-09-01

    Full Text Available A reversed-phase HPLC method which allows the simultaneous assay of (+- catechin, (–-epicatechin, trans-resveratrol, quercetin and quercetin glycosides in grape berries is described. Kromasil 100 served as stationary phase and a gradient of acetic acid, water and methanol was used. The analytical run requires 42 min for complete sample elution. Satisfactory peak resolution was achieved following a novel extraction process and direct injection of a 20 μL sample. The method was used for the analyses of eighteen samples. Linearities were in the range of 0.98 to 0.999 regression coefficient, for all phenolics, while detection limits ranged from 30 μg mL–1 for trans-resveratrol to 1.5 mg mL–1 for (+-catechin. Recoveries ranged from 95.1 to 98.7% while the method provided good precision, with standard deviations between 3.5 and 6.1%, n=5.

  16. Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle

    DEFF Research Database (Denmark)

    Larsen, Lars Erik; Tjørnehøj, Kirsten; Viuff, B.

    1999-01-01

    A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved......, in addition, 10 animals that were negative with the ELISA were positive with the RT-PCR assay. These results indicates that the RT-PCR assay can be a sensitive, reliable alternative to conventional diagnostic procedures....... among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal...

  17. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik

    2014-01-01

    samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... of mutations in exons 18-21 of the EGFR gene, employing the cobas(®) EGFR Tissue Test and cobas(®) EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). RESULTS: Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  18. Comparative analysis of drug resistance mutations in the human immunodeficiency virus reverse transcriptase gene in patients who are non-responsive, responsive and naive to antiretroviral therapy.

    Science.gov (United States)

    Misbah, Mohammad; Roy, Gaurav; Shahid, Mudassar; Nag, Nalin; Kumar, Suresh; Husain, Mohammad

    2016-05-01

    Drug resistance mutations in the Pol gene of human immunodeficiency virus 1 (HIV-1) are one of the critical factors associated with antiretroviral therapy (ART) failure in HIV-1 patients. The issue of resistance to reverse transcriptase inhibitors (RTIs) in HIV infection has not been adequately addressed in the Indian subcontinent. We compared HIV-1 reverse transcriptase (RT) gene sequences to identify mutations present in HIV-1 patients who were ART non-responders, ART responders and drug naive. Genotypic drug resistance testing was performed by sequencing a 655-bp region of the RT gene from 102 HIV-1 patients, consisting of 30 ART-non-responding, 35 ART-responding and 37 drug-naive patients. The Stanford HIV Resistance Database (HIVDBv 6.2), IAS-USA mutation list, ANRS_09/2012 algorithm, and Rega v8.02 algorithm were used to interpret the pattern of drug resistance. The majority of the sequences (96 %) belonged to subtype C, and a few of them (3.9 %) to subtype A1. The frequency of drug resistance mutations observed in ART-non-responding, ART-responding and drug-naive patients was 40.1 %, 10.7 % and 20.58 %, respectively. It was observed that in non-responders, multiple mutations were present in the same patient, while in responders, a single mutation was found. Some of the drug-naive patients had more than one mutation. Thymidine analogue mutations (TAMs), however, were found in non-responders and naive patients but not in responders. Although drug resistance mutations were widely distributed among ART non-responders, the presence of resistance mutations in the viruses of drug-naive patients poses a big concern in the absence of a genotyping resistance test.

  19. Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii

    Science.gov (United States)

    Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2) using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/ CRISPR-associated) hom...

  20. Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens.

    Science.gov (United States)

    Malapelle, Umberto; Mayo-de-Las-Casas, Clara; Molina-Vila, Miguel A; Rosell, Rafael; Savic, Spasenija; Bihl, Michel; Bubendorf, Lukas; Salto-Tellez, Manuel; de Biase, Dario; Tallini, Giovanni; Hwang, David H; Sholl, Lynette M; Luthra, Rajyalakshmi; Weynand, Birgit; Vander Borght, Sara; Missiaglia, Edoardo; Bongiovanni, Massimo; Stieber, Daniel; Vielh, Philippe; Schmitt, Fernando; Rappa, Alessandra; Barberis, Massimo; Pepe, Francesco; Pisapia, Pasquale; Serra, Nicola; Vigliar, Elena; Bellevicine, Claudio; Fassan, Matteo; Rugge, Massimo; de Andrea, Carlos E; Lozano, Maria D; Basolo, Fulvio; Fontanini, Gabriella; Nikiforov, Yuri E; Kamel-Reid, Suzanne; da Cunha Santos, Gilda; Nikiforova, Marina N; Roy-Chowdhuri, Sinchita; Troncone, Giancarlo

    2017-08-01

    Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational

  1. Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin.

    Science.gov (United States)

    Sheehan, Alice; Messer, Andrew E; Papadaki, Maria; Choudhry, Afnan; Kren, Vladimír; Biedermann, David; Blagg, Brian; Khandelwal, Anuj; Marston, Steven B

    2018-01-01

    The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca 2+ regulation via troponin. HCM is usually linked to higher myofilament Ca 2+ -sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca 2+ -sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca 2+ -sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro , independent of the mutation (15 mutations tested). We have labeled this property "re-coupling." The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca 2+ -sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold ( n = 19) compared to 2.0 ± 0.24-fold ( n = 7) in wild-type thin filaments

  2. Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin

    Directory of Open Access Journals (Sweden)

    Alice Sheehan

    2018-03-01

    Full Text Available The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM and dilated cardiomyopathy (DCM are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested. We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19 compared to 2.0 ± 0.24-fold (n = 7 in wild-type thin

  3. Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

    Science.gov (United States)

    Paget, Vincent; Lechevrel, Mathilde; André, Véronique; Le Goff, Jérémie; Pottier, Didier; Billet, Sylvain; Garçon, Guillaume; Shirali, Pirouz; Sichel, François

    2012-01-01

    Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B1 exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B1 and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers. PMID:22319594

  4. Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

    Directory of Open Access Journals (Sweden)

    Rupreht Ruth

    2007-11-01

    Full Text Available Abstract Background Hereditary hemochromatosis (HH is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. Methods Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. Results The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI 11.5 – 14.2%, 1.8% (95% CI 1.4 – 2.5% and 3.6% (95% CI 3.0 – 4.5%, respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. Conclusion The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for

  5. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

    Directory of Open Access Journals (Sweden)

    Isabel Hostettler

    2014-12-01

    Full Text Available Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48–72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

  6. Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

    Directory of Open Access Journals (Sweden)

    Laurent Dacheux

    2016-07-01

    Full Text Available The definitive diagnosis of lyssavirus infection (including rabies in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR and a second reaction using an intercalating dye (SYBR Green to detect other lyssavirus species (pan-lyssa RT-qPCR. The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135 including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5% and saliva (54% samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco. This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for

  7. Dual Combined Real-Time Reverse Transcription Polymerase Chain Reaction Assay for the Diagnosis of Lyssavirus Infection.

    Science.gov (United States)

    Dacheux, Laurent; Larrous, Florence; Lavenir, Rachel; Lepelletier, Anthony; Faouzi, Abdellah; Troupin, Cécile; Nourlil, Jalal; Buchy, Philippe; Bourhy, Herve

    2016-07-01

    The definitive diagnosis of lyssavirus infection (including rabies) in animals and humans is based on laboratory confirmation. The reference techniques for post-mortem rabies diagnosis are still based on direct immunofluorescence and virus isolation, but molecular techniques, such as polymerase chain reaction (PCR) based methods, are increasingly being used and now constitute the principal tools for diagnosing rabies in humans and for epidemiological analyses. However, it remains a key challenge to obtain relevant specificity and sensitivity with these techniques while ensuring that the genetic diversity of lyssaviruses does not compromise detection. We developed a dual combined real-time reverse transcription polymerase chain reaction (combo RT-qPCR) method for pan-lyssavirus detection. This method is based on two complementary technologies: a probe-based (TaqMan) RT-qPCR for detecting the RABV species (pan-RABV RT-qPCR) and a second reaction using an intercalating dye (SYBR Green) to detect other lyssavirus species (pan-lyssa RT-qPCR). The performance parameters of this combined assay were evaluated with a large panel of primary animal samples covering almost all the genetic variability encountered at the viral species level, and they extended to almost all lyssavirus species characterized to date. This method was also evaluated for the diagnosis of human rabies on 211 biological samples (positive n = 76 and negative n = 135) including saliva, skin and brain biopsies. It detected all 41 human cases of rabies tested and confirmed the sensitivity and the interest of skin biopsy (91.5%) and saliva (54%) samples for intra-vitam diagnosis of human rabies. Finally, this method was successfully implemented in two rabies reference laboratories in enzootic countries (Cambodia and Morocco). This combined RT-qPCR method constitutes a relevant, useful, validated tool for the diagnosis of rabies in both humans and animals, and represents a promising tool for lyssavirus

  8. Utility of bronchial lavage fluids for epithelial growth factor receptor mutation assay in lung cancer patients: Comparison between cell pellets, cell blocks and matching tissue specimens

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Nakata, Rie; Negishi, Tatsuya; Yamamoto, Hiroshi; Shiina, Takayuki; Shigeto, Shohei; Matsuda, Kazuyuki; Kobayashi, Yukihiro; Honda, Takayuki

    2018-01-01

    The detection of epidermal growth factor receptor (EGFR) mutations is necessary for the selection of suitable patients with non-small cell lung cancer (NSCLC) for treatment with EGFR tyrosine kinase inhibitors. Cytology specimens are known to be suitable for EGFR mutation detection, although tissue specimens should be prioritized; however, there are limited studies that examine the utility of bronchial lavage fluid (BLF) in mutation detection. The purpose of the present study was to investigate the utility of BLF specimens for the detection of EGFR mutations using a conventional quantitative EGFR polymerase chain reaction (PCR) assay. Initially, quantification cycle (Cq) values of cell pellets, cell-free supernatants and cell blocks obtained from three series of 1% EGFR mutation-positive lung cancer cell line samples were compared for mutation detection. In addition, PCR analysis of BLF specimens obtained from 77 consecutive NSCLC patients, detecting EGFR mutations was validated, and these results were compared with those for the corresponding formalin-fixed paraffin-embedded (FFPE) tissue specimens obtained by surgical resection or biopsy of 49 of these patients. The Cq values for mutation detection were significantly lower in the cell pellet group (average, 29.58) compared with the other groups, followed by those in cell-free supernatants (average, 34.15) and in cell blocks (average, 37.12) for all three series (P<0.05). Mutational status was successfully analyzed in 77 BLF specimens, and the results obtained were concordant with those of the 49 matching FFPE tissue specimens. Notably, EGFR mutations were even detected in 10 cytological specimens that contained insufficient tumor cells. EGFR mutation testing with BLF specimens is therefore a useful and reliable method, particularly when sufficient cancer cells are not obtained. PMID:29399190

  9. Reverse Transcriptase drug resistance mutations in HIV-1 Subtype C infected patients on ART in Karonga District, Malawi

    LENUS (Irish Health Repository)

    Bansode, Vijay B

    2011-10-13

    Abstract Background Drug resistance testing before initiation of, or during, antiretroviral therapy (ART) is not routinely performed in resource-limited settings. High levels of viral resistance circulating within the population will have impact on treatment programs by increasing the chances of transmission of resistant strains and treatment failure. Here, we investigate Drug Resistance Mutations (DRMs) from blood samples obtained at regular intervals from patients on ART (Baseline-22 months) in Karonga District, Malawi. One hundred and forty nine reverse transcriptase (RT) consensus sequences were obtained via nested PCR and automated sequencing from blood samples collected at three-month intervals from 75 HIV-1 subtype C infected individuals in the ART programme. Results Fifteen individuals showed DRMs, and in ten individuals DRMs were seen from baseline samples (reported to be ART naïve). Three individuals in whom no DRMs were observed at baseline showed the emergence of DRMs during ART exposure. Four individuals who did show DRMs at baseline showed additional DRMs at subsequent time points, while two individuals showed evidence of DRMs at baseline and either no DRMs, or different DRMs, at later timepoints. Three individuals had immune failure but none appeared to be failing clinically. Conclusion Despite the presence of DRMs to drugs included in the current regimen in some individuals, and immune failure in three, no signs of clinical failure were seen during this study. This cohort will continue to be monitored as part of the Karonga Prevention Study so that the long-term impact of these mutations can be assessed. Documenting proviral population is also important in monitoring the emergence of drug resistance as selective pressure provided by ART compromises the current plasma population, archived viruses can re-emerge

  10. Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma.

    Science.gov (United States)

    Yi, Ping; Chen, Zhuqin; Yu, Lili; Zheng, Yingru; Liu, Guodong; Xie, Haichang; Zhou, Yuanguo; Zheng, Xiuhui; Han, Jian; Li, Li

    2010-08-01

    Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA. The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples. Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

  11. Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR)

    International Nuclear Information System (INIS)

    Rudnicka, L.; Diaz, A.; Varga, J.; Jimenez, S.A.; Christiano, A.; Uitto, J.

    1995-01-01

    Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary 'epidermilysis bulosa dystrophica' associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by 'Ear I' endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of α 32 P-labelled dCTP. The reaction was followed by 'Ear I' digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR. (author). 12 refs, 3 figs

  12. nr0b1 (DAX1) mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation.

    Science.gov (United States)

    Chen, Sijie; Zhang, Hefei; Wang, Fenghua; Zhang, Wei; Peng, Gang

    2016-09-15

    Sex determinations are diverse in vertebrates. Although many sex-determining genes and pathways are conserved, the mechanistic roles of these genes and pathways in the genetic sex determination are not well understood. DAX1 (encoded by the NR0B1 gene) is a vertebrate specific orphan nuclear receptor that regulates gonadal development and sexual determination. In human, duplication of the NR0B1 gene leads to male-to-female sex reversal. In mice, Nr0b1 shows both pro-testis and anti-testis functions. We generated inheritable nr0b1 mutation in the zebrafish and found the nr0b1 mutation caused homozygous mutants to develop as fertile males due to female-to-male sex reversal. The nr0b1 mutation did not increase Caspase-3 labeling nor tp53 expression in the developing gonads. Introduction of a tp53 mutation into the nr0b1 mutant did not rescue the sex-reversal phenotype. Further examination revealed reduction in cell proliferation and abnormal somatic cell differentiation in the nr0b1 mutant gonads at the undifferentiated and bi-potential ovary stages. Together, our results suggest nr0b1 regulates somatic cell differentiation and cell proliferation to ensure normal sex development in the zebrafish. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

    DEFF Research Database (Denmark)

    Russell, Rodney S; Meunier, Jean-Christophe; Takikawa, Shingo

    2008-01-01

    mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7......The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five...

  14. Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

    Science.gov (United States)

    Ngcapu, Sinaye; Theys, Kristof; Libin, Pieter; Marconi, Vincent C; Sunpath, Henry; Ndung'u, Thumbi; Gordon, Michelle L

    2017-11-08

    The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

  15. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Science.gov (United States)

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-01-01

    Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot

  16. Use of GenoType® MTBDRplus assay to assess drug resistance and mutation patterns of multidrug-resistant tuberculosis isolates in northern India

    Directory of Open Access Journals (Sweden)

    A K Maurya

    2013-01-01

    Full Text Available Purpose: The emergence and spread of multidrug-resistant tuberculosis (MDR-TB is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. The aim of this study was to evaluate drug resistance and mutational patterns in clinical isolates MDR-TB by GenoType® MTBDRplus assay. Material and Methods: A total of 350 non-repeated sputum specimens were collected from highly suspected drug-resistant pulmonary tuberculosis (PTB cases; which were processed by microscopy, culture, differentiation and first line drug susceptibility testing (DST using BacT/ALERT 3D system. Results: Among a total of 125 mycobacterium tuberculosis complex (MTBC strains, readable results were obtained from 120 (96% strains by GenoType® MTBDRplus assay. Only 45 MDR-TB isolates were analysed for the performance, frequency and mutational patterns by GenoType® MTBDRplus assay. The sensitivity of the GenoType® MDRTBplus assay for detecting individual resistance to rifampicin (RIF, isoniazid (INH and multidrug resistance was found to be 95.8%, 96.3% and 97.7%, respectively. Mutation in codon S531L of the rpoB gene and codon S315T1 of katG genes were dominated in MDR-TB strains, respectively (P < 0.05. Conclusions: The GenoType® MTBDRplus assay is highly sensitive with short turnaround times and a rapid test for the detection of the most common mutations conferring resistance in MDR-TB strains that can readily be included in a routine laboratory workflow.

  17. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2018-01-01

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  18. Prognostic value of IDH1 mutations identified with PCR-RFLP assay in acute myeloid leukemia patients

    International Nuclear Information System (INIS)

    Elsayed, Gh.M.; Zaher, A.; Elnoshokaty, E.H.; Nassar, H.R.; Moneer, M.M.

    2014-01-01

    Background: Somatic mutations in isocitrate dehydrogenase 1 (1DH1) gene occur frequently in primary brain tumors. Recently theses mutations were demonstrated in acute myeloid leukemia (AML). So far, assessment of these mutations relied on the DNA sequencing technique. Aim of the work: The aim of this study was to detect somatic mutations in IDH1 gene using mismatched primers suitable for endonuclease based detection, without the need for DNA sequencing, and to estimate its prognostic value, on patients with de novo AML. Methods: Residual DNA extracted from pretreatment bone marrow (BM) samples of 100 patients with de novo AML was used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was adapted to IDHl gene, codon 132 mutations screening. Results: The frequency of IDH1 mutations was 13%. In the non-acute promyelocytic leukemia group (non-APL), IDH1 mutations were significantly associated with FLT3-ITD negative patients (p = 0.03). Patients with 1DH1 mutations did not achieve complete remission (CR). There was a trend for shorter overall survival (OS) in patients with IDH1 mutation compared to those with wild type (p = 0.08). Conclusion: IDH1 mutations are recurring genetic alterations in AML and they may have unfavorable impact on clinical outcome in adult AML. The PCR-RFLP method allows for a fast, inexpensive, and sensitive method for the detection of IDF11 mutations in AML.

  19. Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic.

    Science.gov (United States)

    Cernanská, Dana; Paoletti, Barbara; Králová-Hromadová, Ivica; Iorio, Raffaella; Cudeková, Patrícia; Milillo, Piermarino; Traversa, Donato

    2009-03-09

    Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.

  20. Evaluation of the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction assay and the INFINITI® Respiratory Viral Panel Plus assay for the detection of human metapneumovirus in Kuwait.

    Science.gov (United States)

    Al-Turab, Mariam; Chehadeh, Wassim; Al-Mulla, Fahd; Al-Nakib, Widad

    2012-04-01

    Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI(®) Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI(®) RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI(®) RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI(®) RVP-Plus assay in 33% of hMPV-positive patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. A case report of reversible generalized seizures in a patient with Waardenburg syndrome associated with a novel nonsense mutation in the penultimate exon of SOX10.

    Science.gov (United States)

    Suzuki, Noriomi; Mutai, Hideki; Miya, Fuyuki; Tsunoda, Tatsuhiko; Terashima, Hiroshi; Morimoto, Noriko; Matsunaga, Tatsuo

    2018-05-23

    Waardenburg syndrome type 1 (WS1) can be distinguished from Waardenburg syndrome type 2 (WS2) by the presence of dystopia canthorum. About 96% of WS1 are due to PAX3 mutations, and SOX10 mutations have been reported in 15% of WS2. This report describes a patient with WS1 who harbored a novel SOX10 nonsense mutation (c.652G > T, p.G218*) in exon 3 which is the penultimate exon. The patient had mild prodromal neurological symptoms that were followed by severe attacks of generalized seizures associated with delayed myelination of the brain. The immature myelination recovered later and the neurological symptoms could be improved. This is the first truncating mutation in exon 3 of SOX10 that is associated with neurological symptoms in Waardenburg syndrome. Previous studies reported that the neurological symptoms that associate with WS are congenital and irreversible. These findings suggest that the reversible neurological phenotype may be associated with the nonsense mutation in exon 3 of SOX10. When patients of WS show mild prodromal neurological symptoms, the clinician should be aware of the possibility that severe attacks of generalized seizures may follow, which may be associated with the truncating mutation in exon 3 of SOX10.

  2. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

    Directory of Open Access Journals (Sweden)

    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  3. Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

    Science.gov (United States)

    Ambagala, A; Fisher, M; Goolia, M; Nfon, C; Furukawa-Stoffer, T; Ortega Polo, R; Lung, O

    2017-10-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT ™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco ™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems. © 2016 Her Majesty the Queen in Right of Canada.

  4. Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

    Science.gov (United States)

    Maan, S; Maan, N S; Batra, K; Kumar, A; Gupta, A; Rao, Panduranga P; Hemadri, Divakar; Reddy, Yella Narasimha; Guimera, M; Belaganahalli, M N; Mertens, P P C

    2016-08-01

    Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Pre-existing mutations in reverse transcriptase of hepatitis B virus in treatment-naive Chinese patients with chronic hepatitis B.

    Directory of Open Access Journals (Sweden)

    Jie Xu

    Full Text Available High rate of viral replication and lacking of proofreading activity in hepatitis B virus (HBV polymerase lead to the generation of mutations in HBV virus. Mutations in the reverse transcriptase (RT region of HBV polymerase are demonstrated to be strongly associated with drug resistance during antiviral treatment. However, the presence of mutations as well as its clinical significance in treatment-naïve hepatitis patients (defined as pre-existing mutations need to be further investigated. In the present study, a total of 168 serum samples from treatment-naive chronic hepatitis B (CHB patients were collected, and the RT region of HBV polymerase was sequenced. The results showed that pre-existing mutations in the RT region of HBV polymerase were detected in 43 of 168 (25.6% treatment-naive CHB patients within which there were no well-characterized primary nucleotide analogs (NAs resistance sites. Three dominant sites at rt191, rt207 and rt226 were found mutant in 7(16.28%, 8(18.60%, and 14(32.56% samples respectively among these 43 patients. No significant correlation was found between pre-existing mutations and gender, age, HBV genotype, ALT, HBeAg or HBV DNA loads. However, patients with pre-existing RT mutations under HBeAg sero-negative status exhibited decreased HBV DNA loads, which contributed to the decreased HBV DNA loads in the total HBeAg sero-negative patients. The above investigation indicated that there was a prevalence of pre-existing mutations in RT region of HBV polymerase which might affect the serum HBV DNA level in treatment-naive CHB patients. Its effects on the occurrence of NAs resistance and the prognosis after treatment need to be further investigated.

  6. A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A.

    LENUS (Irish Health Repository)

    Song, Yajun

    2010-08-01

    OBJECTIVES: Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. METHODS: By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (Nal(R)) and\\/or decreased susceptibility to fluoroquinolones. RESULTS: This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (Nal(R) = 223 and Nal(S) = 69) and 106 isolates of Salmonella Paratyphi A (Nal(R) = 24 and Nal(S) = 82). All of the 247 Nal(R) Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143\\/223 for Salmonella Typhi and 18\\/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight Nal(S) Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. CONCLUSIONS: The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.

  7. Mutagenicity of the potent rat hepatocarcinogen 6BT to the liver of transgenic (lacI) rats: consideration of a reduced mutation assay protocol.

    Science.gov (United States)

    Lefevre, P A; Tinwell, H; Ashby, J

    1997-01-01

    6-(p-dimethylaminophenylazo)benzothiazole (6BT) is an unusually potent rat hepatocarcinogen, producing large malignant liver tumours after only 2-3 months of dietary administration in a riboflavin-deficient diet. This azocarcinogen has been evaluated in a Big Blue F344 transgenic rat (lacI) gene mutation assay. In a reproduction of the early stages of the carcinogenesis bioassay of this agent, rats were maintained on a riboflavin-deficient diet and were given 10 consecutive daily doses of 6BT (10 mg/kg) by oral gavage. The animals were killed and the livers examined 11 days after the final dose. The livers of 6BT-treated rats showed evidence of hepatocellular hypertrophy in centrolobular areas, with some indication of an increased incidence of mitotic figures. An approximately 10-fold increase in the mutation frequency of DNA isolated from an aliquot of the combined liver homogenates of 6BT-treated rats was observed over that obtained from an equivalent aliquot from control animals. Examination of DNA samples isolated from the livers of individual animals confirmed that 6BT was mutagenic in Big Blue rat livers. These data extend the sensitivity of this transgenic assay to include azo hepatocarcinogens. The determination of mutation frequencies using pooled tissue samples represented a major resource-saving adaptation of the assay protocol in the present study; the general advantages and disadvantages of this practice are discussed.

  8. [Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

    Science.gov (United States)

    Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

    2017-08-10

    To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

  9. A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells.

    Science.gov (United States)

    Kuijpers, Taco W; van Leeuwen, Ester M M; Barendregt, Barbara H; Klarenbeek, Paul; aan de Kerk, Daan J; Baars, Paul A; Jansen, Machiel H; de Vries, Niek; van Lier, René A W; van der Burg, Mirjam

    2013-07-01

    Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B(+)T(-)NK(-) X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined immunodeficiencies with a variety of clinical presentations. We analyzed peripheral blood mononuclear cells of a 6-year-old boy with normal lymphocyte counts, who suffered from recurrent pneumonia and disseminated mollusca contagiosa. Since proliferative responses of T cells and NK cells to γc -cytokines were severely impaired, we performed IL2RG gene analysis, showing a heterozygous mutation in the presence of a single X-chromosome. Interestingly, an IL2RG reversion to normal predominated in both naïve and antigen-primed CD8(+) T cells and increased over time. Only the revertant CD8(+) T cells showed normal expression of CD132 and the various CD8(+) T cell populations had a different T-cell receptor repertoire. Finally, a fraction of γδ(+) T cells and differentiated CD4(+)CD27(-) effector-memory T cells carried the reversion, whereas NK or B cells were repeatedly negative. In conclusion, in a patient with a novel IL2RG mutation, gene-reverted CD8(+) T cells accumulated over time. Our data indicate that selective outgrowth of particular T-cell subsets may occur following reversion at the level of committed T progenitor cells.

  10. A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

    Energy Technology Data Exchange (ETDEWEB)

    Letant, S E; .Ortiz, J I; Tammero, L; Birch, J M; Derlet, R W; Cohen, S; Manning, D; McBride, M T

    2007-04-11

    We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. This article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.

  11. Analysis of Hexanitrostilbene (HNS) and Dipicryethane (DPE) for Mutagenicity by the Ames/Salmonella Assay

    Energy Technology Data Exchange (ETDEWEB)

    Wu, R; Felton, J

    2007-10-12

    The Ames/Salmonella assay, developed by Professor Bruce Ames at the University of California, Berkeley, is a rapid and sensitive assay for detecting mutagenicity of various chemical compounds (Maron and Ames, 1983). It is a widely accepted short-term assay for detecting chemicals that induce mutations in the histidine (his) gene of Salmonella typhimurium. This is a reverse mutation assay that detects the mutational reversion of his-dependent Salmonella to the his-independent counterpart. Thereby, mutagenic compounds will increase the frequency of occurrence of his-independent bacterial colonies. The assay utilizes the specific genetically constructed strains of bacteria either with or without mammalian metabolic activation enzymes (S9), Aroclor induced rat liver homogenate to assess the mutagenicity of different compounds. In this study, we will use the Ames/Salmonella assay to investigate the mutagenicity of Hexanitrostilbene (HNS) from both Bofors and Pantex, and Dipicryethane (DPE).

  12. Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

    Directory of Open Access Journals (Sweden)

    Stephen B. Hughes

    2013-08-01

    Full Text Available The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation, real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection, and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor. This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1

  13. Nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by. gamma. -rays or ethyl methanesulphonate

    Energy Technology Data Exchange (ETDEWEB)

    Brown, R; Stretch, A; Thacker, J

    1986-04-01

    A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells, were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from ..gamma..-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the ..gamma..-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency. All except 2 of the EMS-induced mutants reverted with ethyl nitrosourea ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of ..gamma..-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. (Auth.). 30 refs.; 6 figs.; 2 tabs.

  14. Detection of knockdown resistance (kdr mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Directory of Open Access Journals (Sweden)

    Ball Amanda

    2007-08-01

    Full Text Available Abstract Background Knockdown resistance (kdr is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1 TaqMan probes and 2 high resolution melt (HRM analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR, Heated Oligonucleotide Ligation Assay (HOLA, Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost, and safety (requirement for hazardous chemicals. Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions and the most specific (with the lowest number of incorrect scores. Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS

  15. Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay.

    Science.gov (United States)

    Das, S; Pingle, M R; Muñoz-Jordán, J; Rundell, M S; Rondini, S; Granger, K; Chang, G-J J; Kelly, E; Spier, E G; Larone, D; Spitzer, E; Barany, F; Golightly, L M

    2008-10-01

    The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

  16. Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.

    Science.gov (United States)

    Martinez-Serra, Jordi; Gutiérrez, Antonio; Marcús, Toni F; Soverini, Simona; Amat, Juan Carlos; Navarro-Palou, María; Ros, Teresa; Bex, Teresa; Ballester, Carmen; Bauça, Josep Miquel; SanFelix, Sara; Novo, Andrés; Vidal, Carmen; Santos, Carmen; Besalduch, Joan

    2012-03-01

    Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method. With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain. Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

    Science.gov (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

    2009-11-01

    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  18. 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.

  19. A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

    Science.gov (United States)

    Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

    2017-09-01

    Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

    Science.gov (United States)

    Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

    2004-01-01

    Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

  1. Transgene silencing of the Hutchinson-Gilford progeria syndrome mutation results in a reversible bone phenotype, whereas resveratrol treatment does not show overall beneficial effects

    DEFF Research Database (Denmark)

    Strandgren, Charlotte; Nasser, Hasina Abdul; McKenna, Tomás

    2015-01-01

    model to study the possibility of recovering from HGPS bone disease upon silencing of the HGPS mutation, and the potential benefits from treatment with resveratrol. We show that complete silencing of the transgenic expression of progerin normalized bone morphology and mineralization already after 7...... weeks. The improvements included lower frequencies of rib fractures and callus formation, an increased number of osteocytes in remodeled bone, and normalized dentinogenesis. The beneficial effects from resveratrol treatment were less significant and to a large extent similar to mice treated with sucrose...... alone. However, the reversal of the dental phenotype of overgrown and laterally displaced lower incisors in HGPS mice could be attributed to resveratrol. Our results indicate that the HGPS bone defects were reversible upon suppressed transgenic expression and suggest that treatments targeting aberrant...

  2. Evaluation of 10 Jet Fuels in the Salmonella-Escherichia coli Mutagenicity Assay

    Science.gov (United States)

    2016-09-07

    Department of Defense, nor the U.S. Government. This work was funded by work unit number 60769 and the Defense Logistics Agency (provided to Dr...mutagenicity in the bacterial reverse mutation assay using Salmonella and E. coli strains. Based on the parameters used in this in vitro study, Citgo JP8 (POSF...471 (Bacterial Reverse Mutation Test) and the U.S. Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.5100 (Bacterial

  3. Detection of single nucleotide polymorphisms in p53 mutation hotspots and expression of mutant p53 in human cell lines using an enzyme-linked electrochemical assay

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Šimková, Eva; Vychodilová, Zdenka; Brázdová, Marie; Fojta, Miroslav

    2009-01-01

    Roč. 21, č. 15 (2009), s. 1723-1729 ISSN 1040-0397 R&D Projects: GA ČR(CZ) GA203/07/1195; GA AV ČR(CZ) IAA400040901; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : enzyme-linked electrochemical assay * SNP typing * p53 mutation Subject RIV: AQ - Safety, Health Protection, Human - Machine Impact factor: 2.630, year: 2009

  4. Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

    Science.gov (United States)

    Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

    2011-07-01

    Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

  5. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

    Directory of Open Access Journals (Sweden)

    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  6. A Novel Mutation in the CYP11B1 Gene Causes Steroid 11β-Hydroxylase Deficient Congenital Adrenal Hyperplasia with Reversible Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Mohammad A. Alqahtani

    2015-01-01

    Full Text Available Congenital adrenal hyperplasia (CAH due to steroid 11β-hydroxylase deficiency is the second most common form of CAH, resulting from a mutation in the CYP11B1 gene. Steroid 11β-hydroxylase deficiency results in excessive mineralcorticoids and androgen production leading to hypertension, precocious puberty with acne, enlarged penis, and hyperpigmentation of scrotum of genetically male infants. In the present study, we reported 3 male cases from a Saudi family who presented with penile enlargement, progressive darkness of skin, hypertension, and cardiomyopathy. The elder patient died due to heart failure and his younger brothers were treated with hydrocortisone and antihypertensive medications. Six months following treatment, cardiomyopathy disappeared with normal blood pressure and improvement in the skin pigmentation. The underlying molecular defect was investigated by PCR-sequencing analysis of all coding exons and intron-exon boundary of the CYP11B1 gene. A novel biallelic mutation c.780 G>A in exon 4 of the CYP11B1 gene was found in the patients. The mutation created a premature stop codon at amino acid 260 (p.W260∗, resulting in a truncated protein devoid of 11β-hydroxylase activity. Interestingly, a somatic mutation at the same codon (c.779 G>A, p.W260∗ was reported in a patient with papillary thyroid cancer (COSMIC database. In conclusion, we have identified a novel nonsense mutation in the CYP11B1 gene that causes classic steroid 11β-hydroxylase deficient CAH. Cardiomyopathy and cardiac failure can be reversed by early diagnosis and treatment.

  7. Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiang; Cox, Jonathan T.; Huang, Weiliang; Kane, Maureen; Tang, Keqi; Bieberich, Charles J.

    2016-12-06

    Reversible protein phosphorylation regulates essentially all cellular activities. Aberrant protein phosphorylation is an etiological factor in a wide array of diseases, including cancer1, diabetes2, and Alzheimer’s3. Given the broad impact of protein phosphorylation on cellular biology and organismal health, understanding how protein phosphorylation is regulated and the consequences of gain and loss of phosphoryl moieties from proteins is of primary importance. Advances in instrumentation, particularly in mass spectrometry, coupled with high throughput approaches have recently yielded large datasets cataloging tens of thousands of protein phosphorylation sites in multiple organisms4-6. While these studies are seminal in term of data collection, our understanding of protein phosphorylation regulation remains largely one-dimensional.

  8. Prognostic role of a multigene reverse transcriptase-PCR assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy.

    Science.gov (United States)

    Esteva, Francisco J; Sahin, Aysegul A; Cristofanilli, Massimo; Coombes, Kevin; Lee, Sang-Joon; Baker, Joffre; Cronin, Maureen; Walker, Michael; Watson, Drew; Shak, Steven; Hortobagyi, Gabriel N

    2005-05-01

    To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene expression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tissue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. We obtained archival paraffin-embedded tissue from patients with invasive breast cancer but no axillary lymph node involvement who had received no adjuvant systemic therapy and been followed for at least 5 years. RNA was extracted from three 10-microm-thick sections. The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the ability of the test to accurately predict distant recurrence-free survival in this population. We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21 gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status was noted. RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to develop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy.

  9. Comparison of the Diagnostic Value Between Real-Time Reverse Transcription-Polymerase Chain Reaction Assay and Histopathologic Examination in Sentinel Lymph Nodes for Patients With Gastric Carcinoma.

    Science.gov (United States)

    Kwak, Yoonjin; Nam, Soo Kyung; Shin, Eun; Ahn, Sang-Hoon; Lee, Hee Eun; Park, Do Joong; Kim, Woo Ho; Kim, Hyung-Ho; Lee, Hye Seung

    2016-05-01

    Sentinel lymph node (SLN)-based diagnosis in gastric cancers has shown varied sensitivities and false-negative rates in several studies. Application of the reverse transcription-polymerase chain reaction (RT-PCR) in SLN diagnosis has recently been proposed. A total of 155 SLNs from 65 patients with cT1-2, N0 gastric cancer were examined. The histopathologic results were compared with results obtained by real-time RT-PCR for detecting molecular RNA (mRNA) of cytokeratin (CK)19, carcinoembryonic antigen (CEA), and CK20. The sensitivity and specificity of the multiple marker RT-PCR assay standardized against the results of the postoperative histological examination were 0.778 (95% confidence interval [CI], 0.577-0.914) and 0.781 (95% CI, 0.700-0.850), respectively. In comparison, the sensitivity and specificity of intraoperative diagnosis were 0.819 (95% CI, 0.619-0.937) and 1.000 (95% CI, 0.972-1.000), respectively. The positive predictive value of the multiple-marker RT-PCR assay was 0.355 (95% CI, 0.192-0.546) for predicting non-SLN metastasis, which was lower than that of intraoperative diagnosis (0.813, 95% CI, 0.544-0.960). The real-time RT-PCR assay could detect SLN metastasis in gastric cancer. However, the predictive value of the real-time RT-PCR assay was lower than that of precise histopathologic examination and did not outweigh that of our intraoperative SLN diagnosis. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii.

    Science.gov (United States)

    Li, Jianwei; Handler, Alfred M

    2017-09-28

    Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) homology-directed repair gene-editing. Ds-tra-2 ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 °C, but at higher restrictive temperatures (26 to 29 °C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-TRA-2 ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

  11. Reverse-Bumpy-Ball-Type-Nanoreactor-Loaded Nylon Membranes as Peroxidase-Mimic Membrane Reactors for a Colorimetric Assay for H₂O₂.

    Science.gov (United States)

    Tong, Ying; Jiao, Xiangyu; Yang, Hankun; Wen, Yongqiang; Su, Lei; Zhang, Xueji

    2016-04-01

    Herein we report for the first time fabrication of reverse bumpy ball (RBB)-type-nanoreactor-based flexible peroxidase-mimic membrane reactors (MRs). The RBB-type nanoreactors with gold nanoparticles embedded in the inner walls of carbon shells were loaded on nylon membranes through a facile filtration approach. The as-prepared flexible catalytic membrane was studied as a peroxidase-mimic MR. It was found that the obtained peroxidase-mimic MR could exhibit several advantages over natural enzymes, such as facile and good recyclability, long-term stability and easy storage. Moreover, the RBB NS-modified nylon MRs as a peroxidase mimic provide a useful colorimetric assay for H₂O₂.

  12. Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

    Science.gov (United States)

    Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

    2007-02-01

    Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

  13. Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue.

    Science.gov (United States)

    Fritsch, Michael K; Bridge, Julia A; Schuster, Amy E; Perlman, Elizabeth J; Argani, Pedram

    2003-01-01

    Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase-polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we

  14. Repair-resistant mutation in Neurospora

    International Nuclear Information System (INIS)

    Stadler, D.; Macleod, H.; Loo, M.

    1987-01-01

    Chronic UV treatment produces severalfold fewer mutations in Neurospora conidia than does the same total dose of acute UV. Experiments were designed to determine the conditions required for chronic UV mutagenesis. Measurement of the coincidence frequency for two independent mutations revealed the existence of a subset of cells which are mutable by chronic UV. Analysis of forward mutation at the mtr locus showed that the genetic alterations produced by chronic UV were virtually all point mutants, even though the assay system could detect alterations or deletions extending into neighboring genes. A significant fraction of the mutants produced by acute UV were multigenic deletions. The size of the dose-rate effect (acute UV mutation frequency divided by chronic UV mutation frequency) was compared for several different mutation assay systems. Forward mutations (recessive lethals and mtr) gave values ranging from four to nine. For events which were restricted to specific molecular sites (specific reversions and nonsense suppressor mutations), there was a wider range of dose-rate ratios. This suggests that chronic UV mutation may be restricted to certain molecular sequences or configurations

  15. WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations

    Directory of Open Access Journals (Sweden)

    Assawamakin Anunchai

    2007-08-01

    Full Text Available Abstract Background Allele-specific (AS Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results. Results This work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG draw that allow users to select SNPs and graphically visualize designed primers and their conditions. Conclusion WASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number

  16. UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast.

    Science.gov (United States)

    Lawrence, C W; O'Brien, T; Bond, J

    1984-01-01

    The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

  17. Adrenal Insufficiency, Sex Reversal, and Angelman Syndrome due to Uniparental Disomy Unmasking a Mutation in CYP11A1.

    Science.gov (United States)

    Kim, Ahlee; Fujimoto, Masanobu; Hwa, Vivian; Backeljauw, Philippe; Dauber, Andrew

    2018-01-01

    Cholesterol side-chain cleavage enzyme (P450scc) deficiency is a rare genetic disorder causing primary adrenal insufficiency with or without a 46,XY disorder of sexual development (DSD). Herein, we report a case of the combination of primary adrenal insufficiency, a DSD (testes with female external genitalia in a setting of a 47,XXY karyotype), and Angelman syndrome. Comprehensive genetic analyses were performed, including a single nucleotide polymorphism microarray and whole-exome sequencing. In vitro studies were performed to evaluate the pathogenicity of the novel mutation that was identified by whole-exome sequencing. The patient was found to have segmental uniparental disomy (UPD) of chromosome 15 explaining her diagnosis of Angelman syndrome. Whole-exome sequencing further revealed a novel homozygous intronic variant in CYP11A1, the gene encoding P450scc, found within the region of UPD. In vitro studies confirmed that this variant led to decreased efficiency of CYP11A1 splicing. We report the first case of the combination of 2 rare genetic disorders, Angelman syndrome, and P450scc deficiency. After 20 years of diagnostic efforts, significant advances in genetic diagnostic technology allowed us to determine that these 2 disorders originate from a unified genetic etiology, segmental UPD unmasking a novel recessive mutation in CYP11A1. © 2018 S. Karger AG, Basel.

  18. A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

    Science.gov (United States)

    Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke

    2015-07-01

    A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

  19. Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

    Science.gov (United States)

    Capucchio, Maria Teresa; Spalenza, Veronica; Biasibetti, Elena; Bottero, Maria Teresa; Rasero, Roberto; Dalmasso, Alessandra; Sacchi, Paola

    2014-02-01

    Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

  20. Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer

    International Nuclear Information System (INIS)

    O’Donnell, Patrick; Shieh, Felice; Wei, Wen; Lawrence, H Jeffrey; Wu, Lin; Schilling, Robert; Bloom, Kenneth; Maltzman, Warren; Anderson, Steven; Soviero, Stephen; Ferguson, Jane; Shyu, Johnny; Current, Robert; Rehage, Taraneh; Tsai, Julie; Christensen, Mari; Tran, Ha Bich; Chien, Sean Shih-Chang

    2013-01-01

    Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test

  1. Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Barbara Ziegler

    2011-11-01

    Full Text Available This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip. Biochip hybridization identified 17 (21% samples to carry a KRAS mutation of which 16 (33% were adenocarcinomas and 1 (3% was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples.

  2. Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection

    Directory of Open Access Journals (Sweden)

    Pedersen Niels C

    2007-04-01

    Full Text Available Abstract Background We reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251 mutants with a K65R mutation in reverse transcriptase (RT, and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what extent findings with SIV can be extrapolated to HIV-1 RT. Accordingly, to model HIV-1 RT responses, 12 macaques were inoculated with RT-SHIV, a chimeric SIV containing HIV-1 RT, and started on prolonged tenofovir therapy 5 months later. Results The early virologic response to tenofovir correlated with baseline viral RNA levels and expression of the MHC class I allele Mamu-A*01. For all animals, sensitive real-time PCR assays detected the transient emergence of K70E RT mutants within 4 weeks of therapy, which were then replaced by K65R mutants within 12 weeks of therapy. For most animals, the occurrence of these mutations preceded a partial rebound of plasma viremia to levels that remained on average 10-fold below baseline values. One animal eventually suppressed K65R viremia to undetectable levels for more than 4 years; sequential experiments using CD8+ cell depletion and tenofovir interruption demonstrated that both CD8+ cells and continued tenofovir therapy were required for sustained suppression of viremia. Conclusion This is the first evidence that tenofovir therapy can select directly for K70E viral mutants in vivo. The observations on the clinical implications of the K65R RT-SHIV mutants were consistent with those of SIVmac251, and suggest that for persons infected with K65R HIV-1 both immune-mediated and drug-dependent antiviral activities play a role in controlling viremia. These findings suggest also that even in the presence of K65R virus, continuation of tenofovir treatment as part of HAART may be

  3. Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

    Directory of Open Access Journals (Sweden)

    A Rasul

    2013-06-01

    Full Text Available Background: The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp. in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.Methods: Reverse line blot (RLB assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.Results: Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.Conclusion: Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001. Risk factor analysis revealed that male (P = 0.03, ani­mals infested by ticks (P = 0.03 and herd composed of sheep only (P = 0.001 were more infected by blood parasites.

  4. Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay.

    Science.gov (United States)

    Del Prete, Raffaele; Di Taranto, Anna Maria; Lipsi, Maria Rosaria; Natalicchio, Maria Iole; Antonetti, Raffaele; Miragliotta, Giuseppe

    2009-04-01

    The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.

  5. Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis

    NARCIS (Netherlands)

    Felten, Sandra; Leutenegger, Christian M.; Balzer, Hans Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman|info:eu-repo/dai/nl/089740890; Hartmann, Katrin

    2017-01-01

    Background: Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse

  6. A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model.

    Science.gov (United States)

    Mohanty, Sujit K; Donnelly, Bryan; Dupree, Phylicia; Lobeck, Inna; Mowery, Sarah; Meller, Jaroslaw; McNeal, Monica; Tiao, Greg

    2017-08-01

    Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRV VP4-R446G ) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRV VP4-R446G ) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro , the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo , it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia. Copyright © 2017 American Society for Microbiology.

  7. Responses of the L51781Y tk/sup +//tk/sup -/ mouse lymphoma cell forward mutation assay: III. 72 coded chemicals

    Energy Technology Data Exchange (ETDEWEB)

    McGregor, D.B.; Brown, A.; Cattanach, P.; Edwards, I.; McBride, D.; Riach, C.; Caspary, W.J.

    1988-01-01

    Seventy-two chemicals were tested for their mutagenic potential in the L51781Y tk/sup +///sup -/ mouse lymphoma cell forward mutation assay, using procedures based upon those described previously. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before planting in soft agar with or without trifluorothymidine (TFT), 3 ..mu..g/ml. The chemicals were tested at least twice. Significant responses were obtained with allyl isothiocyanate, p-benzoquinone dioxime, benzyl acetate, 2-biphenylamine HCl, bis(2-chloro-1-methylethyl)ether, cadmium chloride, chlordane, chlorobenzene, chlorobenzilate, 2-chloroethanol, chlorothalonil, cytarabine x HCl, p,p'-DDE, diazinon, 2,6-dichloro-p-phenylenediamine, N,N-diethylthiourea, diglycidylresorcinol ether, 2,4-dimethoxy aniline x HCl, disperse yellow 3, endosulfan, 1,2-epoxyhexadecane, ethyl acrylate, ethyl benzene, ethylene thiourea, F D and C yellow Number 6, furan, heptachlor, isophorone, mercuric chloride, 4,4'-methylenedianiline x 2 HCl, methyl viologen, nickel sulfate x 6H/sub 2/O, 4,4'-oxydianiline, pentachloroethane, piperonyl butoxide, propyl gallate, quinoline, rotenone, 2,4,5,6-tetrachloro-4-nitro-anisole, 1,1,1,2-tetrachloroethane, trichlorfon, 2,4,6-trichlorophenol, 2,4,5-trimethoxybenzaldehyde, 1,1,3-trimethyl-2-thiourea, 1-vinyl-3-cyclopetene dioxide, vinyl toluene, and ziram. The assay was incapable of providing a clear indication of whether some chemicals were mutagens; these benzyl alcohol, 1,4-dichlorobenzene, phenol, succinic acid-2,2-dimethyl hydrazide, and toluene.

  8. Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

    International Nuclear Information System (INIS)

    Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung; Park, Sun; Shin, Ho-Joon; Kim, Kyongmin

    2008-01-01

    Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate 32 P-ribonucleotides, but not HBV core particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems

  9. A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

    Directory of Open Access Journals (Sweden)

    Crumpacker Clyde S

    2011-01-01

    and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004. Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT. Conclusions The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

  10. Comparison of Two Molecular Assays for Detection and Characterization of Aspergillus fumigatus Triazole Resistance and Cyp51A Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients

    OpenAIRE

    Patricia Postina; Julian Skladny; Tobias Boch; Oliver A. Cornely; Oliver A. Cornely; Axel Hamprecht; Peter-Michael Rath; Jörg Steinmann; Oliver Bader; Thomas Miethke; Anne Dietz; Natalia Merker; Wolf-Karsten Hofmann; Dieter Buchheidt; Birgit Spiess

    2018-01-01

    In hematological patients, the incidence of invasive aspergillosis (IA) caused by azole resistant Aspergillus fumigatus (ARAf) is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR) assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergi...

  11. Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel

    Science.gov (United States)

    Hindiyeh, Musa; Levy, Virginia; Azar, Roberto; Varsano, Noemi; Regev, Liora; Shalev, Yael; Grossman, Zehava; Mendelson, Ella

    2005-01-01

    The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories. PMID:15695650

  12. Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.

    Science.gov (United States)

    Bharathi, Vidhya; Girdhar, Amandeep; Prasad, Archana; Verma, Meenkshi; Taneja, Vibha; Patel, Basant K

    2016-12-01

    Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI + ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi - ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI + ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aβ-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

    Directory of Open Access Journals (Sweden)

    Boerrigter Michaël

    2004-01-01

    Full Text Available Abstract Background Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. Methods Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP, 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls. The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Results Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. Conclusion The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

  14. Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand.

    Science.gov (United States)

    Stenhouse, Steven A; Plernsub, Suriya; Yanola, Jintana; Lumjuan, Nongkran; Dantrakool, Anchalee; Choochote, Wej; Somboon, Pradya

    2013-08-30

    Resistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin. This study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance. The bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance. Our bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some

  15. TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome.

    Science.gov (United States)

    Gray, Phillip N; Tsai, Pei; Chen, Daniel; Wu, Sitao; Hoo, Jayne; Mu, Wenbo; Li, Bing; Vuong, Huy; Lu, Hsiao-Mei; Batth, Navanjot; Willett, Sara; Uyeda, Lisa; Shah, Swati; Gau, Chia-Ling; Umali, Monalyn; Espenschied, Carin; Janicek, Mike; Brown, Sandra; Margileth, David; Dobrea, Lavinia; Wagman, Lawrence; Rana, Huma; Hall, Michael J; Ross, Theodora; Terdiman, Jonathan; Cullinane, Carey; Ries, Savita; Totten, Ellen; Elliott, Aaron M

    2018-04-17

    The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2 , MSH6 , MLH1 , PMS2 and EPCAM . Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases.

  16. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Møller, Michael Boe

    2011-01-01

    The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present ...

  17. Pharmacological modification of multi-drug resistance (MDR) in vitro detected by a novel fluorometric microculture cytotoxicity assay. Reversal of resistance and selective cytotoxic actions of cyclosporin A and verapamil on MDR leukemia T-cells.

    Science.gov (United States)

    Larsson, R; Nygren, P

    1990-07-15

    A novel fluorometric microculture cytotoxicity assay (FMCA), based on measurements of fluorescein diacetate (FDA) hydrolysis and DNA staining by Hoechst 33342, was used for drug sensitivity testing and detection of resistance reversal in acute lymphoblastic leukemia (ALL) cell lines. The 72-hr assay was found to be sensitive, reproducible and linearly related to the number of viable cells within a broad range of cell concentrations. At clinically achievable drug concentrations, the calcium channel blocker Verapamil (ver) and the immunosuppressant Cyclosporin A (csA) were found to partly reverse acquired Vincristine (vcr) resistance in multi-drug resistant (MDR) T-ALL L100 cells with little or no effect on the drug-sensitive parental L0 cell line. By combining the fluorometric indices, we found that low concentrations of csA were growth-inhibitory, whereas higher concentrations (greater than 10 micrograms/ml) were progressively cytotoxic for drug-sensitive L0 cells. In MDR L100 cells, on the other hand, csA produced significant cell kill even at low drug concentrations. Ver had no effects on sensitive L0 cells but showed considerable cytotoxic action towards MDR L100 cells. There was no apparent relationship between drug reversal of vcr resistance and the cytotoxic actions of the drug per se since the calcium channel blocker diltiazem (dil) significantly potentiated the actions of vcr on MDR L100 cells without being more toxic to these cells (compared to vcr-sensitive L0 cells).

  18. Ca2+ toxicity due to reverse Na+/Ca2+ exchange contributes to degeneration of neurites of DRG neurons induced by a neuropathy-associated Nav1.7 mutation

    Science.gov (United States)

    Estacion, M.; Vohra, B. P. S; Liu, S.; Hoeijmakers, J.; Faber, C. G.; Merkies, I. S. J.; Lauria, G.; Black, J. A.

    2015-01-01

    Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na+ concentration ([Na+]) and intracellular [Ca2+] following stimulation with high [K+] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca2+] transients evoked by high [K+] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K+] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K+] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca2+ or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K+] and 2-DG. These results point to [Na+] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca2+ toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy. PMID:26156380

  19. Somatic mosaicism caused by monoallelic reversion of a mutation in T cells of a patient with ADA-SCID and the effects of enzyme replacement therapy on the revertant phenotype.

    Science.gov (United States)

    Moncada-Vélez, M; Vélez-Ortega, A; Orrego, J; Santisteban, I; Jagadeesh, J; Olivares, M; Olaya, N; Hershfield, M; Candotti, F; Franco, J

    2011-11-01

    Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG-ADA. The follow-up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient's clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

  20. A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells

    NARCIS (Netherlands)

    Kuijpers, Taco W.; van Leeuwen, Ester M. M.; Barendregt, Barbara H.; Klarenbeek, Paul; Aan de Kerk, Daan J.; Baars, Paul A.; Jansen, Machiel H.; de Vries, Niek; van Lier, René A. W.; van der Burg, Mirjam

    2013-01-01

    Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B(+)T(-)NK(-) X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined

  1. A Novel Duplex Real-Time Reverse-Transcription PCR Assay for the Detection of Influenza A and the Novel Influenza A(H1N1 Strain

    Directory of Open Access Journals (Sweden)

    Theo P. Sloots

    2009-12-01

    Full Text Available Timely implementation of antiviral treatment and other public health based responses are dependent on accurate and rapid diagnosis of the novel pandemic influenza A(H1N1 strain. In this study we developed a duplex real-time PCR (RT-PCR (dFLU-TM assay for the simultaneous detection of a broad range of influenza A subtypes and specific detection of the novel H1N1 2009 pandemic strain. The assay was compared to the combined results of two previously described monoplex RT-PCR assays using 183 clinical samples and 10 seasonal influenza A isolates. Overall, the results showed that the dFLU-TM RT-PCR method is suitable for detection of influenza A, including the novel H1N1 pandemic strain, in clinical samples.

  2. High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

    Directory of Open Access Journals (Sweden)

    Deguchi Tomonori

    2010-09-01

    Full Text Available Abstract Background During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective. Results In this study, we developed a high resolution melting (HRM assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. Conclusions These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

  3. Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

    Science.gov (United States)

    Kurosaki, Yohei; Magassouba, N’Faly; Oloniniyi, Olamide K.; Cherif, Mahamoud S.; Sakabe, Saori; Takada, Ayato; Hirayama, Kenji; Yasuda, Jiro

    2016-01-01

    Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure. PMID:26900929

  4. Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

    Science.gov (United States)

    Wang, Hye-Young; Ahn, Sungwoo; Park, Sunyoung; Kim, SeungIl; Lee, Hyeyoung

    2017-01-01

    Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression. © 2016 S. Karger AG, Basel.

  5. A highly sensitive, multiplex broad-spectrum PCR-DNA-enzyme immunoassay and reverse hybridization assay for rapid detection and identification of Chlamydia trachomatis serovars.

    NARCIS (Netherlands)

    Quint, K.D.; Doorn, L.J. van; Kleter, B.; Koning, M.N. de; Munckhof, H.A. van den; Morre, S.A.; Harmsel, B. ter; Weiderpass, E.; Harbers, G.; Melchers, W.J.G.; Quint, W.G.V.

    2007-01-01

    Chlamydia trachomatis (Ct) comprises distinct serogroups and serovars. The present study evaluates a novel Ct amplification, detection, and genotyping method (Ct-DT assay). The Ct-DT amplification step is a multiplex broad-spectrum PCR for the cryptic plasmid and the VD2-region of ompl. The Ct-DT

  6. HorTILLUS—A Rich and Renewable Source of Induced Mutations for Forward/Reverse Genetics and Pre-breeding Programs in Barley (Hordeum vulgare L.

    Directory of Open Access Journals (Sweden)

    Miriam E. Szurman-Zubrzycka

    2018-02-01

    Full Text Available TILLING (Targeting Induced Local Lesions IN Genomes is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3 and N-methyl-N-nitrosourea (MNU. The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a

  7. A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21Waf1/Cip1/Sdi1 expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2

    International Nuclear Information System (INIS)

    Okaichi, Kumio; Wang Lihong; Sasaki, Ji-ichiro; Saya, Hideyuki; Tada, Mitsuhiro; Okumura, Yutaka

    1999-01-01

    Purpose: The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected. To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H). Methods and Materials: Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used. The transcriptional activity of mutant p53 was examined using a yeast functional assay. The transformants were examined for the accumulation of p53, the induction of p21 Waf1/Cip1/Sdi1 (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting. Apoptosis was analyzed by detection of DNA fragmentation. Results: The 123A point mutation of p53 was detected as a wild type in the yeast functional assay. The 123A mutant accumulated p53 in response to IR. The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2. The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR. Conclusion: The 123A mutant led to apoptosis, but not p21 expression in response to IR. The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant. The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas

  8. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

    Directory of Open Access Journals (Sweden)

    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  9. Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses.

    NARCIS (Netherlands)

    Heek, N.T. van; Clayton, S.J.; Sturm, P.D.J.; Walker, J.; Gouma, D.J.; Noorduyn, L.A.; Offerhaus, G.J.; Fox, J.C.

    2005-01-01

    BACKGROUND: Extrahepatic biliary stenosis (EBS) has malignant and benign causes. Patients with EBS are at risk of having or developing malignancy. Accurate diagnostic tests for early detection and surveillance are needed. The sensitivity of biliary cytology for malignancy is low. K-ras mutation

  10. Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses

    NARCIS (Netherlands)

    van Heek, N. T.; Clayton, S. J.; Sturm, P. D. J.; Walker, J.; Gouma, D. J.; Noorduyn, L. A.; Offerhaus, G. J. A.; Fox, J. C.

    2005-01-01

    Background: Extrahepatic biliary stenosis (EBS) has malignant and benign causes. Patients with EBS are at risk of having or developing malignancy. Accurate diagnostic tests for early detection and surveillance are needed. The sensitivity of biliary cytology for malignancy is low. K-ras mutation

  11. Mutator activity in Schizophyllum commune

    Energy Technology Data Exchange (ETDEWEB)

    Shneyour, Y.; Koltin, Y. (Tel Aviv Univ. (Israel). Dept. of Microbiology)

    1983-01-01

    A strain with an elevated level of spontaneous mutations and an especially high rate of reversion at a specific locus (pab/sup -/) was identified. The mutator trait is recessive. UV sensitivity and the absence of a UV-specific endonucleolytic activity were associated with the enhancement of the mutation rate in mutator strains. The endonuclease associated with the regulation of the mutation rate also acted on single-stranded DNA. The molecular weight of this enzyme is about 38,000 daltons.

  12. Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays

    DEFF Research Database (Denmark)

    Kabell, Susanne; Handberg, Kurt; Kusk, Mette

    2005-01-01

    Infectious bursal disease (IBD) is a worldwide distributed immunosuppressive viral disease in young chickens, controlled by vaccination. Emergence of several strains of IBD virus (IBDV) has created a demand for strain-specific diagnostic tools. In the present experiment, five different reverse...... included vaccine strain D78, classical strain Faragher 52/70, and the very virulent Danish strain DK01 The presence of the virus infection was confirmed by histopathologic evaluation of bursa lesions. The largest number of positive samples was obtained with a strain-specific two-step multiplex (MPX) RT...

  13. Comparison of Two Molecular Assays for Detection and Characterization of Aspergillus fumigatus Triazole Resistance and Cyp51A Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients

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    Patricia Postina

    2018-03-01

    Full Text Available In hematological patients, the incidence of invasive aspergillosis (IA caused by azole resistant Aspergillus fumigatus (ARAf is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL, 15 cerebrospinal fluid (CSF samples and ARAf isolates (n = 3 of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML. The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant

  14. Activating HER2 mutations in HER2 gene amplification negative breast cancer.

    Science.gov (United States)

    Bose, Ron; Kavuri, Shyam M; Searleman, Adam C; Shen, Wei; Shen, Dong; Koboldt, Daniel C; Monsey, John; Goel, Nicholas; Aronson, Adam B; Li, Shunqiang; Ma, Cynthia X; Ding, Li; Mardis, Elaine R; Ellis, Matthew J

    2013-02-01

    Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.

  15. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  16. A new allelic discrimination assay using locked nucleic acid-modified nucleotides (LNA) probes for detection of JAK2 V617F mutation

    Czech Academy of Sciences Publication Activity Database

    Marková, J.; Průková, Dana; Volková, Z.; Schwarz, J.

    2007-01-01

    Roč. 48, č. 3 (2007), s. 638-641 ISSN 1042-8194 Institutional research plan: CEZ:AV0Z50520514 Keywords : Ph1-negative myeloproliferative disorders * JAK2V617F mutation * allelic discrimination Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.512, year: 2007

  17. Replication-dependent 65R→K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant K65R + L74V

    International Nuclear Information System (INIS)

    Sharma, Prem L.; Nurpeisov, Viktoria; Lee, Kimberly; Skaggs, Sara; Di San Filippo, Christina Amat; Schinazi, Raymond F.

    2004-01-01

    Understanding of the mechanisms of interaction among nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) coding sequence is essential for the design of newer drugs and for enhancing our vision of the structure function relationship among amino acids of the polymerase domain of HIV-1. Although several nucleoside reverse transcriptase inhibitors select RT mutations K65R and L74V, the combination of 65R + 74V is rare in clinics. A novel NRTI (-)-β-D-dioxolane-guanosine (DXG) is known to select in vitro either the 65R or 74V mutant virus (Antimicrob. Agents Chemother. 44 (2000) 1783). These mutations were not selected together during repeated passaging of the HIV-1 in the presence of this drug. To analyze the impact of these RT mutations on viral replication, a double mutant containing K65R + L74V was created by site-directed mutagenesis in a pNL4-3 background. Replication kinetic assays revealed that the mutant K65R + L74V is unstable, and 65R→K reversion occurs during replication of virus in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication kinetic assays in MT-2 cells demonstrated that double mutant 65R + 74V is highly attenuated for replication and the initiation of reversion is related to the increase in RT activity. Additionally, the suppression of viral replication in the presence of DXG or under suboptimal human recombinant interleukin-2 leads to minimal or no 65R→K reversion. These observations provide evidence that 65R→K reversion in the double mutant 65R + 74V is dependent on a specific rate of viral replication in a pNL4-3 background. A similar phenomenon may occur in vivo, which may have implications for treatment management strategies

  18. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation

    International Nuclear Information System (INIS)

    Wiegand, Kimberly C; Lu, Yiling; Zhang, Fan; Anglesio, Michael S; Gilks, Blake; Mills, Gordon B; Huntsman, David G; Carey, Mark S; Hennessy, Bryan T; Leung, Samuel; Wang, Yemin; Ju, Zhenlin; McGahren, Mollianne; Kalloger, Steve E; Finlayson, Sarah; Stemke-Hale, Katherine

    2014-01-01

    Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr 308 and AKT-Ser 473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in

  19. Analysis of the sex-determining region of the Y chromosome (SRY) in sex reversed patients: point-mutation in SRY causing sex-reversion in a 46,XY female

    DEFF Research Database (Denmark)

    Müller, Jørn; Schwartz, M; Skakkebaek, N E

    1992-01-01

    The first and essential step in normal sexual differentiation takes place during the 5th-6th week of gestation. The testis determining factor (TDF) directs the undifferentiated gonad into a testis, which secretes hormones responsible for normal male development. A new candidate for TDF has recently...... been reported, and it has been called the sex determining region of the Y (SRY). The hypothesis has been supported by the finding of XX individuals with SRY, and two females with 46,XY karyotype and a mutation in SRY. However, XX males without SRY has been reported, and the role of SRY still has...... to be determined. We have tested three human females with 46,XY karyotype and gonadal dysgenesis and two 46,XX males for the presence of SRY using the polymerase chain reaction and subsequent DNA sequencing. Both 46,XX males contained SRY, whereas one of the 46,XY females had suffered a point mutation in SRY...

  20. Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin

    Czech Academy of Sciences Publication Activity Database

    Sheehan, A.; Messer, A.E.; Papadaki, M.; Choudhry, A.; Křen, Vladimír; Biedermann, David; Blagg, B.; Khandelwal, A.; Marston, S.B.

    2018-01-01

    Roč. 9, MAR 27 2018 (2018), č. článku 243. ISSN 1664-042X R&D Projects: GA MZd(CZ) NV16-27317A Institutional support: RVO:61388971 Keywords : cardiomyopathy * sarcomeric protein mutations * troponin I phosphorylation OBOR OECD: Cardiac and Cardiovascular systems Impact factor: 4.134, year: 2016

  1. Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay.

    Science.gov (United States)

    Huh, Hee Jae; Kim, Ji-Youn; Lee, Myoung-Keun; Lee, Nam Yong; Kim, Jong-Won; Ki, Chang-Seok

    2016-12-01

    Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90). The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST)

    Science.gov (United States)

    Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

    2014-01-01

    During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase–polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains. PMID:25424931

  3. Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

    Science.gov (United States)

    Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max

    2014-01-01

    During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase-polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.

  4. Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

    Directory of Open Access Journals (Sweden)

    Yasser Baaj

    2009-01-01

    Full Text Available We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes. In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease. DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays. Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio for “homozygous” DNA from healthy individuals. “Heterozygous” mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected. Genotyping by this method was therefore reliable. This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis.

  5. Mutation V111I in HIV-2 reverse transcriptase increases the fitness of the nucleoside analogue-resistant K65R and Q151M viruses

    NARCIS (Netherlands)

    I. Deuzing (Ilona); C. Charpentier (Charlotte); D.J. Wright (David Justin); S. Matheron (Sophie); J. Paton (Jack); D. Frentz (Dineke); D.A.M.C. van de Vijver (David); P.V. Coveney (Peter); D. Descamps (Diane); C.A.B. Boucher (Charles); N. Beerens (Nancy)

    2015-01-01

    textabstractInfection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have

  6. In silico reversal of repeat-induced point mutation (RIP identifies the origins of repeat families and uncovers obscured duplicated genes

    Directory of Open Access Journals (Sweden)

    Hane James K

    2010-11-01

    Full Text Available Abstract Background Repeat-induced point mutation (RIP is a fungal genome defence mechanism guarding against transposon invasion. RIP mutates the sequence of repeated DNA and over time renders the affected regions unrecognisable by similarity search tools such as BLAST. Results DeRIP is a new software tool developed to predict the original sequence of a RIP-mutated region prior to the occurrence of RIP. In this study, we apply deRIP to the genome of the wheat pathogen Stagonospora nodorum SN15 and predict the origin of several previously uncharacterised classes of repetitive DNA. Conclusions Five new classes of transposon repeats and four classes of endogenous gene repeats were identified after deRIP. The deRIP process is a new tool for fungal genomics that facilitates the identification and understanding of the role and origin of fungal repetitive DNA. DeRIP is open-source and is available as part of the RIPCAL suite at http://www.sourceforge.net/projects/ripcal.

  7. Hepatocytes, rather than leukocytes reverse DNA damage in vivo induced by whole body y-irradiation of mice, as shown by the alkaline comet assay

    Directory of Open Access Journals (Sweden)

    JUANA PINCHEIRA

    2008-01-01

    Full Text Available DNA damage repair was assessed in quiescent (G0 leukocytes and in hepatocytes of mice, after 1 and 2 hours recovery from a single whole body y-irradiation with 0.5, 1 or 2 Gy. Evaluation of single-strand breaks (SSB and alkali-labile sites together were carried out by a single-cell electrophoresis at pH>13.0 (alkaline comet assay. In non-irradiated (control mice, the constitutive, endogenous DNA damage (basal was around 1.5 times higher in leukocytes than in hepatocytes. Irradiation immediately increased SSB frequency in both cell types, in a dose-dependent manner. Two sequential phases took place during the in vivo repair of the radio-induced DNA lesions. The earliest one, present in both hepatocytes and leukocytes, further increased the SSB frequency, making evident the processing of some primary lesions in DNA bases into the SSB repair intermediates. In a second phase, SSB frequency decreased because of their removal. In hepatocytes, such a frequency regressed to the constitutive basal level after 2 hours recovery from either 0.5 orí Gy. On the other hand, the SSB repair phase was specifically abrogated in leukocytes, at the doses and recovery times analyzed. Thus, the efficiency of in vivo repair of radio-induced DNA damage in dormant cells (lymphocytes is quite different from that in hepatocytes whose low proliferation activity accounts only for cell renewal.

  8. Preliminary results on ghrelin mRNA quantification in buffalo calves during fasting and refeeding by real-time reverse transcription PCR assay

    Directory of Open Access Journals (Sweden)

    G. Neglia

    2010-02-01

    Full Text Available The aim of this trial was to evaluate ghrelin response to milk administration in 20 days old buffalo calves. The trial was carried out on 5 female buffalo calves with a mean age of 21.2±2.8 days. Five blood samples were collected from each animal into EDTA tubes, starting at 07.00 until 15.00, at 2-h intervals. At 09.00, after the second blood sample, replaced milk was administered to the calves. Blood samples were immediately placed at 4°C until processing, which was performed on the same day. We used real-time reverse transcription PCR system to detect the expression of ghrelin mRNA levels in blood of buffalo calves. Two calves showed a low ghrelin concentration at the start of the trial (Group A = low ghrelin concentration and three calves a high ghrelin concentration (Group B = high ghrelin concentration. Ghrelin expression was significantly higher either two hours (P<0.01 and just before feeding (P<0.05 in Group B vs. Group A. However, in both cases, a significant (P<0.05 difference was observed within each group between -2 and 6 hours after feeding. Therefore, ghrelin concentration tended to increase in animals that showed low levels and, similarly, it lowered in animals that showed high concentration. If these results will be confirmed, may represent the evidence that also in buffalo calves the ghrelin system may affect feed intake. Further studies are needed in order to better evaluate the ghrelin system in buffalo calves.

  9. Novel Tetra-Primer ARMS-PCR Assays for Thiopurine Intolerance Susceptibility Mutations NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C in East Asians

    Directory of Open Access Journals (Sweden)

    Chi-Chun Ho

    2017-10-01

    Full Text Available Thiopurines are clinically useful in the management of diverse immunological and malignant conditions. Nevertheless, these purine analogues can cause lethal myelosuppression, which may be prevented by prospective testing for variants in the thiopurine S-methyltransferase (TPMT and, in East Asians, Nudix hydrolase 15 (NUDT15 genes. Two single-tube, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR assays were developed to genotype the common loss-of-function variants NUDT15 c.415C>T (rs116855232 and TPMT*3C c.719A>G (rs1142345. In a group of 60 unselected patients, one and seven were found to be homozygous and heterozygous, respectively, for NUDT15 c.415C>T; one was found to be heterozygous for TPMT*3C c.719A>G. There was no non-specific amplification, and the genotypes were 100% concordant with Sanger sequencing. Limit-of-detection for both assays was below 1 ng of heterozygous template per reaction. Time- and cost-effective ARMS-PCR assays, suitable for genotyping East-Asian patients for thiopurine intolerance, were successfully developed and validated.

  10. Validation study of a quantitative multigene reverse transcriptase-polymerase chain reaction assay for assessment of recurrence risk in patients with stage II colon cancer.

    Science.gov (United States)

    Gray, Richard G; Quirke, Philip; Handley, Kelly; Lopatin, Margarita; Magill, Laura; Baehner, Frederick L; Beaumont, Claire; Clark-Langone, Kim M; Yoshizawa, Carl N; Lee, Mark; Watson, Drew; Shak, Steven; Kerr, David J

    2011-12-10

    We developed quantitative gene expression assays to assess recurrence risk and benefits from chemotherapy in patients with stage II colon cancer. We sought validation by using RNA extracted from fixed paraffin-embedded primary colon tumor blocks from 1,436 patients with stage II colon cancer in the QUASAR (Quick and Simple and Reliable) study of adjuvant fluoropyrimidine chemotherapy versus surgery alone. A recurrence score (RS) and a treatment score (TS) were calculated from gene expression levels of 13 cancer-related genes (n = 7 recurrence genes and n = 6 treatment benefit genes) and from five reference genes with prespecified algorithms. Cox proportional hazards regression models and log-rank methods were used to analyze the relationship between the RS and risk of recurrence in patients treated with surgery alone and between TS and benefits of chemotherapy. Risk of recurrence was significantly associated with RS (hazard ratio [HR] per interquartile range, 1.38; 95% CI, 1.11 to 1.74; P = .004). Recurrence risks at 3 years were 12%, 18%, and 22% for predefined low, intermediate, and high recurrence risk groups, respectively. T stage (HR, 1.94; P < .001) and mismatch repair (MMR) status (HR, 0.31; P < .001) were the strongest histopathologic prognostic factors. The continuous RS was associated with risk of recurrence (P = .006) beyond these and other covariates. There was no trend for increased benefit from chemotherapy at higher TS (P = .95). The continuous 12-gene RS has been validated in a prospective study for assessment of recurrence risk in patients with stage II colon cancer after surgery and provides prognostic value that complements T stage and MMR. The TS was not predictive of chemotherapy benefit.

  11. Mutations in the catalytic core or the C-terminus of murine leukemia virus (MLV) integrase disrupt virion infectivity and exert diverse effects on reverse transcription

    International Nuclear Information System (INIS)

    Steinrigl, Adolf; Nosek, Dagmara; Ertl, Reinhard; Guenzburg, Walter H.; Salmons, Brian; Klein, Dieter

    2007-01-01

    Understanding of the structures and functions of the retroviral integrase (IN), a key enzyme in the viral replication cycle, is essential for developing antiretroviral treatments and facilitating the development of safer gene therapy vehicles. Thus, four MLV IN-mutants were constructed in the context of a retroviral vector system, harbouring either a substitution in the catalytic centre, deletions in the C-terminus, or combinations of both modifications. IN-mutants were tested for their performance in different stages of the viral replication cycle: RNA-packaging; RT-activity; transient and stable infection efficiency; dynamics of reverse transcription and nuclear entry. All mutant vectors packaged viral RNA with wild-type efficiencies and displayed only slight reductions in RT-activity. Deletion of either the IN C-terminus alone, or in addition to part of the catalytic domain exerted contrasting effects on intracellular viral DNA levels, implying that IN influences reverse transcription in more than one direction

  12. Utility of a multiplex reverse transcriptasepolymerase chain reaction assay (HemaVision in the evaluation of genetic abnormalities in Korean children with acute leukemia: a single institution study

    Directory of Open Access Journals (Sweden)

    Hye-Jin kim

    2013-06-01

    Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; In children with acute leukemia, bone marrow genetic abnormalities (GA have prognostic significance, and may be the basis for minimal residual disease monitoring. Since April 2007, we have used a multiplex reverse transcriptase-polymerase chain reaction tool (HemaVision to detect of GA. &lt;b&gt;Methods:&lt;/b&gt; In this study, we reviewed the results of HemaVision screening in 270 children with acute leukemia, newly diagnosed at The Catholic University of Korea from April 2007 to December 2011, and compared the results with those of fluorescence in situ hybridization (FISH, and G-band karyotyping. &lt;b&gt;Results:&lt;/b&gt; Among the 270 children (153 males, 117 females, 187 acute lymphoblastic leukemia and 74 acute myeloid leukemia patients were identified. Overall, GA was detected in 230 patients (85.2%. HemaVision, FISH, and G-band karyotyping identified GA in 125 (46.3%, 126 (46.7%, and 215 patients (79.6%, respectively. TEL-AML1 (20.9%, 39/187 and AML1-ETO (27%, 20/74 were the most common GA in ALL and AML, respectively. Overall sensitivity of HemaVision was 98.4%, with false-negative results in 2 instances: 1 each for TEL-AML1 and MLL-AF4 . An aggregate of diseasesspecific FISH showed 100% sensitivity in detection of GA covered by HemaVision for actual probes utilized. G-band karyotype revealed GA other than those covered by HemaVison screening in 133 patients (49.3%. Except for hyperdiplody and hypodiploidy, recurrent GA as defined by the World Health Organizationthat were not screened by HemaVision, were absent in the karyotype. &lt;b&gt;Conclusion:&lt;/b&gt; HemaVision, supported by an aggregate of FISH tests for important translocations, may allow for accurate diagnosis of GA in Korean children with acute leukemia.

  13. Detection of cystic fibrosis transmembrane conductance regulator ΔF508 gene mutation using a paper-based nucleic acid hybridization assay and a smartphone camera.

    Science.gov (United States)

    Malhotra, Karan; Noor, M Omair; Krull, Ulrich J

    2018-05-29

    Diagnostic technology that makes use of paper platforms in conjunction with the ubiquitous availability of digital cameras in cellular telephones and personal assistive devices offers opportunities for development of bioassays that are cost effective and widely distributed. Assays that operate effectively in aqueous solution require further development for implementation in paper substrates, overcoming issues associated with surface interactions on a matrix that offers a large surface-to-volume ratio and constraints on convective mixing. This report presents and compares two related methods for determination of oligonucleotides that serve as indicators of cystic fibrosis, differentiating between the normal wild-type sequence, and a mutant-type sequence that has a 3-base replacement. The transduction strategy operates by selective hybridization of oligonucleotide probes that are conjugated to fluorescent quantum dots, where hybridization of target sequences causes a molecular fluorophore to approach the quantum dot and become emissive through fluorescence resonance energy transfer. Detection can rely on hybridization of a target that is labelled with Cy3 fluorophore, or in the presence of an unlabelled target when a sandwich assay format is implemented with a labelled reporter oligonucleotide. Selectivity to determine the presence of mismatched sequences involves appropriate selection of nucleotide sequences to set melt temperatures, in conjunction with control of stringency conditions using formamide as a chaotrope. It was determined that both direct and sandwich assays on paper substrates are able to distinguish between wild-type and mutant-type samples.

  14. A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

    Science.gov (United States)

    Sahli, R; Andrei, G; Estrade, C; Snoeck, R; Meylan, P R

    2000-04-01

    Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

  15. Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

    Science.gov (United States)

    Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

    2017-08-02

    Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

  16. Reverse transcriptase and protease inhibitor resistant mutations in art treatment naïve and treated HIV-1 infected children in India A Short Review

    Directory of Open Access Journals (Sweden)

    Dinesh Bure,

    2016-08-01

    Full Text Available Introduction of first line and second line antiretroviral therapy has dramatically improved the quality of life and survival of the HIV-1 infected individuals. Extension of this therapy in children has similar effect. However the emergence of drug selected resistance has hampered the response to the therapy. A database of prevalence of drug resistance mutations in the Indian children both ART naïve and treated will help in deciding the appropriate regimen for the individual patient as well as formulating the policies regarding the composition of drugs included in the fixed dose combinations and its periodic review by analysis of the information that is made available from time to time. This will enable us to utilize our limited resources in most prudent way.

  17. Mutation induction by and mutational interaction between monochromatic wavelength radiations in the near-ultraviolet and visible ranges

    International Nuclear Information System (INIS)

    Tyrrell, R.M.

    1980-01-01

    The induction of mutations (reversion to tryptophan independence) by various UV (254, 313, 334 and 365 nm) and visible (405 and 434 nm) wavelengths was measured in exponential phase populations of Escherichia coli B/r thy trp and B/r thy trp uvr A by assay of irradiated populations on semi-enriched media. No mutations were induced in the repair proficient strain at wavelengths longer than 313 nm. Mutations were induced to the excisionless strain at wavelengths as long as 405 nm but less than expected from the known amount of DNA damage induced. Irradiation at the long wavelenths (434, 405, 365 and 334 nm) suppressed the appearance of 254- or 313 nm-induced mutations in the repair competent strain but not in the excision deficient strain. The relative dose-requirement for mutation suppression was related to the relative efficiency of these wavelengths in inducing growth delay. These results suggest that the growth delay induced by near-UV and visible wavelenghts allows more time for the 'error-free' excision repair process to act on the potentially mutagenic lesions induced by 254- and 313-nm radiations, thereby reducing the mutation frequency observed in the repair-proficient strain. The level of near-UV mutation induced in the excision deficient strain is lower than expected from the DNA damage known to be induced. It is possible that near-UV radiation induces a class of lethal lesions that are not susceptible to error-prone repair. (author)

  18. Use of the microscreen phage-induction assay to assess the genotoxicity of 14 hazardous industrial wastes

    Energy Technology Data Exchange (ETDEWEB)

    Houk, V.S.; DeMarini, D.M.

    1988-01-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 pg per ml. Comparisons between the ability of these waste samples to induce prophage and their mutagenicity in the Salmonella reverse mutation assay indicate that the phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic five additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed, as are some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

  19. Reverse Algols

    Science.gov (United States)

    Leung, K. C.

    1989-01-01

    Reverse Algols, binary systems with a semidetached configuration in which the more massive component is in contact with the critical equipotential surface, are examined. Observational evidence for reverse Algols is presented and the parameters of seven reverse Algols are listed. The evolution of Algols and reverse Algols is discussed. It is suggested that, because reverse Algols represent the premass-reversal semidetached phase of close binary evolution, the evolutionary time scale between regular and reverse Algols is the ratio of the number of confirmed systems of these two Algol types.

  20. Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

    Science.gov (United States)

    Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

    2017-09-01

    It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

  1. Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose.

    Science.gov (United States)

    Moury, B; Cardin, L; Onesto, J P; Candresse, T; Poupet, A

    2000-05-01

    We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

  2. Reverse Logistics

    OpenAIRE

    Kulikova, Olga

    2016-01-01

    This thesis was focused on the analysis of the concept of reverse logistics and actual reverse processes which are implemented in mining industry and finding solutions for the optimization of reverse logistics in this sphere. The objective of this paper was the assessment of the development of reverse logistics in mining industry on the example of potash production. The theoretical part was based on reverse logistics and mining waste related literature and provided foundations for further...

  3. IDH1/2 Mutations Sensitize Acute Myeloid Leukemia to PARP Inhibition and This Is Reversed by IDH1/2-Mutant Inhibitors.

    Science.gov (United States)

    Molenaar, Remco J; Radivoyevitch, Tomas; Nagata, Yasunobu; Khurshed, Mohammed; Przychodzen, Bartolomiej; Makishima, Hideki; Xu, Mingjiang; Bleeker, Fonnet E; Wilmink, Johanna W; Carraway, Hetty E; Mukherjee, Sudipto; Sekeres, Mikkael A; van Noorden, Cornelis J F; Maciejewski, Jaroslaw P

    2018-04-01

    Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2 MUT enzymes produce D -2-hydroxyglutarate ( D 2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for IDH1/2 MUT AML is not known. Experimental Design: Well-characterized primary IDH1 MUT , IDH2 MUT , and IDH1/2 WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. Results: IDH1/2 MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2 MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2 MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2 MUT cells was caused by D 2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2 MUT cells. Conclusions: IDH1/2 MUT AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2 MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2 MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2 MUT AML. Clin Cancer Res; 24(7); 1705-15. ©2018 AACR . ©2018 American Association for Cancer Research.

  4. Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia.

    Science.gov (United States)

    Zhang, Xianbo; Li, Mengru; Ma, He; Liu, Xingyong; Shi, Hongjuan; Li, Minghui; Wang, Deshou

    2017-08-01

    It is well accepted that Forkhead box protein L2 (Foxl2) and aromatase (Cyp19a1; the enzyme responsible for estrogen synthesis) are critical for ovarian development in vertebrates. Knockouts of Foxl2 and Cyp19a1 in goat, mouse, and zebrafish have revealed similar but not identical functions across species. Functional analyses of these two genes in other animals are needed to elucidate their conserved roles in vertebrate sexual development. In this study, we established foxl2 and cyp19a1a mutant lines in Nile tilapia. Both foxl2-/- and cyp19a1a-/- XX fish displayed female-to-male sex reversal. Sf1, Dmrt1, and Gsdf were upregulated in the foxl2-/- and the cyp19a1a-/- XX gonads. Downregulation of Cyp19a1a and serum estradiol-17β level, and upregulation of Cyp11b2 and serum 11-ketotestosterone level were observed in foxl2-/- XX fish. The mutant phenotype of foxl2-/- XX individuals could be rescued by 17β-estradiol treatment from 5 to 30 days after hatching (dah). Upregulation of Star1, the enzyme involved in androgen production in tilapia, was also observed in the foxl2-/- XX gonad at 30 and 90 dah. In vitro promoter analyses consistently demonstrated that Foxl2 could suppress the transcription of star1 in a dose-dependent manner. In addition, compared with the control XX gonad, fewer germ cells were detected in the foxl2-/- XX, cyp19a1a-/- XX, and control XY gonads 10 dah. These results demonstrate that Foxl2 promotes ovarian development by upregulating Cyp19a1a expression and repressing male pathway gene expression. These results extend the study of Foxl2 and Cyp19a1a loss of function to a commercially important fish species. Copyright © 2017 Endocrine Society.

  5. RBE-LET relationships of high-LET radiations in drosophila mutations

    International Nuclear Information System (INIS)

    Yoshikawa, Isao; Takatsuji, Toshihiro; Nagano, Masaaki; Takada, Jun; Endo, Satoru; Hoshi, Masaharu

    1999-01-01

    The relative biological effectiveness (RBE) of 252 Cf neutrons and synchrotron-generated high-energy charged particles for mutation induction was evaluated as a function of linear energy transfer (LET), using the loss of heterozygosity for wing-hair mutations and the reversion of the mutant white-ivory eye-color in Drosophila melanogaster. Loss of heterozygosity for wing-hair mutations results predominantly from mitotic crossing over induced in wing anlage cells of larvae, while the reverse mutation of eye-color is due to an intragenic structural change (2.96 kb-DNA excision) in the white locus on the X-chromosome. The measurements were performed in a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-color clone can be detected simultaneously in the same individual. Larvae were irradiated at the age of 3 days post oviposition with 252 Cf neutrons, carbon beam or neon beam. For the neutron irradiation, the RBE values for wing-hair mutations were larger than that for eye-color mutation by about 7 fold. The RBE of carbon ions for producing the wing-hair mutations increased with increase in LET. The estimated RBE values were found to be in the range 2 to 6.5 for the wing-hair. For neon beam irradiation, the RBE values for wing-hair mutations peak near 150 keV/μm and decrease with further increase in LET. On the other hand, the RBE values for the induction of the eye-color mutation are nearly unity in 252 Cf neutrons and both ions throughout the LET range irradiated. We discuss the relationships between the initial DNA damage and LET in considering the mechanism of somatic mutation induction. (author)

  6. Strand transfer and elongation of HIV-1 reverse transcription is facilitated by cell factors in vitro.

    Directory of Open Access Journals (Sweden)

    David Warrilow

    Full Text Available Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis.

  7. Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.

    Science.gov (United States)

    Liu, Baoming; Yang, Jing-Xian; Yan, Ling; Zhuang, Hui; Li, Tong

    2018-01-01

    As one of the major global public health concerns, hepatitis B virus (HBV) can be divided into at least eight genotypes, which may be related to disease severity and treatment response. We previously demonstrated that genotypes B and C HBV, with distinct geographical distribution in China, had divergent genotype-dependent amino acid polymorphisms and variations in reverse transcriptase (RT) gene region, a target of antiviral therapy using nucleos(t)ide analogues. Recently recombination between HBV genotypes B and C was reported to occur in the RT region. However, their frequency and clinical significance is poorly understood. Here full-length HBV RT sequences from 201 Chinese chronic hepatitis B (CHB) patients were amplified and sequenced, among which 31.34% (63/201) were genotype B whereas 68.66% (138/201) genotype C. Although no intergenotypic recombination was detected among C-genotype HBV, 38.10% (24/63) of B-genotype HBV had recombination with genotype C in the 3'-terminal RT sequences. The patients with B/C intergenotypic recombinants had significantly (Pdistribution feature in China. Our findings provide novel insight into the virological, clinical and epidemiological features of new HBV B/C intergenotypic recombinants at the 3' end of RT sequences among Chinese CHB patients. The highly complex genetic background of the novel recombinant HBV carrying new mutations affecting RT protein may contribute to an enhanced heterogeneity in treatment response or prognosis among CHB patients. Published by Elsevier B.V.

  8. Reverse Osmosis

    Indian Academy of Sciences (India)

    many applications, one of which is desalination of seawater. The inaugural Nobel Prize in Chemistry was awarded in 1901 to van 't Hoff for his seminal work in this area. The present article explains the principle of osmosis and reverse osmosis. Osmosis and Reverse Osmosis. As the name suggests, reverse osmosis is the ...

  9. Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

    Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

  10. Three novel and two known androgen receptor gene mutations ...

    Indian Academy of Sciences (India)

    gene mutations associated with androgen insensitivity syndrome in sex-reversed XY female patients. J. Genet. ... signal and a C-terminal. Keywords. androgen insensitivity syndrome; androgen receptor; truncation mutation; N-terminal domain; XY sex reversal. .... and an increased risk of gonadal tumour. Mutations in SRY.

  11. Application of a Receptor-Binding Capture Quantitative Reverse Transcription-PCR Assay To Concentrate Human Norovirus from Sewage and To Study the Distribution and Stability of the Virus

    Science.gov (United States)

    Yang, David; Pan, Liangwen; Mandrell, Robert

    2012-01-01

    Water is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method and compared it to the existing negatively charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (CT) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV were measured as average CT values of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE-concentrated HuNoV was not protected from RNase I and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 103 genomic copies per liter (gc/liter) and 4.3 × 104 gc/liter, respectively. No GI and sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage. PMID:22101044

  12. Convergent Evolution of Head Crests in Two Domesticated Columbids Is Associated with Different Missense Mutations in EphB2

    Science.gov (United States)

    Vickrey, Anna I.; Domyan, Eric T.; Horvath, Martin P.; Shapiro, Michael D.

    2015-01-01

    Head crests are important display structures in wild bird species and are also common in domesticated lineages. Many breeds of domestic rock pigeon (Columba livia) have crests of reversed occipital feathers, and this recessive trait is associated with a nonsynonymous coding mutation in the intracellular kinase domain of EphB2 (Ephrin receptor B2). The domestic ringneck dove (Streptopelia risoria) also has a recessive crested morph with reversed occipital feathers, and interspecific crosses between crested doves and pigeons produce crested offspring, suggesting a similar genetic basis for this trait in both species. We therefore investigated EphB2 as a candidate for the head crest phenotype of ringneck doves and identified a nonsynonymous coding mutation in the intracellular kinase domain that is significantly associated with the crested morph. This mutation is over 100 amino acid positions away from the crest mutation found in rock pigeons, yet both mutations are predicted to negatively affect the function of ATP-binding pocket. Furthermore, bacterial toxicity assays suggest that “crest” mutations in both species severely impact kinase activity. We conclude that head crests are associated with different mutations in the same functional domain of the same gene in two different columbid species, thereby representing striking evolutionary convergence in morphology and molecules. PMID:26104009

  13. Adaptive mutation: has the unicorn landed?

    Science.gov (United States)

    Foster, P L

    1998-01-01

    Reversion of an episomal Lac- allele during lactose selection has been studied as a model for adaptive mutation. Although recent results show that the mutations that arise during selection are not "adaptive" in the original sense, the mutagenic mechanism that produces these mutations may nonetheless be of evolutionary significance. In addition, a transient mutational state induced in a subpopulation of starving cells could provide a species with a mechanism for adaptive evolution. PMID:9560365

  14. Adaptive mutation: has the unicorn landed?

    Science.gov (United States)

    Foster, P L

    1998-04-01

    Reversion of an episomal Lac- allele during lactose selection has been studied as a model for adaptive mutation. Although recent results show that the mutations that arise during selection are not "adaptive" in the original sense, the mutagenic mechanism that produces these mutations may nonetheless be of evolutionary significance. In addition, a transient mutational state induced in a subpopulation of starving cells could provide a species with a mechanism for adaptive evolution.

  15. Natural Plant Alkaloid (Emetine Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity

    Directory of Open Access Journals (Sweden)

    Ana Luiza Chaves Valadão

    2015-06-01

    Full Text Available Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine’s potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V. Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.

  16. TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma

    NARCIS (Netherlands)

    M. Remke (Marc); E.A. Ramaswamy; M. Peacock (Munro); D.J.H. Shih (David J.); C. Koelsche (Christian); P.A. Northcott (Paul A.); N. Hill (Nadia); S. Cavalli (Silvia); M. Kool (Marcel); X. Wang (Xin); S. Mack (Stephen); M. Barszczyk (Mark); A.S. Morrissy (A. Sorana); X. Wu (Xiaochong); S. Agnihotri (Sameer); P. Luu (Phan); D. Jones (David); L. Garzia (Livia); A.M. Dubuc (Adrian); N. Zhukova (Nataliya); R. Vanner (Robert); J.M. Kros (Johan); P.J. French (Pim); E.G. van Meir (Erwin); R. Vibhakar (Rajeev); K. Zitterbart (Karel); J.A. Chan (Jennifer); L. Bognár (László); A. Klekner (Almos); B. Lach (Boleslaw); S. Jung (Shin); F. Saad (Fred); L.M. Liau (Linda); S. Albrecht (Steffen); M. Zollo (Maurizio); M.K. Cooper (Michael); R.C. Thompson (Reid); O. Delattre (Olivier); F. Bourdeaut (Franck); F.F. Doz (François); M. Garami (Miklós); P. Hauser (Peter); C.G. Carlotti (Carlos); T.E. Van Meter (Timothy); L. Massimi (Luca); D. Fults (Daniel); L.W. Pomeroy (Laura); T. Kumabe (Toshiro); Y.S. Ra (Young Shin); J.R. Leonard (Jeffrey); S.K. Elbabaa (Samer); J. Mora (Jaume); J.B. Rubin (Joshua); Y.-J. Cho (Yoon-Jae); R.E. McLendon (Roger); D.D. Bigner (Darell); C.G. Eberhart (Charles); M. Fouladi (Maryam); R.J. Wechsler-Reya (Robert); R. Faria (Rui); S.E. Croul (Sidney); A. Huang (Anding); E. Bouffet (Eric); C.E. Hawkins (Cynthia); M. Dirks (Maaike); W.A. Weiss (William); U. Schüller (Ulrich); A. Pollack (Aaron); P. Rutkowski (Piotr); D. Meyronet (David); A. Jouvet (Anne); M. Fèvre-Montange (Michelle); N. Jabado (Nada); M. Perek-Polnik (Marta); W.A. Grajkowska (Wieslawa); S.-K. Kim (Seung-Ki); J.T. Rutka (James); E. Malkin (Elissa); U. Tabori (Uri); S.M. Pfister (Stefan); A. Korshunov (Andrey); A. von Deimling (Andreas); M.D. Taylor (Michael)

    2013-01-01

    textabstractTelomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought

  17. Serial QuantiFERON-TB Gold In-Tube assay and tuberculin skin test to diagnose latent tuberculosis in household Mexican contacts: conversion and reversion rates and associated factors using conventional and borderline zone definitions

    Directory of Open Access Journals (Sweden)

    Joel Monárrez-Espino

    2014-11-01

    Full Text Available A cohort of 123 adult contacts was followed for 18‐24 months (86 completed the follow-up to compare conversion and reversion rates based on two serial measures of QuantiFERON (QFT and tuberculin skin test (TST (PPD from TUBERSOL, Aventis Pasteur, Canada for diagnosing latent tuberculosis (TB in household contacts of TB patients using conventional (C and borderline zone (BZ definitions. Questionnaires were used to obtain information regarding TB exposure, TB risk factors and socio-demographic data. QFT (IU/mL conversion was defined as 0.70 (BZ and reversion was defined as ≥0.35 to 10 (BZ and reversion was defined as ≥5 to <5 (C. The QFT conversion and reversion rates were 10.5% and 7% with C and 8.1% and 4.7% with the BZ definitions, respectively. The TST rates were higher compared with QFT, especially with the C definitions (conversion 23.3%, reversion 9.3%. The QFT conversion and reversion rates were higher for TST ≥5; for TST, both rates were lower for QFT <0.35. No risk factors were associated with the probability of converting or reverting. The inconsistency and apparent randomness of serial testing is confusing and adds to the limitations of these tests and definitions to follow-up close TB contacts.

  18. The loss-of-allele assay for ES cell screening and mouse genotyping.

    Science.gov (United States)

    Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

  19. Three novel and two known androgen receptor gene mutations ...

    Indian Academy of Sciences (India)

    with androgen insensitivity syndrome in sex-reversed XY female patients. BALACHANDRAN .... Three novel AR gene mutations associated with AIS in XY sex-reversed females. Ta b le. 1 . ( contd. ) ..... disease, 1st edition. Springer Science + ...

  20. Novel TGM5 mutations in acral peeling skin syndrome.

    Science.gov (United States)

    van der Velden, Jaap J A J; van Geel, Michel; Nellen, Ruud G L; Jonkman, Marcel F; McGrath, John A; Nanda, Arti; Sprecher, Eli; van Steensel, Maurice A M; McLean, W H Irwin; Cassidy, Andrew J

    2015-04-01

    Acral peeling skin syndrome (APSS, MIM #609796) is a rare autosomal recessive disorder characterized by superficial exfoliation and blistering of the volar and dorsal aspects of hands and feet. The level of separation is at the junction of the stratum granulosum and stratum corneum. APSS is caused by mutations in the TGM5 gene encoding transglutaminase-5, which is important for structural integrity of the outermost epidermal layers. The majority of patients originate from Europe and carry a p.(Gly113Cys) mutation in TGM5. In this study, we report both European and non-European families carrying other mutations in the TGM5 gene. In 5 patients, we found 3 novel mutations: c.1001+2_1001+3del, c.1171G>A and c.1498C>T. To confirm their pathogenicity, we performed functional analyses with a transglutaminase activity assay, determined alternative splicing by reverse-transcribed PCR analysis and used databases and in silico prediction tools. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  2. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  3. Comparison of multiplex reverse transcription-PCR-enzyme ...

    African Journals Online (AJOL)

    Comparison of multiplex reverse transcription-PCR-enzyme hybridization assay with immunofluorescence techniques for the detection of four viral respiratory pathogens in pediatric community acquired pneumonia.

  4. Assay optimization for molecular detection of Zika virus

    NARCIS (Netherlands)

    Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

    2016-01-01

    To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

  5. Reversible infantile mitochondrial diseases.

    Science.gov (United States)

    Boczonadi, Veronika; Bansagi, Boglarka; Horvath, Rita

    2015-05-01

    Mitochondrial diseases are usually severe and progressive conditions; however, there are rare forms that show remarkable spontaneous recoveries. Two homoplasmic mitochondrial tRNA mutations (m.14674T>C/G in mt-tRNA(Glu)) have been reported to cause severe infantile mitochondrial myopathy in the first months of life. If these patients survive the first year of life by extensive life-sustaining measures they usually recover and develop normally. Another mitochondrial disease due to deficiency of the 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) causes severe liver failure in infancy, but similar to the reversible mitochondrial myopathy, within the first year of life these infants may also recover completely. Partial recovery has been noted in some other rare forms of mitochondrial disease due to deficiency of mitochondrial tRNA synthetases and mitochondrial tRNA modifying enzymes. Here we summarize the clinical presentation of these unique reversible mitochondrial diseases and discuss potential molecular mechanisms behind the reversibility. Understanding these mechanisms may provide the key to treatments of potential broader relevance in mitochondrial disease, where for the majority of the patients no effective treatment is currently available.

  6. Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

    Science.gov (United States)

    Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

    2016-07-01

    Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being

  7. Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer

    KAUST Repository

    Roperch, Jean-Pierre

    2016-09-02

    Background: Non-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients. Methods: We used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time\\'s and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination. Results: For Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers\\' methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82. Conclusions: We showed that combined analysis of FGFR3 mutation and DNA methylation markers

  8. Supplementary Material for: Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer

    KAUST Repository

    Roperch, Jean-Pierre; Grandchamp, Bernard; Desgrandchamps, Franç ois; Mongiat-Artus, Pierre; Ravery, Vincent; Ouzaid, Idir; Roupret, Morgan; Phe, Vé ronique; Ciofu, Calin; Tubach, Florence; Cussenot, Olivier; Incitti, Roberto

    2016-01-01

    Abstract Background Non-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients. Methods We used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time’s and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination. Results For Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers’ methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82. Conclusions We showed that combined analysis of FGFR3 mutation and DNA

  9. Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer

    KAUST Repository

    Roperch, Jean-Pierre; Grandchamp, Bernard; Desgrandchamps, Franç ois; Mongiat-Artus, Pierre; Ravery, Vincent; Ouzaid, Idir; Roupret, Morgan; Phe, Vé ronique; Ciofu, Calin; Tubach, Florence; Cussenot, Olivier; Incitti, Roberto

    2016-01-01

    Background: Non-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients. Methods: We used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time's and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination. Results: For Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers' methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82. Conclusions: We showed that combined analysis of FGFR3 mutation and DNA methylation markers on

  10. APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

    Directory of Open Access Journals (Sweden)

    Kate N Bishop

    2008-12-01

    Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

  11. The relationship between quantitative human epidermal growth factor receptor 2 gene expression by the 21-gene reverse transcriptase polymerase chain reaction assay and adjuvant trastuzumab benefit in Alliance N9831.

    Science.gov (United States)

    Perez, Edith A; Baehner, Frederick L; Butler, Steven M; Thompson, E Aubrey; Dueck, Amylou C; Jamshidian, Farid; Cherbavaz, Diana; Yoshizawa, Carl; Shak, Steven; Kaufman, Peter A; Davidson, Nancy E; Gralow, Julie; Asmann, Yan W; Ballman, Karla V

    2015-10-01

    The N9831 trial demonstrated the efficacy of adjuvant trastuzumab for patients with human epidermal growth factor receptor 2 (HER2) locally positive tumors by protein or gene analysis. We used the 21-gene assay to examine the association of quantitative HER2 messenger RNA (mRNA) gene expression and benefit from trastuzumab. N9831 tested the addition of trastuzumab to chemotherapy in stage I-III HER2-positive breast cancer. For two of the arms of the trial, doxorubicin and cyclophosphamide followed by paclitaxel (AC-T) and doxorubicin and cyclophosphamide followed by paclitaxel and trastuzumab concurrent chemotherapy-trastuzumab (AC-TH), recurrence score (RS) and HER2 mRNA expression were determined by the 21-gene assay (Oncotype DX®) (negative 10 % positive cells = positive), 91 % for RT-PCR versus central fluorescence in situ hybridization (FISH) (≥2.0 = positive) and 94 % for central IHC versus central FISH. In the primary analysis, the association of HER2 expression by 21-gene assay with trastuzumab benefit was marginally nonsignificant (nonlinear p = 0.057). In hormone receptor-positive patients (local IHC) the association was significant (p = 0.002). The association was nonlinear with the greatest estimated benefit at lower and higher HER2 expression levels. Concordance among HER2 assessments by central IHC, FISH, and RT-PCR were similar and high. Association of HER2 mRNA expression with trastuzumab benefit as measured by time to distant recurrence was nonsignificant. A consistent benefit of trastuzumab irrespective of mHER2 levels was observed in patients with either IHC-positive or FISH-positive tumors. Trend for benefit was observed also for the small groups of patients with negative results by any or all of the central assays. Clinicaltrials.gov NCT00005970 . Registered 5 July 2000.

  12. Radiation-induced mutation at minisatellite loci

    International Nuclear Information System (INIS)

    Dubrova, Y.E.; Nesterov, V.N.; Krouchinsky, N.G.

    1997-01-01

    We are studying the radiation-induced increase of mutation rate in minisatellite loci in mice and humans. Minisatellite mutations were scored by multilocus DNA fingerprint analysis in the progeny of γ-irradiated and non-irradiated mice. The frequency of mutation in offspring of irradiated males was 1.7 higher that in the control group. Germline mutation at human minisatellite loci was studied among children born in heavily polluted areas of the Mogilev district of Belarus after the Chernobyl accident and in a control population. The frequency of mutation assayed both by DNA fingerprinting and by eight single locus probes was found to be two times higher in the exposed families than in the control group. Furthermore, mutation rate was correlated with the parental radiation dose for chronic exposure 137 Cs, consistent with radiation-induction of germline mutation. The potential use of minisatellites in monitoring germline mutation in humans will be discussed

  13. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    Directory of Open Access Journals (Sweden)

    Kayla A Boortz

    Full Text Available Elevated fasting blood glucose (FBG has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln, rs149663725 (Gly114Arg and rs2232326 (Ser324Pro SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu, rs138726309 (His177Tyr, rs2232323 (Tyr207Ser rs374055555 (Arg293Trp, rs2232326 (Ser324Pro, rs137857125 (Pro313Leu and rs2232327 (Pro340Leu SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

  14. Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

    Science.gov (United States)

    Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

    2016-11-01

    Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for

  15. Estimating the Per-Base-Pair Mutation Rate in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Lang, Gregory I.; Murray, Andrew W.

    2008-01-01

    Although mutation rates are a key determinant of the rate of evolution they are difficult to measure precisely and global mutations rates (mutations per genome per generation) are often extrapolated from the per-base-pair mutation rate assuming that mutation rate is uniform across the genome. Using budding yeast, we describe an improved method for the accurate calculation of mutation rates based on the fluctuation assay. Our analysis suggests that the per-base-pair mutation rates at two genes...

  16. Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study.

    Science.gov (United States)

    Porter, Danielle P; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D; White, Kirsten L

    2015-12-07

    At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study.

  17. Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

    Directory of Open Access Journals (Sweden)

    Danielle P. Porter

    2015-12-01

    Full Text Available At Week 96 of the Single-Tablet Regimen (STaR study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF developed resistance mutations compared to those treated with efavirenz (EFV/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33, as well as baseline samples from control subjects with virologic response (n = 118. Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20% were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study.

  18. A Reverse-Genetics Mutational Analysis of the Barley HvDWARF Gene Results in Identification of a Series of Alleles and Mutants with Short Stature of Various Degree and Disturbance in BR Biosynthesis Allowing a New Insight into the Process.

    Science.gov (United States)

    Gruszka, Damian; Gorniak, Malgorzata; Glodowska, Ewelina; Wierus, Ewa; Oklestkova, Jana; Janeczko, Anna; Maluszynski, Miroslaw; Szarejko, Iwona

    2016-04-22

    Brassinosteroids (BRs) are plant steroid hormones, regulating a broad range of physiological processes. The largest amount of data related with BR biosynthesis has been gathered in Arabidopsis thaliana, however understanding of this process is far less elucidated in monocot crops. Up to now, only four barley genes implicated in BR biosynthesis have been identified. Two of them, HvDWARF and HvBRD, encode BR-6-oxidases catalyzing biosynthesis of castasterone, but their relation is not yet understood. In the present study, the identification of the HvDWARF genomic sequence, its mutational and functional analysis and characterization of new mutants are reported. Various types of mutations located in different positions within functional domains were identified and characterized. Analysis of their impact on phenotype of the mutants was performed. The identified homozygous mutants show reduced height of various degree and disrupted skotomorphogenesis. Mutational analysis of the HvDWARF gene with the "reverse genetics" approach allowed for its detailed functional analysis at the level of protein functional domains. The HvDWARF gene function and mutants' phenotypes were also validated by measurement of endogenous BR concentration. These results allowed a new insight into the BR biosynthesis in barley.

  19. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  20. Modification of mutation frequency in Saccharomyces Cerevisiae

    International Nuclear Information System (INIS)

    Vashishat, R.K.; Kakar, S.N.

    1976-01-01

    In a reverse mutation system, using haploid, histidine-requirinq strain of Saccharomyces cerevisiae, the frequency of uv-induced prototrophs increased if the post-irradiation minimal medium was supplemented with limited amounts of histidine. Addition of natural amino acids or RNA bases in the post-irradiation minimal medium, with or without histidine, also increased the uv-induced mutation frequency. Thus, post-irradiation conditions favouring protein and RNA synthesis, are effective in increasing uv-induced mutations in yeast. As compared to uv light, nitrous acid was more effective in inducing reversions in this strain and the frequency increased if the treated cells were plated on minimal medium supplemented with limited amounts of histidine. However, the addition of amino acids or RNA bases decreased the number of revertants. An additional inclusion of histidine reversed the suppressive effect of these metabolites. The mutation induction processes are thus different or differently modifiable in uv and nitrous acid. (author)

  1. TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA.

    Science.gov (United States)

    Wang, Na; Liu, Tiantian; Sofiadis, Anastasios; Juhlin, C Christofer; Zedenius, Jan; Höög, Anders; Larsson, Catharina; Xu, Dawei

    2014-10-01

    The telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T have been found in many malignancies, including in thyroid carcinomas. However, it is unclear how early these mutations occur in thyroid tumorigenesis. The study included primary tumors from 58 patients initially diagnosed with follicular thyroid adenoma (FTA), a benign entity, 18 with atypical FTA (AFTA) having an uncertain malignant potential, and 52 with follicular thyroid carcinoma (FTC). Sanger sequencing was used to investigate the mutational status of the TERT promoter. Telomere length and TERT messenger RNA (mRNA) expression were determined using quantitative polymerase chain reaction (PCR). Telomerase activity was assessed using a Telomerase PCR enzyme-linked immunosorbent assay kit. The C228T mutation was identified in 1 of 58 FTA (2%) and 3 of 18 AFTA (17%) samples. These 4 tumors all expressed TERT mRNA and telomerase activity, whereas the majority of C228T-negative adenomas lacked TERT expression (C228T versus wild-type, P = .008). The C228T mutation was associated with NRAS gene mutations (P = .016). The patient with C228T-mutated FTA later developed a scar recurrence and died of FTC, whereas none of the remaining 57 patients with FTA had recurrence. No recurrence occurred in 3 patients with AFTA who carried C228T during the follow-up period (36-285 months). Nine of the 52 FTCs (17%) exhibited the TERT mutation (8 of 9 C228T and 1 of 9 C250T), and the presence of the mutation was associated with shorter patient survival. TERT promoter mutations may occur as an early genetic event in thyroid follicular tumors that have not developed malignant features on routine histopathological workup. © 2014 American Cancer Society.

  2. Reverse Zymography: Overview and Pitfalls.

    Science.gov (United States)

    Sharma, Kanika; Bhattacharyya, Debasish

    2017-01-01

    Reverse zymography is a technique by which protease inhibitor(s) in a sample could be electrophoretically separated in a substrate-impregnated acrylamide gel and their relative abundance could be semi-quantified. The gel after electrophoresis is incubated with a protease when the impregnated substrate and all other proteins of the sample are degraded into small peptides except the inhibitor(s) that show clear bands against a white background. Since reverse zymography cannot distinguish between a protease inhibitor and a protein that is resistant against proteolysis, the results should be confirmed from inhibition of protease activity by solution state assay.

  3. Reversible Statistics

    DEFF Research Database (Denmark)

    Tryggestad, Kjell

    2004-01-01

    The study aims is to describe how the inclusion and exclusion of materials and calculative devices construct the boundaries and distinctions between statistical facts and artifacts in economics. My methodological approach is inspired by John Graunt's (1667) Political arithmetic and more recent work...... within constructivism and the field of Science and Technology Studies (STS). The result of this approach is here termed reversible statistics, reconstructing the findings of a statistical study within economics in three different ways. It is argued that all three accounts are quite normal, albeit...... in different ways. The presence and absence of diverse materials, both natural and political, is what distinguishes them from each other. Arguments are presented for a more symmetric relation between the scientific statistical text and the reader. I will argue that a more symmetric relation can be achieved...

  4. A novel reversed-phase HPLC method for the determination of urinary creatinine by pre-column derivatization with ethyl chloroformate: comparative studies with the standard Jaffé and isotope-dilution mass spectrometric assays.

    Science.gov (United States)

    Leung, Elvis M K; Chan, Wan

    2014-02-01

    Creatinine is an important biomarker for renal function diagnosis and normalizing variations in urinary drug/metabolites concentration. Quantification of creatinine in biological fluids such as urine and plasma is important for clinical diagnosis as well as in biomonitoring programs and urinary metabolomics/metabonomics research. Current methods for creatinine determination either are nonselective or involve the use of expensive mass spectrometers. In this paper, a novel reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of creatinine of high hydrophilicity by pre-column derivatization with ethyl chloroformate is presented. N-Ethyloxycarbonylation of creatinine significantly enhanced the hydrophobicity of creatinine, facilitating its chromatographic retention as well as quantification by HPLC. Factors governing the derivatization reaction were studied and optimized. The developed method was validated and applied for the determination of creatinine in rat urine samples. Comparative studies with isotope-dilution mass spectrometric method revealed that the two methods do not yield systematic differences in creatinine concentrations, indicating the HPLC method is suitable for the determination of creatinine in urine samples.

  5. Experimental evolution and the dynamics of genomic mutation rate modifiers.

    Science.gov (United States)

    Raynes, Y; Sniegowski, P D

    2014-11-01

    Because genes that affect mutation rates are themselves subject to mutation, mutation rates can be influenced by natural selection and other evolutionary forces. The population genetics of mutation rate modifier alleles has been a subject of theoretical interest for many decades. Here, we review experimental contributions to our understanding of mutation rate modifier dynamics. Numerous evolution experiments have shown that mutator alleles (modifiers that elevate the genomic mutation rate) can readily rise to high frequencies via genetic hitchhiking in non-recombining microbial populations. Whereas these results certainly provide an explanatory framework for observations of sporadically high mutation rates in pathogenic microbes and in cancer lineages, it is nonetheless true that most natural populations have very low mutation rates. This raises the interesting question of how mutator hitchhiking is suppressed or its phenotypic effect reversed in natural populations. Very little experimental work has addressed this question; with this in mind, we identify some promising areas for future experimental investigation.

  6. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  7. Molecular methods for the detection of mutations.

    Science.gov (United States)

    Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

    2000-01-01

    We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

  8. Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site [v2; ref status: indexed, http://f1000r.es/18y

    Directory of Open Access Journals (Sweden)

    Jasmina Kurepa

    2013-06-01

    Full Text Available Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 (sor1. The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.

  9. Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

    Science.gov (United States)

    Ivanov, E L; Kovaltzova, S V; Korolev, V G

    1989-08-01

    We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

  10. F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

    Science.gov (United States)

    Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

    2014-05-01

    Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

  11. Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform

    International Nuclear Information System (INIS)

    Tian, Hong-Xia; Zhang, Xu-Chao; Wang, Zhen; Chen, Jian-Guang; Chen, Shi-Liang; Guo, Wei-Bang; Wu, Yi-Long

    2016-01-01

    Objective: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on MassARRAY mass spectrometry platform. Methods: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of 99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a LungCarta TM kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with LungCarta TM kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively. Conclusions: The proposed MassARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues

  12. AT-101 enhances gefitinib sensitivity in non-small cell lung cancer with EGFR T790M mutations

    International Nuclear Information System (INIS)

    Zhao, Ren; Zhou, Shun; Xia, Bing; Zhang, Cui-ying; Hai, Ping; Zhe, Hong; Wang, Yan-yang

    2016-01-01

    Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have become the standard care of patients with advanced EGFR-mutant non-small cell lung cancer (NSCLC), development of acquired resistance is inevitable. A secondary mutation of threonine 790 (T790M) is associated with approximately half of the cases of acquired resistance. Strategies or agents to overcome this type of resistance are still limited. In this study, enhanced antitumor effect of AT-101, a-pan-Bcl-2 inhibitor, on gefitinib was explored in NSCLC with T790M mutation. The effect of cotreatment with AT-101 and gefitinib on the viability of NSCLC cell lines harboring acquired T790M mutation was investigated using the MTT assay. The cellular apoptosis of NSCLC cells after cotreatment with AT-101 and gefitinib was assessed by FITC-annexin V/PI assay and Western blots analysis. The potential underlying mechanisms of the enhanced therapeutic effect for AT-101 was also studied using Western blots analysis. The in vivo anti-cancer efficacy of the combination with AT-101 and gefitinib was examined in a mouse xenograft model. In this study, we found that treatment with AT-101 in combination with gefitinib significantly inhibited cell proliferation, as well as promoted apoptosis of EGFR TKIs resistant lung cancer cells. The apoptotic effects of the use of AT-101 was related to the blocking of antiapoptotic protein: Bcl-2, Bcl-xl, and Mcl-1 and downregrulation of the molecules in EGFR pathway. The observed enhancements of tumor growth suppression in xenografts supported the reverse effect of AT-101 in NSCLC with T790M mutation, which has been found in in vitro studies before. AT-101 enhances gefitinib sensitivity in NSCLC with EGFR T790M mutations. The addition of AT-101 to gefitinib is a promising strategy to overcome EGFR TKIs resistance in NSCLC with EGFR T790M mutations

  13. Managing Reverse Logistics or Reversing Logistics Management?

    OpenAIRE

    Brito, Marisa

    2004-01-01

    textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse logistics. The thesis brings insights on reverse logistics decision-making and it lays down theoretical principles for reverse logistics as a research field.In particular it puts together a framework ...

  14. Quinolone Resistance Reversion by Targeting the SOS Response.

    Science.gov (United States)

    Recacha, E; Machuca, J; Díaz de Alba, P; Ramos-Güelfo, M; Docobo-Pérez, F; Rodriguez-Beltrán, J; Blázquez, J; Pascual, A; Rodríguez-Martínez, J M

    2017-10-10

    Suppression of the SOS response has been postulated as a therapeutic strategy for potentiating antimicrobial agents. We aimed to evaluate the impact of its suppression on reversing resistance using a model of isogenic strains of Escherichia coli representing multiple levels of quinolone resistance. E. coli mutants exhibiting a spectrum of SOS activity were constructed from isogenic strains carrying quinolone resistance mechanisms with susceptible and resistant phenotypes. Changes in susceptibility were evaluated by static (MICs) and dynamic (killing curves or flow cytometry) methodologies. A peritoneal sepsis murine model was used to evaluate in vivo impact. Suppression of the SOS response was capable of resensitizing mutant strains with genes encoding three or four different resistance mechanisms (up to 15-fold reductions in MICs). Killing curve assays showed a clear disadvantage for survival (Δlog 10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Δlog 10 CFU/g of 1.76 log units) in resistant strains with a suppressed SOS response. This effect was evident even after short periods (60 min) of exposure. Suppression of the SOS response reverses antimicrobial resistance across a range of E. coli phenotypes from reduced susceptibility to highly resistant, playing a significant role in increasing the in vivo efficacy. IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this

  15. Managing Reverse Logistics or Reversing Logistics Management?

    NARCIS (Netherlands)

    M.P. de Brito (Marisa)

    2004-01-01

    textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse

  16. Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

    2018-05-03

    Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

  17. Distinct difference in relative biological effectiveness of 252Cf neutrons for the induction of mitotic crossing over and intragenic reversion of the white-ivory allele in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Yoshikawa, Isao; Hoshi, Masaharu; Ikenaga, Mituo

    1996-01-01

    The relative biological effectiveness (RBE) of 252 Cf neutrons was determined for two different types of somatic mutations, i.e., loss of heterozygosity for wing-hair mutations and reversion of the mutant white-ivory eye-color, in Drosophila melanogaster. Loss of heterozygosity for wing-hair mutations results predominantly from mitotic crossing over induced in wing anlage cells of larvae, while the reverse mutation of eye-color is due to an intragenic structural change in the white locus on the X-chromosome. For a quantitative comparison of RBE values for these events, we have constructed a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-color clones can be detected simultaneously in the same individuals. Larvae were irradiated at the age of 80±4 h post-oviposition with 252 Cf neutrons or 137 Cs γ-rays, and male adult flies were examined under the microscope for the presence of the two types of clonal mosaic spots appearing. The induction of wing-hair spots per dose unit was much greater for 252 Cf neutrons than for 137 Cs γ -rays, whereas the frequencies of eye-color reversion were similar for neutrons and γ-rays. The estimated RBE values of neutrons were 8.5 and 1.2 for the induction of mutant wing-hair spots and revertant eye-color spots, respectively. These results indicate that the RBE of neutrons is much greater for mitotic crossing over in comparison to the intragenic white-ivory reversion events. Possible causes for the difference in RBE are discussed

  18. Reversible Thermoset Adhesives

    Science.gov (United States)

    Mac Murray, Benjamin C. (Inventor); Tong, Tat H. (Inventor); Hreha, Richard D. (Inventor)

    2016-01-01

    Embodiments of a reversible thermoset adhesive formed by incorporating thermally-reversible cross-linking units and a method for making the reversible thermoset adhesive are provided. One approach to formulating reversible thermoset adhesives includes incorporating dienes, such as furans, and dienophiles, such as maleimides, into a polymer network as reversible covalent cross-links using Diels Alder cross-link formation between the diene and dienophile. The chemical components may be selected based on their compatibility with adhesive chemistry as well as their ability to undergo controlled, reversible cross-linking chemistry.

  19. Forward and reverse mutagenesis in C. elegans

    Science.gov (United States)

    Kutscher, Lena M.; Shaham, Shai

    2014-01-01

    Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

  20. Revertant mutation releases confined lethal mutation, opening Pandora's box: a novel genetic pathogenesis.

    Directory of Open Access Journals (Sweden)

    Yasushi Ogawa

    2014-05-01

    Full Text Available When two mutations, one dominant pathogenic and the other "confining" nonsense, coexist in the same allele, theoretically, reversion of the latter may elicit a disease, like the opening of Pandora's box. However, cases of this hypothetical pathogenic mechanism have never been reported. We describe a lethal form of keratitis-ichthyosis-deafness (KID syndrome caused by the reversion of the GJB2 nonsense mutation p.Tyr136X that would otherwise have confined the effect of another dominant lethal mutation, p.Gly45Glu, in the same allele. The patient's mother had the identical misssense mutation which was confined by the nonsense mutation. The biological relationship between the parents and the child was confirmed by genotyping of 15 short tandem repeat loci. Haplotype analysis using 40 SNPs spanning the >39 kbp region surrounding the GJB2 gene and an extended SNP microarray analysis spanning 83,483 SNPs throughout chromosome 13 in the family showed that an allelic recombination event involving the maternal allele carrying the mutations generated the pathogenic allele unique to the patient, although the possibility of coincidental accumulation of spontaneous point mutations cannot be completely excluded. Previous reports and our mutation screening support that p.Gly45Glu is in complete linkage disequilibrium with p.Tyr136X in the Japanese population. Estimated from statisitics in the literature, there may be approximately 11,000 p.Gly45Glu carriers in the Japanese population who have this second-site confining mutation, which acts as natural genetic protection from the lethal disease. The reversion-triggered onset of the disesase shown in this study is a previously unreported genetic pathogenesis based on Mendelian inheritance.

  1. Tubal Ligation Reversal

    Science.gov (United States)

    ... seal off the fallopian tubes, such as the Essure or Adiana systems, generally aren't reversible. Why ... electrocautery). Some types of sterilization, such as the Essure or Adiana systems, aren't considered reversible. Risks ...

  2. Reverse logistics - a framework

    NARCIS (Netherlands)

    M.P. de Brito (Marisa); R. Dekker (Rommert)

    2002-01-01

    textabstractIn this paper we define and compare Reverse Logistics definitions. We start by giving an understanding framework of Reverse Logistics: the why-what-how. By this means, we put in context the driving forces for Reverse Logistics, a typology of return reasons, a classification of

  3. New mutations affecting induced mutagenesis in yeast.

    Science.gov (United States)

    Lawrence, C W; Krauss, B R; Christensen, R B

    1985-01-01

    Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

  4. Reversible flowchart languages and the structured reversible program theorem

    DEFF Research Database (Denmark)

    Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

    2008-01-01

    Many irreversible computation models have reversible counterparts, but these are poorly understood at present. We introduce reversible flowcharts with an assertion operator and show that any reversible flowchart can be simulated by a structured reversible flowchart using only three control flow...... operators. Reversible flowcharts are r- Turing-complete, meaning that they can simuluate reversible Turing machines without garbage data. We also demonstrate the injectivization of classical flowcharts into reversible flowcharts. The reversible flowchart computation model provides a theoretical...

  5. GERMLINE MOLECULAR MUTATION ASSAY IN DROSOPHILA MELANOGASTER. (R825810)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  6. 40 CFR 799.9510 - TSCA bacterial reverse mutation test.

    Science.gov (United States)

    2010-07-01

    ..., and hydrazines. Such substances may be detected by E. coli WP2 strains or S. typhimurium TA102 (see... or TA97 or TA97a. (iii) S. typhimurium TA98. (iv) S. typhimurium TA100. (v) E. coli WP2 uvrA, or E... preferable to include TA102 or to add a DNA repair-proficient strain of E.coli [e.g. E.coli WP2 or E.coli WP2...

  7. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  8. Introduction to reversible computing

    CERN Document Server

    Perumalla, Kalyan S

    2013-01-01

    Few books comprehensively cover the software and programming aspects of reversible computing. Filling this gap, Introduction to Reversible Computing offers an expanded view of the field that includes the traditional energy-motivated hardware viewpoint as well as the emerging application-motivated software approach. Collecting scattered knowledge into one coherent account, the book provides a compendium of both classical and recently developed results on reversible computing. It explores up-and-coming theories, techniques, and tools for the application of rever

  9. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  10. Rapid Identification of Dengue Virus by Reverse Transcription-Polymerase Chain Reaction Using Field-Deployable Instrumentation

    National Research Council Canada - National Science Library

    McAvin, James C; Escamilla, Elizabeth M; Blow, James A; Turell, Micahel J; Quintana, Miguel; Bowles, David E; Swaby, James A; Barnes, William J; Huff, William B; Lahman, Kenton L

    2005-01-01

    ...) reverse transcription-polymerase chain reaction assays were developed for screening and seroype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler...

  11. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  12. BRAF mutation testing in solid tumors: a methodological comparison.

    Science.gov (United States)

    Weyant, Grace W; Wisotzkey, Jeffrey D; Benko, Floyd A; Donaldson, Keri J

    2014-09-01

    Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAF status with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAF alleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAF alleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration-approved companion diagnostic assay for the detection of BRAF mutations. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  13. Validated reverse transcription droplet digital PCR serves as a higher order method for absolute quantification of Potato virus Y strains.

    Science.gov (United States)

    Mehle, Nataša; Dobnik, David; Ravnikar, Maja; Pompe Novak, Maruša

    2018-05-03

    RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis. To achieve this, we initially transferred the PVY universal RT-qPCR assay to a reverse transcription droplet digital PCR (RT-ddPCR) format. RT-ddPCR is an absolute quantification method, where a calibration curve is not needed, and it is less prone to inhibitors. The RT-ddPCR developed and validated in this study achieved a dynamic range of quantification over five orders of magnitude, and in terms of its sensitivity, it was comparable to, or even better than, RT-qPCR. RT-ddPCR showed lower measurement variability. We have shown that RT-ddPCR can be used as a reference tool for the evaluation of different RT-qPCR assays. In addition, it can be used for quantification of RNA based on in-house reference materials that can then be used as calibrators in diagnostic laboratories.

  14. Reversibility of female sterilization.

    Science.gov (United States)

    Siegler, A M; Hulka, J; Peretz, A

    1985-04-01

    The discussion considers the current status of reversibility of sterilization in the US and describes clinical and experimental efforts for developing techniques designed for reversibility. It focuses on regret following sterilization, reversal potential of current sterilization techniques, patient selection, current reversal techniques, results of sterilization procedures, experimental approaches to reversal of current techniques of sterilization, and sterilization procedures devised for reversibility, in humans and in animals. Request is the 1st stage of reversal, but a request for sterilization reversal (SR) does not always mean regret for a decision made at the time. Frequently it is a wish to restore fertility because life circumstances have changed after a sterilization that was ppropriate at the time it was performed. Schwyhart and Kutner reviewed 22 studies published between 1949-69 in which they found that the percentage of patients regretting the procedure ranged from 1.3-15%. Requests for reversal remain low in most countries, but if sterilization becomes a more popular method of contraception, requests will also increase. The ideal operation considered as a reversaible method of sterilization should include an easy, reliable outpatient method of tubal occlusion with miniml risk or patient discomfort that subsequently could be reversed without the need for a major surgical intervention. Endoscopic methods have progressed toward the 1st objective. A recent search of the literature uncovered few series of SR of more than 50 cases. The 767 operations found were analyzed with regard to pregnancy outcome. The precent of live births varied from 74-78.8%, and the occurance of tubal pregnancies ranged from 1.7-6.5%. All of the confounding variables in patient selection and small numbers of reported procedures preclude any conclusion about the different techniques or the number of operations that give a surgeon a level of expertise. Few authors classify their

  15. Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

    Science.gov (United States)

    Pliaka, V; Kyriakopoulou, Z; Tsakogiannis, D; Ruether, I G A; Gartzonika, C; Levidiotou-Stefanou, S; Krikelis, A; Markoulatos, P

    2010-12-01

    Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

  16. Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

    Science.gov (United States)

    Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

    2012-08-01

    Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or 15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

  17. Quantum reverse hypercontractivity

    Energy Technology Data Exchange (ETDEWEB)

    Cubitt, Toby [Department of Computer Science, University College London, London, United Kingdom and Centre for Quantum Information and Foundations, DAMTP, University of Cambridge, Cambridge (United Kingdom); Kastoryano, Michael [NBIA, Niels Bohr Institute, University of Copenhagen, 2100 Copenhagen (Denmark); Montanaro, Ashley [School of Mathematics, University of Bristol, Bristol (United Kingdom); Temme, Kristan [Institute for Quantum Information and Matter, California Institute of Technology, Pasadena, California 91125 (United States)

    2015-10-15

    We develop reverse versions of hypercontractive inequalities for quantum channels. By generalizing classical techniques, we prove a reverse hypercontractive inequality for tensor products of qubit depolarizing channels. We apply this to obtain a rapid mixing result for depolarizing noise applied to large subspaces and to prove bounds on a quantum generalization of non-interactive correlation distillation.

  18. Atrioventricular Pacemaker Lead Reversal

    Directory of Open Access Journals (Sweden)

    Mehmet K Aktas, MD

    2007-01-01

    Full Text Available During cardiac surgery temporary epicardial atrial and ventricular leads are placed in case cardiac pacing is required postoperatively. We present the first reported series of patients with reversal of atrioventricular electrodes in the temporary pacemaker without any consequent deleterious hemodynamic effect. We review the electrocardiographic findings and discuss the findings that lead to the discovery of atrioventricular lead reversal.

  19. 2-Hydroxypyridine-N-oxide (HOPO): Equivocal in the ames assay.

    Science.gov (United States)

    Dobo, Krista L; Cheung, Jennifer R; Gunther, William C; Kenyon, Michelle O

    2018-05-01

    2-Hydroxypyridine-N-oxide (HOPO) is a useful coupling reagent for synthesis of active pharmaceutical ingredients. It has been reported to be weakly mutagenic in the Ames assay (Ding W et al. []: J Chromatogr A 1386:47-52). According to the ICH M7 guidance (2014) regarding control of mutagenic impurities to limit potential carcinogenic risk, mutagens require control in drug substances such that exposure not exceeds the threshold of toxicological concern. Given the weak response observed in the Ames assay and the lack of any obvious structural features that could confer DNA reactivity we were interested to determine if the results were reproducible and investigate the role of potentially confounding experimental parameters. Specifically, Ames tests were conducted to assess the influence of compound purity, solvent choice, dose spacing, toxicity, type of S9 (aroclor vs phenobarbital/β-napthoflavone), and lot variability on the frequency of HOPO induced revertant colonies. Initial extensive testing using one lot of HOPO produced no evidence of mutagenic potential in the Ames assays. Subsequent studies with four additional lots produced conflicting results, with an ∼2.0-fold increase in revertant colonies observed. Given the rigor of the current investigation, lack of reproducibility between lots, and the weak increase in revertants, it is concluded that HOPO is equivocal in the bacterial reverse mutation assay. It is highly unlikely that HOPO poses a mutagenic risk in vivo; therefore, when it is used as a reagent in pharmaceutical synthesis, it should not be regarded as a mutagenic impurity, but rather a normal process related impurity. Environ. Mol. Mutagen. 59:312-321, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  20. Diploid yeast cells yield homozygous spontaneous mutations

    Science.gov (United States)

    Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

    1993-01-01

    A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

  1. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  2. Use of γ-ray-induced mutations in the genome era in rice

    International Nuclear Information System (INIS)

    Kusaba, Makoto

    2007-01-01

    Ionizing radiation has been used for inducing mutations and improving crops since the discovery by STADLER (1928) that X-rays could induce mutations in barley. At the end of 2004, the whole genome sequence of rice was determined (INTERNATIONAL RICE GENOME SEQUENCING PROJECT, 2005). What can γ-ray-induced mutations contribute now that this has been achieved? One answer could be the elucidation of the functions of the numerous genes revealed by the complete sequence of the rice genome. This includes identification of mutants through reverse genetics and the isolation of genes containing mutations through forward genetics using molecular markers and sequence information. Another answer could be mutation breeding using reverse genetics. But first we must know what kind of DNA lesions are caused by γ-rays. In this article, I describe the production of DNA lesions, and then discuss how γ-ray-induced mutations can contribute to the elucidation of gene function and to mutation breeding. (author)

  3. Mutation induction in Haemophilus influenzae by ICR-191. Pt. 1

    International Nuclear Information System (INIS)

    Perdue, S.W.; Kimball, R.F.; McGray, P.C.; Tennessee Univ., Oak Ridge

    1981-01-01

    The investigation of mutagenic mechanisms in Haemophilus influenzae has been confined until now to mutagens that normally produce mainly base pair substitutions. This paper describes the development of a system suitable for detecting frameshift mutations induced by ICR-191. The system involves reversions from thymidine dependence to thymidine independence. Evidence is presented from a comparison of the responses to ICR-191 and to N-methyl-N'-nitro-N-nitrosoguanidine that the system is specific for frameshift mutations. The genetic recombination involved in transformation leads to a marked increase in spontaneous reversion of the frameshift mutations but not of the base substitution mutations. Presumably, this is a consequence of mispairing, with consequent change in the number of bases, during the recombination. (orig.)

  4. Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR.

    Science.gov (United States)

    Decraene, Charles; Silveira, Amanda B; Bidard, François-Clément; Vallée, Audrey; Michel, Marc; Melaabi, Samia; Vincent-Salomon, Anne; Saliou, Adrien; Houy, Alexandre; Milder, Maud; Lantz, Olivier; Ychou, Marc; Denis, Marc G; Pierga, Jean-Yves; Stern, Marc-Henri; Proudhon, Charlotte

    2018-02-01

    Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan ® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy. © 2017 American Association for Clinical Chemistry.

  5. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  6. Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

    International Nuclear Information System (INIS)

    Kato, T.; Nakano, E.

    1981-01-01

    The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

  7. An algebra of reversible computation.

    Science.gov (United States)

    Wang, Yong

    2016-01-01

    We design an axiomatization for reversible computation called reversible ACP (RACP). It has four extendible modules: basic reversible processes algebra, algebra of reversible communicating processes, recursion and abstraction. Just like process algebra ACP in classical computing, RACP can be treated as an axiomatization foundation for reversible computation.

  8. Economic and agricultural impact of mutation breeding in fruit trees

    International Nuclear Information System (INIS)

    Spiegel Roy, P.

    1990-01-01

    Constraints of conventional cross breeding in fruit trees, wide market acceptance of definite cultivars, especially in apple, pear, citrus and wine grape, and the increased impact of natural mutants provide incentives for mutation breeding. Only few induced mutants in fruit trees have been commercialized and are being planted on a large scale. The main method followed in mutation breeding of tree fruit has been acute irradiation of meristematic multicellular buds but, Chimera formation and reversion present a serious problem. 87 refs, 4 tabs

  9. Solid phase assays

    International Nuclear Information System (INIS)

    Reese, M.G.; Johnson, L.R.; Ransom, D.K.

    1980-01-01

    In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

  10. N348I in the connection domain of HIV-1 reverse transcriptase confers zidovudine and nevirapine resistance.

    Directory of Open Access Journals (Sweden)

    Soo-Huey Yap

    2007-12-01

    Full Text Available The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1 reverse transcriptase (RT consists of DNA polymerase, connection, and ribonuclease H (RNase H domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 x 10(-12. N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs M41L and T215Y/F (p < 0.001, the lamivudine resistance mutations M184V/I (p < 0.001, and non-nucleoside RTI (NNRTI resistance mutations K103N and Y181C/I (p < 0.001. The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43-4.81. The appearance of N348I was associated with a significant increase in viral load (p < 0.001, which

  11. Deletion mutations of bacteriophage

    International Nuclear Information System (INIS)

    Ryo, Yeikou

    1975-01-01

    Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

  12. Sex reversal in vertebrates

    OpenAIRE

    2016-01-01

    This special topic issue of Sexual Development gives an overview of sex reversal in vertebrates, from fishes naturally changing their sex, to rodents escaping the mammalian SRY-determining system. It offers eight up-to-date reviews on specific subjects in sex reversal, considering fishes, amphibians, reptiles, birds, marsupials, and placental mammals, including humans. The broad scope of represented animals makes this ideal for students and researchers, especially those interested in the...

  13. Factor IX assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  14. Factor VIII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  15. Factor II assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003674.htm Factor II assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  16. Factor VII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003676.htm Factor VII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  17. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

    2013-01-01

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

  18. Phenylketonuria mutation analysis in Northern Ireland: A rapid stepwise approach

    Energy Technology Data Exchange (ETDEWEB)

    Zschocke, J.; Graham, C.A.; Nevin, N.C. [Queen`s Univ., Belfast (Australia)] [and others

    1995-12-01

    We present a multistep approach for the rapid analysis of phenylketonuria (PKU) mutations. In the first step, three common mutations and a polymorphic short tandem repeat (STR) system are rapidly analyzed with a fluorescent multiplex assay. In the second step, minihaplotypes combining STR and VNTR data are used to determine rare mutations likely to be present in an investigated patient, which are then confirmed by restriction enzyme analysis. The remaining mutations are analyzed with denaturant gradient-gel electrophoresis and sequencing. The first two steps together identify both mutations in 90%-95% of PKU patients, and results can be obtained within 2 d. We have investigated 121 Northern Irish families with hyperphenylalaninemia, including virtually all patients born since 1972, and have found 34 different mutations on 241 of the 242 mutant alleles. Three mutations (R408W, 165T, and F39L) account for 57.5% of mutations, while 14 mutations occur with a frequency of 1%-6%. The present analysis system is efficient and inexpensive and is particularly well suited to routine mutation analysis in a diagnostic setting. 19 refs., 5 tabs.

  19. On thermodynamic and microscopic reversibility

    International Nuclear Information System (INIS)

    Crooks, Gavin E

    2011-01-01

    The word 'reversible' has two (apparently) distinct applications in statistical thermodynamics. A thermodynamically reversible process indicates an experimental protocol for which the entropy change is zero, whereas the principle of microscopic reversibility asserts that the probability of any trajectory of a system through phase space equals that of the time reversed trajectory. However, these two terms are actually synonymous: a thermodynamically reversible process is microscopically reversible, and vice versa

  20. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  1. Application of statistical process control to qualitative molecular diagnostic assays

    LENUS (Irish Health Repository)

    O'Brien, Cathal P.

    2014-11-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  2. Application of statistical process control to qualitative molecular diagnostic assays.

    Science.gov (United States)

    O'Brien, Cathal P; Finn, Stephen P

    2014-01-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  3. Reversible Communicating Processes

    Directory of Open Access Journals (Sweden)

    Geoffrey Brown

    2016-02-01

    Full Text Available Reversible distributed programs have the ability to abort unproductive computation paths and backtrack, while unwinding communication that occurred in the aborted paths. While it is natural to assume that reversibility implies full state recovery (as with traditional roll-back recovery protocols, an interesting alternative is to separate backtracking from local state recovery. For example, such a model could be used to create complex transactions out of nested compensable transactions where a programmer-supplied compensation defines the work required to "unwind" a transaction. Reversible distributed computing has received considerable theoretical attention, but little reduction to practice; the few published implementations of languages supporting reversibility depend upon a high degree of central control. The objective of this paper is to demonstrate that a practical reversible distributed language can be efficiently implemented in a fully distributed manner. We discuss such a language, supporting CSP-style synchronous communication, embedded in Scala. While this language provided the motivation for the work described in this paper, our focus is upon the distributed implementation. In particular, we demonstrate that a "high-level" semantic model can be implemented using a simple point-to-point protocol.

  4. Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the SPIRIT Study.

    Science.gov (United States)

    Porter, Danielle P; Toma, Jonathan; Tan, Yuping; Solberg, Owen; Cai, Suqin; Kulkarni, Rima; Andreatta, Kristen; Lie, Yolanda; Chuck, Susan K; Palella, Frank; Miller, Michael D; White, Kirsten L

    2016-02-01

    Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48. Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed. Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance

  5. Assay method and compositions

    International Nuclear Information System (INIS)

    1977-01-01

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  6. Hidden Randomness between Fitness Landscapes Limits Reverse Evolution

    Science.gov (United States)

    Tan, Longzhi; Serene, Stephen; Xiao Chao, Hui; Gore, Jeff

    2012-02-01

    Natural populations must constantly adapt to the ever-changing environment. A fundamental question in evolutionary biology is whether adaptations can be reversed by returning the population to its ancestral environment. Traditionally, reverse evolution is defined as restoring an ancestral phenotype (physical characteristics such as body size), and the classic Dollo's Law has hypothesized the impossibility of reversing complex adaptations. However, this ``law'' remains ambiguous unless reverse evolution can be studied at the level of genotypes (the underlying genome sequence). We measured the fitness landscapes of a bacterial antibiotic-resistance gene and analyzed the reversibility of evolution as a global, statistical feature of the landscapes. In both experiments and simulations, we find that an adaptation's reversibility declines as the number of mutations it involves increases, suggesting a probabilistic form of Dollo's Law at the molecular level. We also show computationally that slowly switching between environments facilitates reverse evolution in small populations, where clonal interference is negligible or moderate. This is an analogy to thermodynamics, where the reversibility of a physical process is maximized when conditions are modified infinitely slowly.

  7. Economic impact of reversion

    International Nuclear Information System (INIS)

    2005-01-01

    Estimations of the Norwegian hydropower production and various reversion models' market value have been made. The value of the Norwegian hydropower production until 01.01.2007 is estimated to about Nok 289 billion after taxes, or about 2,42 Nok/kWh medium production, given an expected future electricity price of around 0,25 Nok/kWh and a discount rate at 6,5 percent in nominal terms after taxes. The estimate is slightly above the level of prices for Norwegian hydropower plants in the last 8-10 years. The value of reversion in private plants which today have a limited licence time is estimated to Nok 5,5 billion. The value of reversion in public-owned Norwegian hydropower plants are about Nok 21 billion with a 60 year licence period from 01.01.2007, and about 12 billion for 75 years (ml)

  8. Better plants through mutations

    International Nuclear Information System (INIS)

    1988-01-01

    This is a public relations film describing problems associated with the genetic improvement of crop plants through induced mutations. Mutations are the ultimate source of genetic variation in plants. Mutation induction is now established as a practical tool in plant breeding. The Joint FAO/IAEA Division and the IAEA's laboratory at Seibersdorf have supported research and practical implementation of mutation breeding of both seed propagated and vegetatively propagated plants. Plant biotechnology based on in vitro culture and recombinant DNA technology will make a further significant contribution to plant breeding

  9. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  10. Rover waste assay system

    International Nuclear Information System (INIS)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-01-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  11. Radioreceptor assay for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

    1975-04-01

    Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

  12. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  13. Reversible deep disposal

    International Nuclear Information System (INIS)

    2009-10-01

    This presentation, given by the national agency of radioactive waste management (ANDRA) at the meeting of October 8, 2009 of the high committee for the nuclear safety transparency and information (HCTISN), describes the concept of deep reversible disposal for high level/long living radioactive wastes, as considered by the ANDRA in the framework of the program law of June 28, 2006 about the sustainable management of radioactive materials and wastes. The document presents the social and political reasons of reversibility, the technical means considered (containers, disposal cavities, monitoring system, test facilities and industrial prototypes), the decisional process (progressive development and blocked off of the facility, public information and debate). (J.S.)

  14. Common Variable Immunodeficiency Caused by FANC Mutations.

    Science.gov (United States)

    Sekinaka, Yujin; Mitsuiki, Noriko; Imai, Kohsuke; Yabe, Miharu; Yabe, Hiromasa; Mitsui-Sekinaka, Kanako; Honma, Kenichi; Takagi, Masatoshi; Arai, Ayako; Yoshida, Kenichi; Okuno, Yusuke; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Muramatsu, Hideki; Kojima, Seiji; Hira, Asuka; Takata, Minoru; Ohara, Osamu; Ogawa, Seishi; Morio, Tomohiro; Nonoyama, Shigeaki

    2017-07-01

    Common variable immunodeficiency (CVID) is the most common adult-onset primary antibody deficiency disease due to various causative genes. Several genes, which are known to be the cause of different diseases, have recently been reported as the cause of CVID in patients by performing whole exome sequencing (WES) analysis. Here, we found FANC gene mutations as a cause of adult-onset CVID in two patients. B cells were absent and CD4 + T cells were skewed toward CD45RO + memory T cells. T-cell receptor excision circles (TRECs) and signal joint kappa-deleting recombination excision circles (sjKRECs) were undetectable in both patients. Both patients had no anemia, neutropenia, or thrombocytopenia. Using WES, we identified compound heterozygous mutations of FANCE in one patient and homozygous mutation of FANCA in another patient. The impaired function of FANC protein complex was confirmed by a monoubiquitination assay and by chromosome fragility test. We then performed several immunological evaluations including quantitative lymphocyte analysis and TRECs/sjKRECs analysis for 32 individuals with Fanconi anemia (FA). In total, 22 FA patients (68.8%) were found to have immunological abnormalities, suggesting that such immunological findings may be common in FA patients. These data indicate that FANC mutations are involved in impaired lymphogenesis probably by the accumulation of DNA replication stress, leading to CVID. It is important to diagnose FA because it drastically changes clinical management. We propose that FANC mutations can cause isolated immunodeficiency in addition to bone marrow failure and malignancy.

  15. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  16. Assays for calcitonin receptors

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

  17. Determination of somatic mutations in human erythrocytes by cytometry

    International Nuclear Information System (INIS)

    Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

    1985-01-01

    Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab

  18. Determination of somatic mutations in human erythrocytes by cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

    1985-06-21

    Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab.

  19. Assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

    International Nuclear Information System (INIS)

    Mehdi, S.; Wiseman, J.S.

    1989-01-01

    An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection

  20. Detection of hepatitis C virus RNA using reverse transcription PCR

    International Nuclear Information System (INIS)

    Yap, S.F.

    1998-01-01

    Detection of the viral genome (HCV RNA) is by a combination of cDNA synthesis and PCR followed by gel analysis and/or hybridization assay. In principle, cDNA is synthesized using the viral RNA as template and the enzyme, reverse transcriptase. The cDNA is then amplified by PCR and the product detected. Agarose gel electrophoresis provides a rapid and simple detection method; however, it is non-quantitative. The assay protocol described in this paper is adapted from that published by Chan et al. Comments on various aspects of the assay are based on experience with the method in our laboratory

  1. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  2. Thermosensory reversal effect quantified

    NARCIS (Netherlands)

    Bergmann Tiest, W.M.; Kappers, A.M.L.

    2008-01-01

    At room temperature, some materials feel colder than others due to differences in thermal conductivity, heat capacity and geometry. When the ambient temperature is well above skin temperature, the roles of 'cold' and 'warm' materials are reversed. In this paper, this effect is quantified by

  3. Thermosensory reversal effect quantified

    NARCIS (Netherlands)

    Bergmann Tiest, W.M.; Kappers, A.M.L.

    2008-01-01

    At room temperature, some materials feel colder than others due to differences in thermal conductivity, heat capacity and geometry. When the ambient temperature is well above skin temperature, the roles of ‘cold’ and ‘warm’ materials are reversed. In this paper, this effect is quantified by

  4. Time reversal communication system

    Science.gov (United States)

    Candy, James V.; Meyer, Alan W.

    2008-12-02

    A system of transmitting a signal through a channel medium comprises digitizing the signal, time-reversing the digitized signal, and transmitting the signal through the channel medium. The channel medium may be air, earth, water, tissue, metal, and/or non-metal.

  5. Engineering Encounters: Reverse Engineering

    Science.gov (United States)

    McGowan, Veronica Cassone; Ventura, Marcia; Bell, Philip

    2017-01-01

    This column presents ideas and techniques to enhance your science teaching. This month's issue shares information on how students' everyday experiences can support science learning through engineering design. In this article, the authors outline a reverse-engineering model of instruction and describe one example of how it looked in our fifth-grade…

  6. Sex Reversal in Birds.

    Science.gov (United States)

    Major, Andrew T; Smith, Craig A

    2016-01-01

    Sexual differentiation in birds is controlled genetically as in mammals, although the sex chromosomes are different. Males have a ZZ sex chromosome constitution, while females are ZW. Gene(s) on the sex chromosomes must initiate gonadal sex differentiation during embryonic life, inducing paired testes in ZZ individuals and unilateral ovaries in ZW individuals. The traditional view of avian sexual differentiation aligns with that expounded for other vertebrates; upon sexual differentiation, the gonads secrete sex steroid hormones that masculinise or feminise the rest of the body. However, recent studies on naturally occurring or experimentally induced avian sex reversal suggest a significant role for direct genetic factors, in addition to sex hormones, in regulating sexual differentiation of the soma in birds. This review will provide an overview of sex determination in birds and both naturally and experimentally induced sex reversal, with emphasis on the key role of oestrogen. We then consider how recent studies on sex reversal and gynandromorphic birds (half male:half female) are shaping our understanding of sexual differentiation in avians and in vertebrates more broadly. Current evidence shows that sexual differentiation in birds is a mix of direct genetic and hormonal mechanisms. Perturbation of either of these components may lead to sex reversal. © 2016 S. Karger AG, Basel.

  7. Elastomers with Reversible Nanoporosity

    DEFF Research Database (Denmark)

    Szewczykowski, Piotr Przemyslaw; Andersen, K.; Schulte, Lars

    2009-01-01

    nanostructure and displays liquid-filled cavities. Upon several cycles of swelling and drying the cavities open and close in a reversible fashion. When exposed to a nonsolvent, the material remains collapsed. This discriminating behavior of liquid-material interaction holds potential for the use...

  8. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  9. Microchemiluminescent assay system

    Energy Technology Data Exchange (ETDEWEB)

    Kiel, J.L.

    1986-04-09

    The patent concerns a microchemiluminescent assay system, which can be used to detect ionizing radiation, heat or specific substances. The method involves the use of a complex formed from serum albumin and a luminescer which, in the presence of ionizing radiation (heat, or a specific analyte), will emit light in an amount proportional to the amount of radiation, etc. (U.K.).

  10. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  11. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  12. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a...

  13. Evaluation of a multiple-cycle, recombinant virus, growth competition assay that uses flow cytometry to measure replication efficiency of human immunodeficiency virus type 1 in cell culture.

    Science.gov (United States)

    Dykes, Carrie; Wang, Jiong; Jin, Xia; Planelles, Vicente; An, Dong Sung; Tallo, Amanda; Huang, Yangxin; Wu, Hulin; Demeter, Lisa M

    2006-06-01

    Human immunodeficiency virus type 1 (HIV-1) replication efficiency or fitness, as measured in cell culture, has been postulated to correlate with clinical outcome of HIV infection, although this is still controversial. One limitation is the lack of high-throughput assays that can measure replication efficiency over multiple rounds of replication. We have developed a multiple-cycle growth competition assay to measure HIV-1 replication efficiency that uses flow cytometry to determine the relative proportions of test and reference viruses, each of which expresses a different reporter gene in place of nef. The reporter genes are expressed on the surface of infected cells and are detected by commercially available fluorescence-labeled antibodies. This method is less labor-intensive than those that require isolation and amplification of nucleic acids. The two reporter gene products are detected with similar specificity and sensitivity, and the proportion of infected cells in culture correlates with the amount of viral p24 antigen produced in the culture supernatant. HIV replication efficiencies of six different drug-resistant site-directed mutants were reproducibly quantified and were similar to those obtained with a growth competition assay in which the relative proportion of each variant was measured by sequence analysis, indicating that recombination between the pol and reporter genes was negligible. This assay also reproducibly quantified the relative fitness conferred by protease and reverse transcriptase sequences containing multiple drug resistance mutations, amplified from patient plasma. This flow cytometry-based growth competition assay offers advantages over current assays for HIV replication efficiency and should prove useful for the evaluation of patient samples in clinical trials.

  14. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

    Directory of Open Access Journals (Sweden)

    Viteri Santiago

    2010-12-01

    Full Text Available Abstract Background Immunohistochemistry (IHC with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93% patients with exon 21 EGFR mutations (all with L858R but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients.

  15. Determination of Letrozole in Tablet Formulations by Reversed ...

    African Journals Online (AJOL)

    Determination of Letrozole in Tablet Formulations by Reversed Phase High Performance Liquid Chromatography. ... The assay values for the two branded letrozole tablets tested were 99.2 and 100.2 %, respectively with % relative standard deviation (RSD) of 0.781 and 0.568, respectively. The bench top stability data of the ...

  16. EFFECT OF THE ANTIMUTAGENS VANILLIN AND CINNAMALDEHYDE ON THE SPONTANEOUS MUTATION SPECTRA OF SALMONELLA TA104

    Science.gov (United States)

    Effect of the Antimutagens Vanillin and Cinnamaldehyde on the / Spontaneous Mutation Spectra of Salmonella TAlO4 Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that, when added to assay plates, reduced the spontaneous mutant frequency in Salmonella typhi...

  17. A Novel Method to Screen for Dominant Negative ATM Mutations in Familial Breast Cancer

    National Research Council Canada - National Science Library

    Khanna, Kum K; Chenevix-Trench, Georgia; Grimmond, Sean

    2005-01-01

    The aim of this proposal is to identify families carrying potentially pathogenic A TM mutations by assaying for ATM kinase activity in cell lines derived from individuals with multiple cases of breast...

  18. Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting.

    Directory of Open Access Journals (Sweden)

    Steven Y C Tong

    Full Text Available We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR, high-resolution melting (HRM assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T was 32.98 would have an H275Y assay C(T>30. Analysis of the TaqMan C(T values for 609 consecutive clinical samples predicted that 207 (34% of the samples would result in an HRM assay C(T>30 and therefore not be amenable to the HRM assay.The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m against C(T. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.

  19. Mutation and premating isolation

    Science.gov (United States)

    Woodruff, R. C.; Thompson, J. N. Jr

    2002-01-01

    While premating isolation might be traceable to different genetic mechanisms in different species, evidence supports the idea that as few as one or two genes may often be sufficient to initiate isolation. Thus, new mutation can theoretically play a key role in the process. But it has long been thought that a new isolation mutation would fail, because there would be no other individuals for the isolation-mutation-carrier to mate with. We now realize that premeiotic mutations are very common and will yield a cluster of progeny carrying the same new mutant allele. In this paper, we discuss the evidence for genetically simple premating isolation barriers and the role that clusters of an isolation mutation may play in initiating allopatric, and even sympatric, species divisions.

  20. Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.

    Directory of Open Access Journals (Sweden)

    Surinder M Singh

    Full Text Available Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM. However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD. We created and expressed the wild-type (WT N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts, and unfolds faster than the WT (stopped-flow kinetics. Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments. These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.

  1. Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

    Science.gov (United States)

    Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis

    2013-12-23

    At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

  2. SPOP Mutations in Prostate Cancer across Demographically Diverse Patient Cohorts

    Directory of Open Access Journals (Sweden)

    Mirjam Blattner

    2014-01-01

    Full Text Available BACKGROUND: Recurrent mutations in the Speckle-Type POZ Protein (SPOP gene occur in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unknown. OBJECTIVE: To investigate SPOP mutations across diverse cohorts and validate a series of assays employing high-resolution melting (HRM analysis and Sanger sequencing for mutational analysis of formalin-fixed paraffin-embedded material. DESIGN, SETTING, AND PARTICIPANTS: 720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both prostate-specific antigen-screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, and CHD1 deletion as well as clinical and pathologic features. RESULTS AND LIMITATIONS: Overall frequency of SPOP mutations was 8.1% (4.6% to 14.4%, SPOP mutation was inversely associated with ERG rearrangement (P < .01, and SPOP mutant (SPOPmut cancers had higher rates of CHD1 deletions (P < .01. There were no significant differences in biochemical recurrence in SPOPmut cancers. Limitations of this study include missing mutational data due to sample quality and lack of power to identify a difference in clinical outcomes. CONCLUSION: SPOP is mutated in 4.6% to 14.4% of patients with prostate cancer across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical, or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer.

  3. Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Flávia S Donaires

    Full Text Available Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3% in patients as compared to controls (p = 0.02. Three of the four variants (T726M, A1062T, and V1090M were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis. A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

  4. Mutagenicity of irradiated food in the host mediated assay system

    International Nuclear Information System (INIS)

    Johnston-Arthur, T.; Turanitz, K.; Hruby, R.; Stehlik, G.; Brena-Valle, M.

    1975-01-01

    Groups of Swiss albino mice (SPF) fed with normal and gamma-irradiated food at doses of 0.75, 1.5 and 3.0 Mrad, were injected intraperitoneally with SALMONELLA TYPHIMURIUM TA 1530 for the host mediated assay test of mutagenesis. The mutation frequency was calculated in terms of the number of mutant colonies per unit number of surviving cells. The results indicate that there is a significant increase in mutation frequency induced by the 3 Mrad sterilized food. No difference was observed in the 0.75 Mrad dose when compared with the control

  5. Mutations and epimutations in the origin of cancer

    Energy Technology Data Exchange (ETDEWEB)

    Peltomaeki, Paeivi, E-mail: Paivi.Peltomaki@Helsinki.Fi

    2012-02-15

    Cancer is traditionally viewed as a disease of abnormal cell proliferation controlled by a series of mutations. Mutations typically affect oncogenes or tumor suppressor genes thereby conferring growth advantage. Genomic instability facilitates mutation accumulation. Recent findings demonstrate that activation of oncogenes and inactivation of tumor suppressor genes, as well as genomic instability, can be achieved by epigenetic mechanisms as well. Unlike genetic mutations, epimutations do not change the base sequence of DNA and are potentially reversible. Similar to genetic mutations, epimutations are associated with specific patterns of gene expression that are heritable through cell divisions. Knudson's hypothesis postulates that inactivation of tumor suppressor genes requires two hits, with the first hit occurring either in somatic cells (sporadic cancer) or in the germline (hereditary cancer) and the second one always being somatic. Studies on hereditary and sporadic forms of colorectal carcinoma have made it evident that, apart from genetic mutations, epimutations may serve as either hit or both. Furthermore, recent next-generation sequencing studies show that epigenetic genes, such as those encoding histone modifying enzymes and subunits for chromatin remodeling systems, are themselves frequent targets of somatic mutations in cancer and can act like tumor suppressor genes or oncogenes. This review discusses genetic vs. epigenetic origin of cancer, including cancer susceptibility, in light of recent discoveries. Situations in which mutations and epimutations occur to serve analogous purposes are highlighted.

  6. Reversed field pinch diagnostics

    International Nuclear Information System (INIS)

    Weber, P.G.

    1986-01-01

    The Reversed Field Pinch (RFP) is a toroidal, axisymmetric magnetic confinement configuration characterized by a magnetic field configuration in which the toroidal magnetic field is of similar strength to the poloidal field, and is reversed at the edge compared to the center. The RFP routinely operates at high beta, and is a strong candidate for a compact fusion device. Relevant attributes of the configuration will be presented, together with an overview of present and planned experiments and their diagnostics. RFP diagnostics are in many ways similar to those of other magnetic confinement devices (such as tokamaks); these lectures will point out pertinent differences, and will present some diagnostics which provide special insights into unique attributes of the RFP

  7. Posterior Reversible Encephalopathy (PRES)

    International Nuclear Information System (INIS)

    Moron E, Fanny E; Diaz Marchan, Pedro

    2005-01-01

    The Posterior Reversible Encephalopathy Syndrome (PRES) is a clinical Syndrome composed of cephalea, alteration in vision and convulsions, usually observed in patients with sudden elevation of arterial pressure. The imagenologic evidence shows reversible vasogenic brain edema without stroke. Its location is predominantly posterior; it affects the cortex and the subcortical white matter of the occipital, parietal and temporal lobes. The treatment with antihypertensive drugs and the removing of immunosupressor medication are generally associated with complete neurological recovery; this is reflected also in the images which return to their basal condition. The untreated hypertension, on the other side, can result in a progressive defect of the autoregulation system of the central nervous system with cerebral hemorrhage, irreversible brain stroke, coma and death

  8. Radioreceptor assay for oxyphenonium

    International Nuclear Information System (INIS)

    Ensing, K.; Zeeuw, R.A. de

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

  9. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  10. Lack of Mutation-histopathology Correlation in a Patient with Proteus Syndrome

    OpenAIRE

    Doucet, Meggie E.; Bloomhardt, Hadley M.; Moroz, Krzysztof; Lindhurst, Marjorie J.; Biesecker, Leslie G.

    2016-01-01

    Proteus syndrome (PS) is characterized by progressive, disproportionate, segmental overgrowth and tumor susceptibility caused by a somatic mosaic AKT1 activating mutation. Each individual has unique manifestations making this disorder extremely heterogeneous. We correlated three variables in 38 tissue samples from a patient who died with PS: the gross affection status, the microscopic affection status, and the mutation level. The AKT1 mutation was measured using a PCR-based RFLP assay. Thirte...

  11. Positioning paper on reversibility

    International Nuclear Information System (INIS)

    2016-01-01

    After having recalled the legal framework adopted in 2006 for the deep geological storage of radioactive wastes, and briefly introduced the concept of reversibility, this publication presents the principle of geological storage, presents high and medium level and long life wastes, highlights the ethical necessity to deal with these radioactive wastes, outlines that geological storage is the generally admitted and adopted solution at the international level, and presents additional means implemented for radioactive waste management. It presents the Cigeo project as the technical answer to the issue of radioactive waste storage, describes the Cigeo development process, its current status and its development planning, and justifies the choice of this technical solution, notably from an ethical point of view. It addresses the issue of reversibility and proposes an overview of the various tools and means which aim at guaranteeing this reversibility. Appendices propose figures illustrating the Cigeo project and its development process, and a rather detailed Power Point presentation of the project by the ANDRA (history, object, planning, installations, and so on)

  12. Induction of spontaneous and UV-induced mutations during commitment to meiosis in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Machida, I.; Nakai, S.

    1980-01-01

    Inductions of reversions of nonsense, missense and frameshift-type mutations were investigated in a diploid cell population of Saccharomyces cerevisiae during commitment to meiosis, by using the medium-transfer technique from sporulation medium to vegetative medium. The yields of spontaneous reverse mutations obtained from the cells that were committed to different stages during meiosis were rather constant irrespective of the alleles tested, although the yields of both intergenic and intragenic recombinations markedly increased. The susceptibilities to UV-induced reverse mutations examined during commitment to meiosis were not changed appreciably. It is concluded that induction of base-change-type mutations in meiosis is not essentially different from that in mitosis. (orig.)

  13. Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

    Directory of Open Access Journals (Sweden)

    Lena Poulsen

    Full Text Available BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%. Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

  14. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

    Directory of Open Access Journals (Sweden)

    Swati Tomar

    Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

  15. Screening for duplications, deletions and a common intronic mutation detects 35% of second mutations in patients with USH2A monoallelic mutations on Sanger sequencing.

    Science.gov (United States)

    Steele-Stallard, Heather B; Le Quesne Stabej, Polona; Lenassi, Eva; Luxon, Linda M; Claustres, Mireille; Roux, Anne-Francoise; Webster, Andrew R; Bitner-Glindzicz, Maria

    2013-08-08

    Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G. Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints. Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome. We found that 8 of

  16. Status of time reversal invariance

    International Nuclear Information System (INIS)

    Henley, E.M.

    1989-01-01

    Time Reversal Invariance is introduced, and theories for its violation are reviewed. The present experimental and theoretical status of Time Reversal Invariance and tests thereof will be presented. Possible future tests will be discussed. 30 refs., 2 figs., 1 tab

  17. Introduction to time reversal theory

    International Nuclear Information System (INIS)

    Henley, E.M.

    1987-01-01

    Theory and reaction mechanisms relevant to time reversal invariance are reviewed. Consequences of time reversal invariance are presented under the headings of CP tests, electromagnetic moments, weak emissions or absorptions, and scattering reactions. 8 refs., 4 figs

  18. Genetic Mutations in Cancer

    Science.gov (United States)

    Many different types of genetic mutations are found in cancer cells. This infographic outlines certain types of alterations that are present in cancer, such as missense, nonsense, frameshift, and chromosome rearrangements.

  19. AIP mutations and gigantism.

    Science.gov (United States)

    Rostomyan, Liliya; Potorac, Iulia; Beckers, Pablo; Daly, Adrian F; Beckers, Albert

    2017-06-01

    AIP mutations are rare in sporadic acromegaly but they are seen at a higher frequency among certain specific populations of pituitary adenoma patients (pituitary gigantism cases, familial isolated pituitary adenoma (FIPA) kindreds, and patients with macroadenomas who are diagnosed ≤30 years). AIP mutations are most prevalent in patients with pituitary gigantism (29% of this group were found to have mutations in AIP gene). These data support targeted genetic screening for AIP mutations/deletions in these groups of pituitary adenoma patients. Earlier diagnosis of AIP-related acromegaly-gigantism cases enables timely clinical evaluation and treatment, thereby improving outcomes in terms of excessive linear growth and acromegaly comorbidities. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. The Causes of Preference Reversal.

    OpenAIRE

    Tversky, Amos; Slovic, Paul; Kahneman, Daniel

    1990-01-01

    Observed preference reversal cannot be adequately explained by violations of independence, the reduction axiom, or transitivity. The primary cause of preference reversal is the failure of procedure invariance, especially the overpricing of low-probability, high-payoff bets. This result violates regret theory and generalized (nonindependent) utility models. Preference reversal and a new reversal involving time preferences are explained by scale compatibility, which implies that payoffs are wei...

  1. Geomagnetic Field During a Reversal

    Science.gov (United States)

    Heirtzler, J. R.

    2003-01-01

    It has frequently been suggested that only the geomagnetic dipole, rather than higher order poles, reverse during a geomagnetic field reversal. Under this assumption the geomagnetic field strength has been calculated for the surface of the Earth for various steps of the reversal process. Even without an eminent a reversal of the field, extrapolation of the present secular change (although problematic) shows that the field strength may become zero in some geographic areas within a few hundred years.

  2. A Study on Reverse Logistics

    OpenAIRE

    Reddy, Dhananjaya

    2011-01-01

    In the competitive world of manufacturing, companies are often searching for new ways to improve their process, customer satisfaction and stay ahead in the game with their competitors. Reverse logistics has been considered a strategy to bring these things to life for the past decade or so. This thesis work tries to shed some light on the basics of reverse logistics and how reverse logistics can be used as a management strategy. This paper points out the fundamentals of reverse logistics and l...

  3. The neural basis of reversal learning: An updated perspective

    Science.gov (United States)

    Izquierdo, Alicia; Brigman, Jonathan L.; Radke, Anna K.; Rudebeck, Peter H.; Holmes, Andrew

    2016-01-01

    Reversal learning paradigms are among the most widely used tests of cognitive flexibility and have been used as assays, across species, for altered cognitive processes in a host of neuropsychiatric conditions. Based on recent studies in humans, non-human primates, and rodents, the notion that reversal learning tasks primarily measure response inhibition, has been revised. In this review, we describe how cognitive flexibility is measured by reversal learning and discuss new definitions of the construct validity of the task that are serving as an heuristic to guide future research in this field. We also provide an update on the available evidence implicating certain cortical and subcortical brain regions in the mediation of reversal learning, and an overview of the principle neurotransmitter systems involved. PMID:26979052

  4. Mutation breeding in peas

    Energy Technology Data Exchange (ETDEWEB)

    Jaranowski, J [Institute of Genetics and Plant Breeding, Academy of Agriculture, Poznan (Poland); Micke, A [Joint FAO/IAEA Division of Isotope and Radiation Applications of Atomic Energy for Food and Agricultural Development, International Atomic Energy Agency, Vienna (Austria)

    1985-02-01

    The pea as an ancient crop plant still today has wide uses and is an import source of food protein. It is also an important object for genetic studies and as such has been widely used in mutation induction experiments. However, in comparison with cereals this ancient crop plant (like several other grain legumes) has gained relatively little from advances in breeding. The review focuses on the prospects of genetic improvement of pea by induced mutations, discusses principles and gives methodological information. (author)

  5. Mutation breeding in peas

    International Nuclear Information System (INIS)

    Jaranowski, J.; Micke, A.

    1985-01-01

    The pea as an ancient crop plant still today has wide uses and is an import source of food protein. It is also an important object for genetic studies and as such has been widely used in mutation induction experiments. However, in comparison with cereals this ancient crop plant (like several other grain legumes) has gained relatively little from advances in breeding. The review focuses on the prospects of genetic improvement of pea by induced mutations, discusses principles and gives methodological information. (author)

  6. Geomagnetic Reversals during the Phanerozoic.

    Science.gov (United States)

    McElhinny, M W

    1971-04-09

    An antalysis of worldwide paleomagnetic measurements suggests a periodicity of 350 x 10(6) years in the polarity of the geomagnetic field. During the Mesozoic it is predominantly normal, whereas during the Upper Paleozoic it is predominantly reversed. Although geomagnetic reversals occur at different rates throughout the Phanerozoic, there appeaars to be no clear correlation between biological evolutionary rates and reversal frequency.

  7. Radiorespirometic assay device

    International Nuclear Information System (INIS)

    Levin, G.V.; Straat, P.A.

    1981-01-01

    A radiorespirometic assay device is described in which the presence of microorganisms in a sample is determined by placing the sample in contact with a metabolisable radioactive labelled substrate, collecting any gas evolved, exposing a photosensitive material to the gas and determining if a spot is produced on the material. A spot indicates the presence of radioactivity showing that the substrate has been metabolized by a microorganism. Bacteria may be detected in body fluids, hospital operating rooms, water, food, cosmetics and drugs. (U.K.)

  8. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  9. A BRCA2 mutation incorrectly mapped in the original BRCA2 reference sequence, is a common West Danish founder mutation disrupting mRNA splicing

    DEFF Research Database (Denmark)

    Thomassen, Mads; Pedersen, Inge Søkilde; Vogel, Ida

    2011-01-01

    Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose carriers to breast and ovarian cancer. The authors have identified a mutation in BRCA2, 7845+1G>A (c.7617+1G>A), not previously regarded as deleterious because of incorrect mapping of the splice junction in the originally...... published genomic reference sequence. This reference sequence is generally used in many laboratories and it maps the mutation 16 base pairs inside intron 15. However, according to the recent reference sequences the mutation is located in the consensus donor splice sequence. By reverse transcriptase analysis......, loss of exon 15 in the final transcript interrupting the open reading frame was demonstrated. Furthermore, the mutation segregates with a cancer phenotype in 18 Danish families. By genetic analysis of more than 3,500 Danish breast/ovarian cancer risk families, the mutation was identified as the most...

  10. Reversal Strategies for NOACs

    DEFF Research Database (Denmark)

    Husted, Steen; Verheugt, Freek; Comuth, Willemijn

    2015-01-01

    , coagulation factor concentrates or NOAC-specific antidotes could be used. Coagulation factor concentrates can be used in patients with haemophilia and to reverse the effect of VKAs but, in NOAC-treated patients, results are inconsistent and these agents could potentially have pro-thrombotic effects. Specific...... antidotes for NOACs are expected to be on the market soon. Phase III clinical trials with a humanized antibody fragment directed against dabigatran (idarucizumab) and recombinant, modified factor Xa (andexanet alfa) are ongoing. A molecule (aripazine) with broad activity against various anticoagulants...

  11. Reversible brazing process

    Science.gov (United States)

    Pierce, Jim D.; Stephens, John J.; Walker, Charles A.

    1999-01-01

    A method of reversibly brazing surfaces together. An interface is affixed to each surface. The interfaces can be affixed by processes such as mechanical joining, welding, or brazing. The two interfaces are then brazed together using a brazing process that does not defeat the surface to interface joint. Interfaces of materials such as Ni-200 can be affixed to metallic surfaces by welding or by brazing with a first braze alloy. The Ni-200 interfaces can then be brazed together using a second braze alloy. The second braze alloy can be chosen so that it minimally alters the properties of the interfaces to allow multiple braze, heat and disassemble, rebraze cycles.

  12. RAS - Screens & Assays - Drug Discovery

    Science.gov (United States)

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  13. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  14. Improving shuffler assay accuracy

    International Nuclear Information System (INIS)

    Rinard, P.M.

    1995-01-01

    Drums of uranium waste should be disposed of in an economical and environmentally sound manner. The most accurate possible assays of the uranium masses in the drums are required for proper disposal. The accuracies of assays from a shuffler are affected by the type of matrix material in the drums. Non-hydrogenous matrices have little effect on neutron transport and accuracies are very good. If self-shielding is known to be a minor problem, good accuracies are also obtained with hydrogenous matrices when a polyethylene sleeve is placed around the drums. But for those cases where self-shielding may be a problem, matrices are hydrogenous, and uranium distributions are non-uniform throughout the drums, the accuracies are degraded. They can be greatly improved by determining the distributions of the uranium and then applying correction factors based on the distributions. This paper describes a technique for determining uranium distributions by using the neutron count rates in detector banks around the waste drum and solving a set of overdetermined linear equations. Other approaches were studied to determine the distributions and are described briefly. Implementation of this correction is anticipated on an existing shuffler next year

  15. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  16. Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays

    DEFF Research Database (Denmark)

    Poulsen, Lena; Søe, Martin Jensen; Moller, Lisbeth Birk

    2011-01-01

    Background: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions...

  17. An evolutionary reduction principle for mutation rates at multiple Loci.

    Science.gov (United States)

    Altenberg, Lee

    2011-06-01

    A model of mutation rate evolution for multiple loci under arbitrary selection is analyzed. Results are obtained using techniques from Karlin (Evolutionary Biology, vol. 14, pp. 61-204, 1982) that overcome the weak selection constraints needed for tractability in prior studies of multilocus event models.A multivariate form of the reduction principle is found: reduction results at individual loci combine topologically to produce a surface of mutation rate alterations that are neutral for a new modifier allele. New mutation rates survive if and only if they fall below this surface-a generalization of the hyperplane found by Zhivotovsky et al. (Proc. Natl. Acad. Sci. USA 91, 1079-1083, 1994) for a multilocus recombination modifier. Increases in mutation rates at some loci may evolve if compensated for by decreases at other loci. The strength of selection on the modifier scales in proportion to the number of germline cell divisions, and increases with the number of loci affected. Loci that do not make a difference to marginal fitnesses at equilibrium are not subject to the reduction principle, and under fine tuning of mutation rates would be expected to have higher mutation rates than loci in mutation-selection balance.Other results include the nonexistence of 'viability analogous, Hardy-Weinberg' modifier polymorphisms under multiplicative mutation, and the sufficiency of average transmission rates to encapsulate the effect of modifier polymorphisms on the transmission of loci under selection. A conjecture is offered regarding situations, like recombination in the presence of mutation, that exhibit departures from the reduction principle. Constraints for tractability are: tight linkage of all loci, initial fixation at the modifier locus, and mutation distributions comprising transition probabilities of reversible Markov chains.

  18. Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rattray, Alison; Santoyo, Gustavo; Shafer, Brenda; Strathern, Jeffrey N

    2015-01-01

    Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

  19. Novel mutations underlying argininosuccinic aciduria in Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Rashed Mohamed S

    2010-03-01

    Full Text Available Abstract Background Argininosuccinic aciduria (ASAuria is an autosomal recessive disorder of the urea cycle relatively common in Saudi Arabia as a consequence of extensive consanguinity. It is the most common urea cycle disorder identified in the Saudi population, which therefore prioritizes the need to delineate the underlying molecular defects leading to disease. Findings We utilized Whole Genome Amplification (WGA, PCR and direct sequencing to identify mutations underlying ASAuria cases diagnosed by our institution. A missense mutation that accounts for 50% of Saudi ASAuria patients was recently reported by our laboratory. In this study we report a further six novel mutations (and one previously reported found in Saudi patients with ASAuria. The novel four missense, one nonsense and one splice-site mutation were confirmed by their absence in >300 chromosomes from the normal population. Pathogenicity of the novel splice-site mutation was also confirmed using reverse transcriptase-PCR analysis. Cross species amino acid conservation at the substituted residues described were observed in some but not all instances. Conclusions Together, the eight mutations described by our laboratory, encompass >90% of ASAuria patients in Saudi Arabia and add to about 45 other ASAuria mutations reported worldwide.

  20. Common mutations of hepatitis B virus and their clinical significance

    Directory of Open Access Journals (Sweden)

    HU Airong

    2016-06-01

    Full Text Available Hepatitis B virus (HBV tends to mutate easily due to its special structure and life cycle. Mutation changes the biological behavior of HBV and its sensitivity to antiviral drugs and even affects therapeutic effect and accelerate disease progression. The point mutations are commonly see in the pre-S/S open reading frame (ORF, which may be associated with immune escape and occult HBV infection. The G1896A mutation is often observed in the pre-C/C-ORF and is associated with the development of HBeAg-negative chronic hepatitis B (CHB, hepatocellular carcinoma (HCC, and severe chronic hepatitis (liver failure. The mutations in P-ORF mainly occur in the reverse transcriptase (RT domain and are closely related to the resistance to nucleos(tide analogues. The A1762T and G1764A mutations occur in the basal core promoter (BCP, which overlaps with X-ORF, and may be associated with HBeAg-negative CHB, HCC, and severe chronic hepatitis (liver failure. Clarification of the association between these mutations and diseases helps to develop tailor-made diagnostic and therapeutic regimens for patients with HBV infection.

  1. Reversibly Bistable Flexible Electronics

    KAUST Repository

    Alfaraj, Nasir

    2015-05-01

    Introducing the notion of transformational silicon electronics has paved the way for integrating various applications with silicon-based, modern, high-performance electronic circuits that are mechanically flexible and optically semitransparent. While maintaining large-scale production and prototyping rapidity, this flexible and translucent scheme demonstrates the potential to transform conventionally stiff electronic devices into thin and foldable ones without compromising long-term performance and reliability. In this work, we report on the fabrication and characterization of reversibly bistable flexible electronic switches that utilize flexible n-channel metal-oxide-semiconductor field-effect transistors. The transistors are fabricated initially on rigid (100) silicon substrates before they are peeled off. They can be used to control flexible batches of light-emitting diodes, demonstrating both the relative ease of scaling at minimum cost and maximum reliability and the feasibility of integration. The peeled-off silicon fabric is about 25 µm thick. The fabricated devices are transferred to a reversibly bistable flexible platform through which, for example, a flexible smartphone can be wrapped around a user’s wrist and can also be set back to its original mechanical position. Buckling and cyclic bending of such host platforms brings a completely new dimension to the development of flexible electronics, especially rollable displays.

  2. Introducing a frameshift mutation to the POL sequence of HIV-1 provirus and evaluation of the immunogenic characteristics of the mutated virions (RINNL4-3).

    Science.gov (United States)

    Zabihollahi, Rezvan; Sadat, Seyed Mehdi; Vahabpour, Rouhollah; Salehi, Mansoor; Azadmanesh, Kayhan; Siadat, Seyed Davar; Saraji, Ali Reza Azizi; Pouriavali, Mohamamd Hassan; Momen, Seyed Bahman; Aghasadeghi, Mohamad Reza

    2012-01-01

    Inactivation of the reverse transcriptase (RT) and integrase (IN) enzymes can abolish the replication of the human immunodeficiency virus (HIV) and, thus, its infectivity. Here, inactivated HIV particles convenient for designing virus-like particle (VLP) based vaccines have been produced. Inactivated HIV-provirus was created by introducing a frame shift mutation. HIV provirus DNA was cut in the pol region by Age I restriction enzyme, followed by filling of sticky ends using the Klenow fragment before ligation. The resulting plasmid was named as pRINNL4-3. HEK-293T cells were used as producer, after being transfected with the modified plasmid. Viral particle production and biological activity were assayed by virus capsid protein (p24) quantification and syncytium formation in MT2 cells, respectively. The immunogenicity of the RINNL4-3 virions was investigated in a mouse model. The mutation was expected to inactivate the virus RT and IN enzymes. The results showed that the VLPs were assembled, as measured by the p24 load of the culture supernatant, and contained functional envelope proteins (Env) as monitored by the syncytium formation. However, these VLPs had no ability to infect target MT2 cells, as well as their VSVG (vesicular stomatitis virus-glycoprotein) pseudotyped counterparts infected HEK-293T cells. A high level of antibody response was observed in immunized mice. Since RINNL4-3 virions are replication incompetent, they are convenient for production and use in biomedical studies. Also, RINNL4-3 is a candidate for a vaccine development due to it contains envelope and structural virus proteins which are crucial for triggering neutralizing antibodies and the cellular immune response.

  3. The molecular anatomy of spontaneous germline mutations in human testes.

    Directory of Open Access Journals (Sweden)

    Jian Qin

    2007-09-01

    Full Text Available The frequency of the most common sporadic Apert syndrome mutation (C755G in the human fibroblast growth factor receptor 2 gene (FGFR2 is 100-1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10(3 to >10(4 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10(-6 the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model. This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation

  4. BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

    LENUS (Irish Health Repository)

    Stordal, Britta

    2013-06-01

    Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

  5. A reversed-phase compatible thin-layer chromatography autography for the detection of acetylcholinesterase inhibitors.

    Science.gov (United States)

    Ramallo, I Ayelen; García, Paula; Furlan, Ricardo L E

    2015-11-01

    A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed-phase or normal-phase thin-layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed-phase thin-layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed-phase thin-layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10(-4) μg. The results can be read by a change in color and/or a change in fluorescence. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The Impact of ESR1 Mutations on the Treatment of Metastatic Breast Cancer.

    Science.gov (United States)

    Pejerrey, Sasha M; Dustin, Derek; Kim, Jin-Ah; Gu, Guowei; Rechoum, Yassine; Fuqua, Suzanne A W

    2018-05-07

    After nearly 20 years of research, it is now established that mutations within the estrogen receptor (ER) gene, ESR1, frequently occur in metastatic breast cancer and influence response to hormone therapy. Though early studies presented differing results, sensitive sequencing techniques now show that ESR1 mutations occur at a frequency between 20 and 40% depending on the assay method. Recent studies have focused on several "hot spot mutations," a cluster of mutations found in the hormone-binding domain of the ESR1 gene. Throughout the course of treatment, tumor evolution can occur, and ESR1 mutations emerge and become enriched in the metastatic setting. Sensitive techniques to continually monitor mutant burden in vivo are needed to effectively treat patients with mutant ESR1. The full impact of these mutations on tumor response to different therapies remains to be determined. However, recent studies indicate that mutant-bearing tumors may be less responsive to specific hormonal therapies, and suggest that aromatase inhibitor (AI) therapy may select for the emergence of ESR1 mutations. Additionally, different mutations may respond discretely to targeted therapies. The need for more preclinical mechanistic studies on ESR1 mutations and the development of better agents to target these mutations are urgently needed. In the future, sequential monitoring of ESR1 mutational status will likely direct personalized therapeutic regimens appropriate to each tumor's unique mutational landscape.

  7. Detection of MPL mutations by a novel allele-specific PCR-based strategy.

    Science.gov (United States)

    Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2013-11-01

    MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  8. Effective multiple oral administration of reverse genetics engineered infectious bursal disease virus in mice in the presence of neutralizing antibodies.

    Science.gov (United States)

    Hornyák, Ákos; Lipinski, Kai S; Bakonyi, Tamás; Forgách, Petra; Horváth, Ernő; Farsang, Attila; Hedley, Susan J; Palya, Vilmos; Bakács, Tibor; Kovesdi, Imre

    2015-01-01

    Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained. To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively. Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro. Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use. Copyright © 2015 John Wiley & Sons, Ltd.

  9. An acoustic prion assay

    Directory of Open Access Journals (Sweden)

    Gordon Hayward

    2016-12-01

    Full Text Available An acoustic prion assay has been demonstrated for sheep brain samples. Only five false positives and no false negatives were observed in a test of 45 positive and 45 negative samples. The acoustic prion sensor was constructed using a thickness shear mode quartz resonator coated with a covalently bound recombinant prion protein. The characteristic indicator of a scrapie infected sheep brain sample was an observed shoulder in the frequency decrease in response to a sample.The response of the sensor aligns with a conformational shift in the surface protein and with the propagation mechanism of the disease. This alignment is evident in the response timing and shape, dependence on concentration, cross species behaviour and impact of blood plasma. This alignment is far from sufficient to prove the mechanism of the sensor but it does offer the possibility of a rapid and inexpensive additional tool to explore prion disease. Keywords: Prions, Thickness shear mode quartz sensor

  10. Assay of oestrogen

    International Nuclear Information System (INIS)

    Edwards, J.C.

    1981-01-01

    A particular problem with the direct radioimmunoassay of unconjugated oestriol in pregnancy is caused by the increased amount of steroid-binding proteins present in pregnancy serum and plasma. The steroid-binding proteins react with oestriol and 125 I-labelled oestriol during the assay procedure and the steroid-protein bound 125 I-labelled oestriol is precipitated along with the antibody-bound 125 I-labelled oestriol by the ammonium sulphate solution separation system. A novel method is described whereby progesterone (1-20 μg/ml) is used to block the action of steroid-binding proteins in pregnancy serum and plasma samples, thus minimizing interference in a direct radioimmunoassay for unconjugated oestriol using a specific anti-oestriol serum. (U.K.)

  11. Reversible posterior leukoencephalopathy syndrome

    International Nuclear Information System (INIS)

    Lee, Eun Ja; Yu, Won Jong; Ahn, Kook Jin; Jung, So Lyung; Lee, Yeon Soo; Kim, Ji Chang; Kang, Si Won; Song, Chang Joon; Song, Soon-Young; Koo, Ja Hong; Kim, Man Deuk

    2001-01-01

    To review reversible posterior leukoencephalopathy syndrome. We reviewed 22 patients (M:F=3:19; age, 17-46 years) with the characteristic clinical and imaging features of reversible posterior leukoencephalopathy syndrome. All underwent brain MRI, and in three cases both CT and MRI were performed. In one, MRA was obtained, and in eleven, follow-up MR images were obtained. We evaluated the causes of this syndrome, its clinical manifestations, and MR findings including the locations of lesions, the presence or absence of contrast enhancement, and the changes seen at follow-up MRI. Of the 22 patients, 13 had eclampsia (six during pregnancy and seven during puerperium). Four were receiving immunosuppressive therapy (three, cyclosporine ; one, FK 506). Four suffered renal failure and one had complicated migraine. The clinical manifestations included headache (n=12), visual disturbance (n=13), seizure (n=15), focal neurologic sign (n=3), and altered mental status (n=2). Fifteen patients had hypertension and the others normotension. MRI revealed that lesions were bilateral (n=20) or unilateral (n=2). In all patients the lesion was found in the cortical and subcortical areas of the parieto-occipital lobes ; other locations were the basal ganglia (n=9), posterior temporal lobe (n=8), frontal lobe (n=5), cerebellum (n=5), pons (n=2), and thalamus (n=1). All lesions were of high signal intensity on T2-weighted images, and of iso to low intensity on T1-weighted images. One was combined with acute hematoma in the left basal ganglia. In eight of 11 patients who underwent postcontrast T1-weighted MRI, there was no definite enhancement ; in one, enhancement was mild, and in tow, patchy. CT studies showed low attenuation, and MRA revealed mild vasospasm. The symptoms of all patients improved. Follow-up MRI in nine of 11 patients depicted complete resolution of the lesions ; in two, small infarctions remained but the extent of the lesions had decreased. Reversible posterior

  12. Reversible posterior leukoencephalopathy syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Ja; Yu, Won Jong; Ahn, Kook Jin; Jung, So Lyung; Lee, Yeon Soo; Kim, Ji Chang; Kang, Si Won [The Catholic Univ. of Korea, Taejon (Korea, Republic of); Song, Chang Joon [Chungnam National Univ. School of Medicine, Cheonju (Korea, Republic of); Song, Soon-Young; Koo, Ja Hong [Kwandong Univ. College of Medicine, Myungji Hospital, Seoul (Korea, Republic of); Kim, Man Deuk [College of Medicine Pochon CHA Univ., Seoul (Korea, Republic of)

    2001-10-01

    To review reversible posterior leukoencephalopathy syndrome. We reviewed 22 patients (M:F=3:19; age, 17-46 years) with the characteristic clinical and imaging features of reversible posterior leukoencephalopathy syndrome. All underwent brain MRI, and in three cases both CT and MRI were performed. In one, MRA was obtained, and in eleven, follow-up MR images were obtained. We evaluated the causes of this syndrome, its clinical manifestations, and MR findings including the locations of lesions, the presence or absence of contrast enhancement, and the changes seen at follow-up MRI. Of the 22 patients, 13 had eclampsia (six during pregnancy and seven during puerperium). Four were receiving immunosuppressive therapy (three, cyclosporine ; one, FK 506). Four suffered renal failure and one had complicated migraine. The clinical manifestations included headache (n=12), visual disturbance (n=13), seizure (n=15), focal neurologic sign (n=3), and altered mental status (n=2). Fifteen patients had hypertension and the others normotension. MRI revealed that lesions were bilateral (n=20) or unilateral (n=2). In all patients the lesion was found in the cortical and subcortical areas of the parieto-occipital lobes ; other locations were the basal ganglia (n=9), posterior temporal lobe (n=8), frontal lobe (n=5), cerebellum (n=5), pons (n=2), and thalamus (n=1). All lesions were of high signal intensity on T2-weighted images, and of iso to low intensity on T1-weighted images. One was combined with acute hematoma in the left basal ganglia. In eight of 11 patients who underwent postcontrast T1-weighted MRI, there was no definite enhancement ; in one, enhancement was mild, and in tow, patchy. CT studies showed low attenuation, and MRA revealed mild vasospasm. The symptoms of all patients improved. Follow-up MRI in nine of 11 patients depicted complete resolution of the lesions ; in two, small infarctions remained but the extent of the lesions had decreased. Reversible posterior

  13. A novel AMELX mutation causes hypoplastic amelogenesis imperfecta.

    Science.gov (United States)

    Kim, Young-Jae; Kim, Youn Jung; Kang, Jenny; Shin, Teo Jeon; Hyun, Hong-Keun; Lee, Sang-Hoon; Lee, Zang Hee; Kim, Jung-Wook

    2017-04-01

    Amelogenesis imperfecta (AI) is a hereditary genetic defect affecting tooth enamel. AI is heterogeneous in clinical phenotype as well as in genetic etiology. To date, more than 10 genes have been associated with the etiology of AI. Amelogenin is the most abundant enamel matrix protein, most of which is encoded by the amelogenin gene in the X-chromosome (AMELX). More than 16 alternative splicing transcripts have been identified in the murine Amelx gene. The purpose of this study was to identify the genetic cause of an AI family. We recruited a family with hypoplastic AI and performed mutational analysis on the candidate gene based on the clinical phenotype. Mutational analysis revealed a missense mutation in exon 6 (NM_182680.1; c.242C > T), which changes a sequence in a highly conserved amino acid (NP_872621.1; p.Pro81Leu). Furthermore, a splicing assay using a minigene displayed that the mutation changed the mRNA splicing repertory. In this study, we identified a novel AMELX missense mutation causing hypoplastic AI, and this mutation also resulted in altered mRNA splicing. These results will not only expand the mutation spectrum causing AI but also broaden our understanding of the biological mechanism of enamel formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. The Versatile Mutational Resistome of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Carla López-Causapé

    2018-04-01

    Full Text Available One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  15. The Versatile Mutational Resistome of Pseudomonas aeruginosa.

    Science.gov (United States)

    López-Causapé, Carla; Cabot, Gabriel; Del Barrio-Tofiño, Ester; Oliver, Antonio

    2018-01-01

    One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS) data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility) between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  16. Prevalence of drug resistance mutations and non-B subtypes in newly diagnosed HIV-1 patients in Denmark

    DEFF Research Database (Denmark)

    Jørgensen, Louise B; Christensen, Marianne B; Gerstoft, Jan

    2003-01-01

    The aim of this study was to monitor the prevalence of drug resistance mutations in newly diagnosed HIV-1 positive individuals in Denmark. In addition we assessed the prevalence of non-B subtypes based on phylogenetic analysis of the pol gene. Plasma samples from 104 newly diagnosed HIV-1 positive...... patients were obtained in the year 2000. The entire protease gene and 320 amino acids of the reverse transcriptase gene were genotyped. Sequences were obtained from 97 patients. No subjects displayed primary resistance mutations in the protease gene, whereas all carried 1 or more secondary mutations....... Resistance mutations in the RT-gene associated with NRTI-resistance were found in 1 patient, who was infected with zidovudine resistant HIV-1 harbouring the M41L mutation in combination with T215S and L210S. The T215S mutation has been showed to be associated with reversion of zidovudine resistance. The T215...

  17. Reverse osmosis application studies

    International Nuclear Information System (INIS)

    Golomb, A.

    1982-02-01

    To assess the feasibility of applying reverse osmosis (RO) and ultrafiltration (UF) for effective treatment of process and waste streams from operations at Ontario Hydro's thermal and nuclear stations, an extensive literature survey has been carried out. It is concluded that RO is not at present economic for pretreatment of Great Lakes water prior to ion exchange demineralization for boiler makeup. Using both conventional and novel commercial membrane modules, RO pilot studies are recommended for treatment of boiler cleaning wastes, fly ash leachates, and flue gas desulphurization scrubber discharges for removal of heavy metals. Volume reduction and decontamination of nuclear station low-level active liquid waste streams by RO/UF also appear promising. Research programmes are proposed

  18. Reverse photoacoustic standoff spectroscopy

    Science.gov (United States)

    Van Neste, Charles W [Kingston, TN; Senesac, Lawrence R [Knoxville, TN; Thundat, Thomas G [Knoxville, TN

    2011-04-12

    A system and method are disclosed for generating a reversed photoacoustic spectrum at a greater distance. A source may emit a beam to a target and a detector measures signals generated as a result of the beam being emitted on the target. By emitting a chopped/pulsed light beam to the target, it may be possible to determine the target's optical absorbance by monitoring the intensity of light collected at the detector at different wavelengths. As the wavelength of light is changed, the target may absorb or reject each optical frequency. Rejection may increase the intensity at the sensing element and absorption may decrease the intensity. Accordingly, an identifying spectrum of the target may be made with the intensity variation of the detector as a function of illuminating wavelength.

  19. Reverse Osmosis Optimization

    Energy Technology Data Exchange (ETDEWEB)

    McMordie Stoughton, Kate; Duan, Xiaoli; Wendel, Emily M.

    2013-08-26

    This technology evaluation was prepared by Pacific Northwest National Laboratory on behalf of the U.S. Department of Energy’s Federal Energy Management Program (FEMP). ¬The technology evaluation assesses techniques for optimizing reverse osmosis (RO) systems to increase RO system performance and water efficiency. This evaluation provides a general description of RO systems, the influence of RO systems on water use, and key areas where RO systems can be optimized to reduce water and energy consumption. The evaluation is intended to help facility managers at Federal sites understand the basic concepts of the RO process and system optimization options, enabling them to make informed decisions during the system design process for either new projects or recommissioning of existing equipment. This evaluation is focused on commercial-sized RO systems generally treating more than 80 gallons per hour.¬

  20. Reverse Osmosis Optimization

    Energy Technology Data Exchange (ETDEWEB)

    None

    2013-08-01

    This technology evaluation was prepared by Pacific Northwest National Laboratory on behalf of the U.S. Department of Energy’s Federal Energy Management Program (FEMP). The technology evaluation assesses techniques for optimizing reverse osmosis (RO) systems to increase RO system performance and water efficiency. This evaluation provides a general description of RO systems, the influence of RO systems on water use, and key areas where RO systems can be optimized to reduce water and energy consumption. The evaluation is intended to help facility managers at Federal sites understand the basic concepts of the RO process and system optimization options, enabling them to make informed decisions during the system design process for either new projects or recommissioning of existing equipment. This evaluation is focused on commercial-sized RO systems generally treating more than 80 gallons per hour.

  1. Sex Reversal in Amphibians.

    Science.gov (United States)

    Flament, Stéphane

    2016-01-01

    Amphibians have been widely used to study developmental biology due to the fact that embryo development takes place independently of the maternal organism and that observations and experimental approaches are easy. Some amphibians like Xenopus became model organisms in this field. In the first part of this article, the differentiation of the gonads in amphibians and the mechanisms governing this process are reviewed. In the second part, the state of the art about sex reversal, which can be induced by steroid hormones in general and by temperature in some species, is presented. Also information about pollutants found in the environment that could interfere with the development of the amphibian reproductive apparatus or with their reproductive physiology is given. Such compounds could play a part in the amphibian decline, since in the wild, many amphibians are endangered species. © 2016 S. Karger AG, Basel.

  2. Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based or remarkably insensitive (antibody-based. Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A

  3. Single assay for simultaneous detection and differential identification of human and avian influenza virus types, subtypes, and emergent variants.

    Science.gov (United States)

    Metzgar, David; Myers, Christopher A; Russell, Kevin L; Faix, Dennis; Blair, Patrick J; Brown, Jason; Vo, Scott; Swayne, David E; Thomas, Colleen; Stenger, David A; Lin, Baochuan; Malanoski, Anthony P; Wang, Zheng; Blaney, Kate M; Long, Nina C; Schnur, Joel M; Saad, Magdi D; Borsuk, Lisa A; Lichanska, Agnieszka M; Lorence, Matthew C; Weslowski, Brian; Schafer, Klaus O; Tibbetts, Clark

    2010-02-03

    For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence

  4. HPRT gene locus mutation in peripheral blood lymphocytes induced by internal exposure to radionuclides

    Energy Technology Data Exchange (ETDEWEB)

    Jingyong, Zhao; Yongzhong, Xu; Tao, Zhao; Fengmei, Cui; Liuyi, Wang; Qinhua, Lao [Suzhou Univ., Suzhou (China). Radiation Medicine Department

    2001-07-01

    HPRT gene locus mutation in peripheral blood lymphocytes induced by internal exposure to radionuclides was performed and the relationships between mutation frequency and dose were studied. Rats were injected intravenously with radionuclides, the blood was sampled at different time after injection; HPRT gene locus mutation frequency (GMF) were examined by methods of multi-nucleus cell and Brdurd assay, working out the Dose-response function. GMF rose with the increase of dose and dose-rates and were clearly interrelated. The HPRT gene locus mutation is very sensitive to radiation and may be used as a biological dosimeter.

  5. Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

    Directory of Open Access Journals (Sweden)

    Leary Jeffry J

    2002-05-01

    Full Text Available Abstract Background The thymidine kinase (tk mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. Methods A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. Results Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. Conclusions This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a mutations may be modulated by other viral polypeptides cooperating with Pol, and (b the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

  6. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  7. MUTATIONS IN CALMODULIN GENES

    DEFF Research Database (Denmark)

    2013-01-01

    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  8. Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast. III. rev 3 mutant strains

    International Nuclear Information System (INIS)

    Lawrence, C.W.; Crhistensen, R.B.

    1979-01-01

    The role of rev3 gene function in uv-induced mutagenesis in the yeast Saccharomyces cerevisiae has been examined by determining the reversion of 12 well-defined cyc1 mutations in diploid strains homozygous for the rev3-1 or rev3-3 allale. The 12 cyc1 alleles include one ochre, one amber, four initiation, two proline missense, and four frameshift mutations. We find that the rev3 mutations reduce the frequency of UV-induced reversion of all of the cyc1 alleles, though different classes of alleles respond to a different extent. These results imply that the rev3 gene function is required for the production of a wide variety of mutational events, though probably not all, and show that each of the three rev loci have different mutational phenotypes. Such diverse phenotypes are not predicted by the unitary model for bacterial mutagenes, suggesting that this is at best an incomplete description of eukaryotic mutagenesis

  9. Novel mutations and mutation combinations of ryanodine receptor in a chlorantraniliprole resistant population of Plutella xylostella (L.)

    Science.gov (United States)

    Guo, Lei; Liang, Pei; Zhou, Xuguo; Gao, Xiwu

    2014-01-01

    A previous study documented a glycine to glutamic acid mutation (G4946E) in ryanodine receptor (RyR) was highly correlated to diamide insecticide resistance in field populations of Plutella xylostella (Lepidoptera: Plutellidae). In this study, a field population collected in Yunnan province, China, exhibited a 2128-fold resistance to chlorantraniliprole. Sequence comparison between resistant and susceptible P. xylostella revealed three novel mutations including a glutamic acid to valine substitution (E1338D), a glutamine to leucine substitution (Q4594L) and an isoleucine to methionine substitution (I4790M) in highly conserved regions of RyR. Frequency analysis of all four mutations in this field population showed that the three new mutations showed a high frequency of 100%, while the G4946E had a frequency of 20%. Furthermore, the florescent ligand binding assay revealed that the RyR containing multiple mutations displayed a significantly lower affinity to the chlorantraniliprole. The combined results suggested that the co-existence of different combinations of the four mutations was involved in the chlorantraniliprole resistance. An allele-specific PCR based method was developed for the diagnosis of the four mutations in the field populations of P. xylostella. PMID:25377064

  10. Clinical Outcomes of Lung Transplantation in Patients with Telomerase Mutations

    Science.gov (United States)

    Tokman, Sofya; Singer, Jonathan P.; Devine, Megan S.; Westall, Glen P.; Aubert, John-David; Tamm, Michael; Snell, Gregory I.; Lee, Joyce S.; Goldberg, Hilary J.; Kukreja, Jasleen; Golden, Jeffrey A.; Leard, Lorriana E.; Garcia, Christine K.; Hays, Steven R.

    2017-01-01

    Background Successful lung transplantation (LT) for patients with pulmonary fibrosis from telomerase mutations is limited by systemic complications of telomerase dysfunction including myelosuppression, cirrhosis, and malignancy. We describe clinical outcomes among 14 LT recipients with telomerase mutations. Methods Subjects underwent LT between February 2005 and April 2014 at 5 LT centers. We abstracted data from medical records, focusing on outcomes reflecting post-LT treatment effects likely to be complicated by telomerase mutations. Results The median age of subjects was 60.5 years (IQR 52.0–62.0), 64.3% were male, and the mean post-LT observation time was 3.2 years (SD ±2.9). Eleven subjects had a mutation in telomerase reverse transcriptase, 2 in telomerase RNA component, and 1 had an uncharacterized mutation. Ten subjects were leukopenic post-LT; leukopenia prompted cessation of mycophenolate mofetil in 5 and treatment with filgrastim in 4. Six subjects had recurrent lower respiratory tract infections (LRTI), 7 had acute cellular rejection (ACR) (A1), and 4 developed chronic lung allograft dysfunction (CLAD). Ten LT recipients developed chronic renal insufficiency and 8 experienced acute, reversible renal failure. Three developed cancer, none had cirrhosis. Thirteen subjects were alive at data censorship. Conclusions The clinical course for LT recipients with telomerase mutations is complicated by renal disease, leukopenia prompting a change in the immunosuppressive regimen, and recurrent LTRI. In contrast, cirrhosis was absent, ACR was mild, and development of CLAD was comparable to other LT populations. While posing challenges, lung transplantation may be feasible for patients with pulmonary fibrosis due to telomerase mutations. PMID:26169663

  11. Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

    Science.gov (United States)

    Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

    2017-10-27

    Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Neighborhood properties are important determinants of temperature sensitive mutations.

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available Temperature-sensitive (TS mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method-logistic regression-to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

  13. In vivo somatic mutation systems in the mouse

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    In an effort to meet the need for a fast and cheap in vivo prescreen for inherited mammalian point mutations, a somatic forward-mutation method, originally developed in an x-ray experiment, has more recently been tested in work with chemical mutagens. The method makes use of coat-color mutations because the gene product is usually locally expressed, mosaics can be detected with minimal effort, and opportunities for making comparison with induction of germinal point mutations are greatest.--Following treatment of embryos that are heterozygous at specific coat-color loci, various induced genetic changes can result in expression of the recessive (RS) in clones derived from mutant melanocyte precursor cells. However, other events, such as decrease in the number of precursor cells, or disturbed differentiation, can also result in spots, which with careful classification can usually be distinguished from RS's on the basis of their location and color. When this is done, the relative RS frequencies for a series of compounds at least roughly parallel the relative spermatogonial mutation rates. The fact that easily measurable (though low) RS rates are obtained with compounds that have yielded negative results in spermatogonial tests is not surprising in view of the fact that RS's can be caused by several mechanisms besides point mutation.--In spite of the parallelism observed in one laboratory, the usefulness of the in vivo somatic mutation method as a prescreen could come to be doubted because of major discrepancies between results of similar experiments at different laboratories. However, It appears probable that at least some of these discrepancies are due to failure to discriminate between spots that probably resulted from melanocyte insufficiency and spots that resulted from expression of the recessive.--Reverse somatic mutation systems can potentially avoid some of the pitfalls of forward mutation systems. Such system are still in developmental stages

  14. Universal fieldable assay with unassisted visual detection

    Science.gov (United States)

    Chelyapov, Nicolas (Inventor)

    2012-01-01

    A universal detection system based on allosteric aptamers, signal amplification cascade, and eye-detectable phrase transition. A broadly applicable homogeneous detection system is provided. It utilizes components of the blood coagulation cascade in the presence of polystyrene microspheres (MS) as a signal amplifier. Russell's viper venom factor X activator (RVV-X) triggers the cascade, which results in an eye-visible phase transition--precipitation of MS bound to clotted fibrin. An allosteric RNA aptamer, RNA132, with affinity for RVV-X and human vascular endothelial growth factor (VEGF.sub.165) was created. RNA132 inhibits enzymatic activity of RVV-X. The effector molecule, VEGF.sub.165, reverses the inhibitory activity of RNA132 on RVV-X and restores its enzymatic activity, thus triggering the cascade and enabling the phase transition. Similar results were obtained for another allosteric aptamer modulated by a protein tyrosine phosphatase. The assay is instrumentation-free for both processing and readout.

  15. 49 CFR 230.89 - Reverse gear.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Reverse gear. 230.89 Section 230.89 Transportation... Reversing Gear § 230.89 Reverse gear. (a) General provisions. Reverse gear, reverse levers, and quadrants... quadrant. Proper counterbalance shall be provided for the valve gear. (b) Air-operated power reverse gear...

  16. Mutation, somatic mutation and diseases of man

    International Nuclear Information System (INIS)

    Burnet, F.M.

    1976-01-01

    The relevance of the intrinsic mutagenesis for the evolution process, genetic diseases and the process of aging is exemplified. The fundamental reaction is the function of the DNA and the DNA-enzymes like the DNA-polymerases in replication, repair, and transcription. These defects are responsible for the mutation frequency and the genetic drift in the evolution process. They cause genetic diseases like Xeroderma pigmentosum which is described here in detail. The accumulation of structural and functional mistakes leads to diseases of old age, for example to autoimmune diseases and immune suppression. There is a proportionality between the duration of life and the frequency of mistakes in the enzymatic repair system. No possibility of prophylaxis or therapy is seen. Methods for prognosis could be developed. (AJ) [de

  17. Mutations and chromosomal aberrations

    International Nuclear Information System (INIS)

    Kihlman, B.A.

    1977-01-01

    The genetic changes of mutations and chromosomal aberrations are discussed. The consequences of both depend not only on the type of genetic change produced but also on the type of cell that is affected and on the development stage of the organism. (C.F.)

  18. Mutations in GABRB3

    DEFF Research Database (Denmark)

    Møller, Rikke S; Wuttke, Thomas V; Helbig, Ingo

    2017-01-01

    OBJECTIVE: To examine the role of mutations in GABRB3 encoding the β3 subunit of the GABAA receptor in individual patients with epilepsy with regard to causality, the spectrum of genetic variants, their pathophysiology, and associated phenotypes. METHODS: We performed massive parallel sequencing ...

  19. Kin Selection - Mutation Balance

    DEFF Research Database (Denmark)

    Dyken, J. David Van; Linksvayer, Timothy Arnold; Wade, Michael J.

    2011-01-01

    selection-mutation balance, which provides an evolutionary null hypothesis for the statics and dynamics of cheating. When social interactions have linear fitness effects and Hamilton´s rule is satisfied, selection is never strong enough to eliminate recurrent cheater mutants from a population, but cheater...

  20. E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient.

    Science.gov (United States)

    L'Huillier, Arnaud G; Abed, Yacine; Petty, Tom J; Cordey, Samuel; Thomas, Yves; Bouhy, Xavier; Schibler, Manuel; Simon, Audrey; Chalandon, Yves; van Delden, Christian; Zdobnov, Evgeny; Boquete-Suter, Patricia; Boivin, Guy; Kaiser, Laurent

    2015-12-01

    An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategies. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization

    International Nuclear Information System (INIS)

    Moreau, Karen; Faure, Claudine; Violot, Sebastien; Gouet, Patrice; Verdier, Gerard; Ronfort, Corinne

    2004-01-01

    During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme

  2. Mutations in galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Reichardt, J.K.V. [Univ. of Southern California School of Medicine, Los Angeles, CA (United States)

    1995-10-01

    This Letter raises four issues concerning two papers on galactosemia published in the March 1995 of the Journal. First, table 2 in the paper by Elsas et al. incorrectly attributes seven galactose-l-phosphate uridyl transferase (GALT) mutations (S135L, L195P, K285N, N314D, R333W, R333G, and K334R). The table also fails to mention that others have reported the same two findings attributed to {open_quotes}Leslie et al.; Elsas et al. and in press{close_quotes} and {open_quotes}Leslie et al.; Elsas et al.{close_quotes} The first finding on the prevalence of the Q188R galactosemia mutation in the G/G Caucasian population has also been described by Ng et al., and the second finding on the correlation of the N314D GALT mutation with the Duarte variant was reported by Lin et al. Second, Elsas et al. suggest that the E203K and N314D mutations may {open_quotes}produce intra-allelic complementation when in cis{close_quotes}. This speculation is supported by the activity data of individual III-2 but is inconsistent with the activities of three other individuals I-1, II-1, and III-1 of the same pedigree. The GALT activity measured in these three individuals suggests a dominant negative effect of E203K in E203K-N314D chromosomes, since they all have less than normal activity. Thus, the preponderance of the data in this paper is at odds with the authors speculation. It is worth recalling that Lin et al. also identified four N314D GALT mutations on 95 galactosemic chromosomes examined. A similar situation also appears to be the case in proband III-1 (with genotype E203K-N314D/IVSC) in the Elsas et al. paper. 9 refs.

  3. Heterozygous RTEL1 mutations are associated with familial pulmonary fibrosis.

    Science.gov (United States)

    Kannengiesser, Caroline; Borie, Raphael; Ménard, Christelle; Réocreux, Marion; Nitschké, Patrick; Gazal, Steven; Mal, Hervé; Taillé, Camille; Cadranel, Jacques; Nunes, Hilario; Valeyre, Dominique; Cordier, Jean François; Callebaut, Isabelle; Boileau, Catherine; Cottin, Vincent; Grandchamp, Bernard; Revy, Patrick; Crestani, Bruno

    2015-08-01

    Pulmonary fibrosis is a fatal disease with progressive loss of respiratory function. Defective telomere maintenance leading to telomere shortening is a cause of pulmonary fibrosis, as mutations in the telomerase component genes TERT (reverse transcriptase) and TERC (RNA component) are found in 15% of familial pulmonary fibrosis (FPF) cases. However, so far, about 85% of FPF remain genetically uncharacterised.Here, in order to identify new genetic causes of FPF, we performed whole-exome sequencing, with a candidate-gene approach, of 47 affected subjects from 35 families with FPF without TERT and TERC mutations.We identified heterozygous mutations in regulator of telomere elongation helicase 1 (RTEL1) in four families. RTEL1 is a DNA helicase with roles in DNA replication, genome stability, DNA repair and telomere maintenance. The heterozygous RTEL1 mutations segregated as an autosomal dominant trait in FPF, and were predicted by structural analyses to severely affect the function and/or stability of RTEL1. In agreement with this, RTEL1-mutated patients exhibited short telomeres in comparison with age-matched controls.Our results provide evidence that heterozygous RTEL1 mutations are responsible for FPF and, thereby, extend the clinical spectrum of RTEL1 deficiency. Thus, RTEL1 enlarges the number of telomere-associated genes implicated in FPF. Copyright ©ERS 2015.

  4. Field reversal experiments (FRX)

    International Nuclear Information System (INIS)

    Linford, R.K.; Armstrong, W.T.; Platts, D.A.; Sherwood, E.G.

    1978-01-01

    The equilibrium, confinement, and stability properties of the reversed-field configuration (RFC) are being studied in two theta-pinch facilities. The RFC is an elongated toroidal plasma confined in a purely poloidal field geometry. The open field lines of the linear theta pinch support the closed-field RFC much like the vertical field centers the toroidal plasma in a tokamak. Depending on stability and confinement properties, the RFC might be used to greatly reduce the axial losses in linear fusion devices such as mirrors, theta pinches, and liners. The FRX systems produce RFC's with a major radius R = 2-6 cm, minor radius a approximately 2 cm, and a total length l approximately 35 cm. The observed temperatures are T/sub e/ approximately 100 eV and T/sub i/ = 150-350 eV with a peak density n approximately 2 x 10 15 cm -3 . After the plasma reaches equilibrium, the RFC remains stable for up to 30 μs followed by the rapid growth of the rotational m = 2 instability, which terminates the confinement. During the stable equilibrium, the particle and energy confinement times are more than 10 times longer than in an open-field system. The behavior of the m = 2 mode qualitatively agrees with the theoretically predicted instability for rotational velocities exceeding some critical value

  5. Field reversal experiments (FRX)

    International Nuclear Information System (INIS)

    Linford, R.K.; Armstrong, W.T.; Platts, D.A.; Sherwood, E.G.

    1979-01-01

    The equilibrium, confinement, and stability properties of the reversed-field configuration (RFC) are being studied in two theta-pinch facilities. The RFC is an elongated toroidal plasma confined in a purely poloidal field geometry. The open field lines of the linear theta pinch support the closed-field RFC much like the vertical field centres the toroidal plasma in a tokamak. Depending on stability and confinement properties, the RFC might be used to greatly reduce the axial losses in linear fusion devices such as mirrors, theta pinches, and liners. The FRX systems produce RFCs with a major radius R=2-6cm, a minor radius a approximately 2cm, and a total length l approximately 35cm. The observed temperatures are Tsub(e) approximately 100eV and Tsub(i)=150-350eV with a peak density n approximately 2x10 15 cm -3 . After the plasma has reached equilibrium, the RFC remains stable for up to 30μs, followed by the rapid growth of the rotational m=2 instability, which terminates the confinement. During the stable equilibrium, the particle and energy confinement times are more than 10 times longer than in an open-field system. The behaviour of the m=2 mode agrees qualitatively with the theoretically predicted instability for rotational velocities exceeding some critical value. (author)

  6. Mutation breeding newsletter. No. 45

    International Nuclear Information System (INIS)

    2001-07-01

    This issue of the Mutation Breeding newsletter contains 39 articles dealing with radiation induced mutations and chemical mutagenesis techniques in plant breeding programs with the aims of improving crop productivity and disease resistance as well as exploring genetic variabilities

  7. Mutation breeding newsletter. No. 33

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1989-01-01

    This issue of the newsletter reports a number of research news and research abstracts on application of radiation induced mutation techniques to increase mutagenesis and mutation frequency in plant breeding projects.

  8. Mutation breeding newsletter. No. 33

    International Nuclear Information System (INIS)

    1989-01-01

    This issue of the newsletter reports a number of research news and research abstracts on application of radiation induced mutation techniques to increase mutagenesis and mutation frequency in plant breeding projects

  9. EG-08IDH MUTATIONS IN GLIOMAS ASSOCIATED WITH ENCHONDROMATOSIS

    Science.gov (United States)

    Nicholas, M. Kelly; Joseph, Loren; Venneti, Sriram; Daher, Ahmad; Pytel, Peter

    2014-01-01

    The enchondromatoses, Ollier's disease and Maffucci syndrome, are non-heritable developmental disorders characterized by multiple enchondromas (Olllier's) in association with hemangiomas (Maffucci). Glial neoplasms are reported in both disorders but a pathogenic mechanism underlying this association has not been identified. We report a case of anaplastic astrocytoma in a 23 year old man with Maffucci syndrome whose tumor carried a substitution mutation of arginine for cysteine at position 132 (R132C) of the isocitrate dehydrogenase 1 (IDH1) protein. This mutation, commonly found in Maffucci-associated enchondromas and hemangiomas, was not detected on routine immunohistochemical (IHC) analysis of the astrocytoma using the R132H mutation-specific antibody, commonly applied in clinical laboratories. The R132C mutation was detected by polymerase chain reaction (PCR) and subsequently confirmed using a SNaPshot assay. Because somatic mosaic IDH mutations are associated with enchondromas and hemangiomas in Maffucci syndrome, we looked for the R132C mutation in a hemangioma, peripheral blood mononuclear cells (PBMNC) and histologically normal brain surrounding the tumor from this patient. The mutation was present in the hemangioma, absent in PBMNC, and present in 2% of alleles in ‘normal’ brain. The low level in surrounding brain tissue is consistent with tumor cell infiltration, not mosaicism, as a S173T p53 mutation in the tumor showed similar results. Using IHC, we further demonstrated that the mutant IDH1 protein in this glioma functions as an oncometabolite. Two repressive histone trimethylation marks were strongly positive in the tumor, supporting a role for 2-hydroxyglutarate in the inhibition of histone demethylation. Together, these data demonstrate that an IDH1 mutation common in enchodromatoses underlies the association of glial tumors reported in both Ollier's disease and Maffucci syndrome.

  10. KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Yu-Lin Lin

    Full Text Available Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC, but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V by small interfering RNAs (siRNA and overexpressed in KRAS-wild-type CRC cells (COLO320DM by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR. In KRAS-wild-type CRC cells (COLO320DM, KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1 downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1(G13D and SW480(G12V, KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.

  11. Effect of genes controlling radiation sensitivity on chemically induced mutations in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Prakash, L.

    1976-01-01

    The effect of 16 different genes (rad) conferring radiation sensitivity on chemically induced reversion in the yeast Saccharomyces cerevisiae was determined. The site of reversion used was a well-defined chain initiation mutant mapping in the structural gene coding for iso-1-cytochrome c. High doses of EMS and HNO 2 resulted in decreased reversion of cyc1-131 in rad6, rad9 and rad15 strains compared to the normal RAD + strains. In addition, rad52 greatly decreased EMS reversion of cyc1-131 but had no effect on HNO 2 -induced reversion; rad18, on the other hand, increased HNO 2 -induced reversion but did not alter EMS-induced reversion. When NQO was used as the mutagen, every rad gene tested, except for rad18, had an effect on reversion; rad6, rad9, rad15, rad17, rad18, rad22, rev1, rev2, and rev3 lowered NQO reversion while rad1, rad2, rad3, rad4, rad10, rad12, and rad16 increased it compared to the RAD + strain. The effect of rad genes on chemical mutagenesis is discussed in terms of their effect on uv mutagenesis. It is concluded that although the nature of the repair pathways may differ for uv- and chemically-induced mutations in yeast, a functional repair system is required for the induction of mutation by the chemical agents NQO, EMS, and HNO 2

  12. ENU-induced phenovariance in mice: inferences from 587 mutations

    Directory of Open Access Journals (Sweden)

    Arnold Carrie N

    2012-10-01

    Full Text Available Abstract Background We present a compendium of N-ethyl-N-nitrosourea (ENU-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1 to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2 to assess the characteristics of these mutations; and 3 to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by

  13. Towards a reversible functional language

    DEFF Research Database (Denmark)

    Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

    2012-01-01

    /equality operator also simplifies inverse computation and program inversion. We discuss the advantages of a reversible functional language using example programs, including run-length encoding. Program inversion is seen to be as lightweight as for imperative reversible languages and realized by recursive descent...

  14. Reverse engineering of RFID devices

    NARCIS (Netherlands)

    Bokslag, W.

    2015-01-01

    This paper discusses the relevance and potential impact of both RFID and reverse engineering of RFID technology, followed by a discussion of common protocols and internals of RFID technology. The focus of the paper is on providing an overview of the different approaches to reverse engineering RFID

  15. How decision reversibility affects motivation

    NARCIS (Netherlands)

    Bullens, L.; van Harreveld, F.; Förster, J.; Higgins, T.E.

    2014-01-01

    The present research examined how decision reversibility can affect motivation. On the basis of extant findings, it was suggested that 1 way it could affect motivation would be to strengthen different regulatory foci, with reversible decision making, compared to irreversible decision making,

  16. Enzymatic reactions in reversed micelles

    NARCIS (Netherlands)

    Hilhorst, M.H.

    1984-01-01

    It has been recognised that enzymes in reversed micelles have potential for application in chemical synthesis. Before these expectations will be realised many problems must be overcome. This thesis deals with some of them.
    In Chapter 1 the present knowledge about reversed micelles and

  17. Reversible networks in supramolecular polymers

    NARCIS (Netherlands)

    Havermans - van Beek, D.J.M.

    2007-01-01

    Non–covalent interactions between low molecular weight polymers form the basis of supramolecular polymers. The material properties of such polymers are determined by the strength and lifetime of the non–covalent reversible interactions. Due to the reversibility of the interactions between the low

  18. Reverse genetics of avian metapneumoviruses

    Science.gov (United States)

    An overview of avian metapneumovirus (aMPV) infection in turkeys and development of a reverse genetics system for aMPV subgroup C (aMPV-C) virus will be presented. By using reverse genetics technology, we generated recombinant aMPV-C viruses containing a different length of glycoprotein (G) gene or...

  19. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

  20. A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

    Science.gov (United States)

    Lucarelli, Marco; Porcaro, Luigi; Biffignandi, Alice; Costantino, Lucy; Giannone, Valentina; Alberti, Luisella; Bruno, Sabina Maria; Corbetta, Carlo; Torresani, Erminio; Colombo, Carla; Seia, Manuela

    2017-09-01

    Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  1. MODELS OF PROJECT REVERSE ENGINEERING

    Directory of Open Access Journals (Sweden)

    Віктор Володимирович ІВАНОВ

    2017-03-01

    Full Text Available Reverse engineering decided important scientific and technical problems of increasing the cost of the existing technical product by transforming it into a product with other features or design. Search ideas of the new application of existing products on the base of heuristic analysis were created. The concept of reverse engineering and its division into three types: conceptual, aggregate and complete was expanded. The use of heuristic methods for reverse engineering concept was showed. The modification model of Reverse engineering based on the model of РМВОК was developed. Our model includes two new phases: identification and transformation. At the identification phase, technical control is made. At the transformation phase, search heuristic idea of the new applied existing technical product was made. The model of execution phase that included heuristic methods, metrological equipment, and CAD/CAM/CAE program complex was created. The model that connected economic indicators of reverse engineering project was developed.

  2. What do reversible programs compute?

    DEFF Research Database (Denmark)

    Axelsen, Holger Bock; Glück, Robert

    2011-01-01

    Reversible computing is the study of computation models that exhibit both forward and backward determinism. Understanding the fundamental properties of such models is not only relevant for reversible programming, but has also been found important in other fields, e.g., bidirectional model...... transformation, program transformations such as inversion, and general static prediction of program properties. Historically, work on reversible computing has focussed on reversible simulations of irreversible computations. Here, we take the viewpoint that the property of reversibility itself should...... are not strictly classically universal, but that they support another notion of universality; we call this RTM-universality. Thus, even though the RTMs are sub-universal in the classical sense, they are powerful enough as to include a self-interpreter. Lifting this to other computation models, we propose r...

  3. Fundamentals of reversible flowchart languages

    DEFF Research Database (Denmark)

    Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

    2016-01-01

    Abstract This paper presents the fundamentals of reversible flowcharts. They are intended to naturally represent the structure and control flow of reversible (imperative) programming languages in a simple computation model, in the same way classical flowcharts do for conventional languages......, structured reversible flowcharts are as expressive as unstructured ones, as shown by a reversible version of the classic Structured Program Theorem. We illustrate how reversible flowcharts can be concretized with two example programming languages, complete with syntax and semantics: a low-level unstructured...... language and a high-level structured language. We introduce concrete tools such as program inverters and translators for both languages, which follow the structure suggested by the flowchart model. To further illustrate the different concepts and tools brought together in this paper, we present two major...

  4. The comet assay in testing the potential genotoxicity of nanomaterials

    Directory of Open Access Journals (Sweden)

    Amaya Azqueta

    2015-06-01

    Full Text Available In the last two decades the production and use of nanomaterials (NMs has impressively increased. Their small size, given a mass equal to that of the corresponding bulk material, implies an increase in the surface area and consequently in the number of atoms that can be reactive. They possess different physical, chemical and biological properties compared to bulk materials of the same composition, which makes them very interesting and valuable for many different applications in technology, energy, construction, electronics, agriculture, optics, paints, textiles, food, cosmetics, medicine... Toxicological assessment of NMs is crucial; the same properties that make them interesting also make them potentially harmful for health and the environment. However, the term NM covers many different kinds of particle , and so there is no simple, standard approach to assessing their toxicity. NMs can enter the cell, interact with cell components and even penetrate the nucleus and interfere with the genetic material. Among the different branches of toxicology, genotoxicity is a main area of concern since