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Sample records for mutation confers increased

  1. Cooperativity of Negative Autoregulation Confers Increased Mutational Robustness

    PubMed Central

    Marciano, David C.; Lua, Rhonald C.; Herman, Christophe; Lichtarge, Olivier

    2016-01-01

    Negative autoregulation is universally found across organisms. In the bacterium Escherichia coli, transcription factors often repress their own expression to form a negative feedback network motif that enables robustness to changes in biochemical parameters. Here we present a simple phenomenological model of a negative feedback transcription factor repressing both itself and another target gene. The strength of the negative feedback is characterized by three parameters: the cooperativity in self-repression, the maximal expression rate of the transcription factor, and the apparent dissociation constant of the transcription factor binding to its own promoter. Analysis of the model shows that the target gene levels are robust to mutations in the transcription factor, and that the robustness improves as the degree of cooperativity in self-repression increases. The prediction is tested in the LexA transcriptional network of E. coli by altering cooperativity in self-repression and promoter strength. Indeed, we find robustness is correlated with the former. Considering the proposed importance of gene regulation in speciation, parameters governing a transcription factor’s robustness to mutation may have significant influence on a cell or organism’s capacity to evolve. PMID:27391757

  2. Cooperativity of Negative Autoregulation Confers Increased Mutational Robustness

    NASA Astrophysics Data System (ADS)

    Marciano, David C.; Lua, Rhonald C.; Herman, Christophe; Lichtarge, Olivier

    2016-06-01

    Negative autoregulation is universally found across organisms. In the bacterium Escherichia coli, transcription factors often repress their own expression to form a negative feedback network motif that enables robustness to changes in biochemical parameters. Here we present a simple phenomenological model of a negative feedback transcription factor repressing both itself and another target gene. The strength of the negative feedback is characterized by three parameters: the cooperativity in self-repression, the maximal expression rate of the transcription factor, and the apparent dissociation constant of the transcription factor binding to its own promoter. Analysis of the model shows that the target gene levels are robust to mutations in the transcription factor, and that the robustness improves as the degree of cooperativity in self-repression increases. The prediction is tested in the LexA transcriptional network of E. coli by altering cooperativity in self-repression and promoter strength. Indeed, we find robustness is correlated with the former. Considering the proposed importance of gene regulation in speciation, parameters governing a transcription factor's robustness to mutation may have significant influence on a cell or organism's capacity to evolve.

  3. Registration of Durum Wheat Germplasm Lines with Combined Mutations in SBEIIa and SBEIIb Genes Conferring Increased Amylose and Resistant Starch.

    PubMed

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Dubcovsky, Jorge

    2014-08-25

    Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations in the linked SBEIIa and SBEIIb paralogs were combined on chromosomes 2A (SBEIIa/b-A; Reg. No. GP-968, PI 670159), 2B (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and on both chromosomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160) in the tetraploid wheat cultivar Kronos, a semidwarf durum wheat cultivar that has high yield potential and excellent pasta quality. These three double and quadruple SBEII-mutant lines were compared with a control sib line with no SBEII mutations in two field locations in California. The SBEIIa/b-AB line with four mutations showed dramatic increases in amylose (average 66%) and resistant starch (average 753%) relative to the control. However, the SBEIIa/b-AB line also showed an average 7% decrease in total starch and an 8% decrease in kernel weight. The release by the University of California-Davis of the durum wheat germplasm combining four SBEIIa and SBEIIb mutations will accelerate the deployment of these mutations in durum wheat breeding programs and the development of durum wheat varieties with increased resistant starch.

  4. Registration of Durum Wheat Germplasm Lines with Combined Mutations in SBEIIa and SBEIIb Genes Conferring Increased Amylose and Resistant Starch

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Dubcovsky, Jorge

    2016-01-01

    Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.], used in pasta, couscous, and flatbread production, is an important source of starch food products worldwide. The amylose portion of the starch forms resistant starch complexes that resist digestion and contribute to dietary fiber. Increasing the amount of amylose and resistant starch in wheat by mutating the STARCH BRANCHING ENZYME II (SBEII) genes has potential to provide human health benefits. Ethyl methane sulfonate mutations in the linked SBEIIa and SBEIIb paralogs were combined on chromosomes 2A (SBEIIa/b-A; Reg. No. GP-968, PI 670159), 2B (SBEIIa/b-B; Reg. No. GP-970, PI 670161), and on both chromosomes (SBEIIa/b-AB; Reg. No. GP-969, PI 670160) in the tetraploid wheat cultivar Kronos, a semidwarf durum wheat cultivar that has high yield potential and excellent pasta quality. These three double and quadruple SBEII-mutant lines were compared with a control sib line with no SBEII mutations in two field locations in California. The SBEIIa/b-AB line with four mutations showed dramatic increases in amylose (average 66%) and resistant starch (average 753%) relative to the control. However, the SBEIIa/b-AB line also showed an average 7% decrease in total starch and an 8% decrease in kernel weight. The release by the University of California–Davis of the durum wheat germplasm combining four SBEIIa and SBEIIb mutations will accelerate the deployment of these mutations in durum wheat breeding programs and the development of durum wheat varieties with increased resistant starch. PMID:27110322

  5. Registration of Common Wheat Germplasm with Mutations in SBEII Genes Conferring Increased Grain Amylose and Resistant Starch Content

    PubMed Central

    Schönhofen, André; Hazard, Brittany; Zhang, Xiaoqin; Dubcovsky, Jorge

    2016-01-01

    Starch present in the endosperm of common wheat (Triticum aestivum L.) grains is an important source of carbohydrates worldwide. Starches with a greater proportion of amylose have increased levels of resistant starch, a dietary fiber that can provide human health benefits. Induced mutations in STARCH BRANCHING ENZYME II (SBEII) genes in wheat are associated with increased amylose and resistant starch. Ethyl methane sulfonate mutations in SBEIIa and SBEIIb paralogs were combined in the hexaploid wheat cultivar Lassik. Four mutant combinations were generated: SBEIIa/b-AB (Reg. No. GP-997, PI 675644); SBEIIa/b-A, SBEIIa-D (Reg. No. GP-998, PI 675645); SBEIIa/b-B, SBEIIa-D (Reg. No. GP-999, PI 675646); and SBEIIa/b-AB, SBEIIa-D (Reg. No. GP-1000, PI 675647). The SBEII mutant lines were compared with a wild-type control in a greenhouse and field experiment. The quintuple mutant line (SBEIIa/b-AB, SBEIIa-D) presented significant increases in both amylose (51% greenhouse; 63% field) and resistant starch (947% greenhouse; 1057% field) relative to the control. A decrease in total starch content (7.8%) was observed in the field experiment. The quintuple mutant also differed in starch viscosity parameters. Registration of the hexaploid wheat SBEII-mutant lines by University of California, Davis can help expedite the development of common wheat cultivars with increased amylose and resistant starch content. PMID:27818720

  6. The mthA Mutation Conferring Low-Level Resistance to Streptomycin Enhances Antibiotic Production in Bacillus subtilis by Increasing the S-Adenosylmethionine Pool Size

    PubMed Central

    Tojo, Shigeo; Kim, Ji-Yun; Tanaka, Yukinori; Inaoka, Takashi; Hiraga, Yoshikazu

    2014-01-01

    Certain Strr mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel+ but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 μg/ml, an antibiotic not produced (<1 μg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues. PMID:24509311

  7. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  8. Increased pilus production conferred by a naturally occurring mutation alters host-pathogen interaction in favor of carriage in Streptococcus pyogenes.

    PubMed

    Flores, Anthony R; Olsen, Randall J; Cantu, Concepcion; Pallister, Kyler B; Guerra, Fermin E; Voyich, Jovanka M; Musser, James M

    2017-03-06

    Studies of the human pathogen group A Streptococcus (GAS) define the carrier phenotype as increased ability to adhere to and persist on epithelial surfaces and decreased ability to cause disease. We tested the hypothesis that a single amino acid change (Arg135Gly) in a highly conserved sensor kinase (LiaS) of a poorly defined GAS regulatory system contributes to a carrier phenotype through increased pilus production. When introduced into an emm serotype-matched invasive strain, the carrier allele (liaS(R135G)) recapitulated a carrier phenotype defined by increased ability to adhere to mucosal surfaces and decreased ability to cause disease. Gene transcript analyses revealed that the liaS mutation significantly altered transcription of the genes encoding pilus when in the presence of bacitracin. Elimination of pilus production in the isogenic carrier mutant decreased ability to colonize the mouse nasopharynx, adhere to and be internalized by cultured human epithelial cells, and restored a virulence phenotype in a mouse model of necrotizing fasciitis. We also observed significantly reduced survival of the isogenic carrier mutant compared to the parental invasive strain after exposure to human neutrophils. Elimination of pilus in the isogenic carrier mutant increased neutrophil survival to the parental invasive strain level. Together, our data demonstrate that the carrier mutation (liaS(R135G)) affects pilus expression. Our data suggest new mechanisms of pilus gene regulation in GAS and differs from the enhanced invasiveness associated with increased pilus production in other bacterial pathogens.

  9. Heterozygosity increases microsatellite mutation rate

    PubMed Central

    Amos, William

    2016-01-01

    Whole genome sequencing of families of Arabidopsis has recently lent strong support to the heterozygote instability (HI) hypothesis that heterozygosity locally increases mutation rate. However, there is an important theoretical difference between the impact on base substitutions, where mutation rate increases in regions surrounding a heterozygous site, and the impact of HI on sequences such as microsatellites, where mutations are likely to occur at the heterozygous site itself. At microsatellite loci, HI should create a positive feedback loop, with heterozygosity and mutation rate mutually increasing each other. Direct support for HI acting on microsatellites is limited and contradictory. I therefore analysed AC microsatellites in 1163 genome sequences from the 1000 genomes project. I used the presence of rare alleles, which are likely to be very recent in origin, as a surrogate measure of mutation rate. I show that rare alleles are more likely to occur at locus-population combinations with higher heterozygosity even when all populations carry exactly the same number of alleles. PMID:26740567

  10. A mutation in the uvi4 gene promotes progression of endo-reduplication and confers increased tolerance towards ultraviolet B light.

    PubMed

    Hase, Yoshihiro; Trung, Khuat Huu; Matsunaga, Tsukasa; Tanaka, Atsushi

    2006-04-01

    We have isolated and characterized a new ultraviolet B (UV-B)-resistant mutant, uvi4 (UV-B-insensitive 4), of Arabidopsis. The fresh weight (FW) of uvi4 plants grown under supplemental UV-B light was more than twice that of the wild-type. No significant difference was found in their ability to repair the UV-B-induced cyclobutane pyrimidine dimers, or in the amount of UV-B absorptive compounds, both of which are well-known factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. We found that the hypocotyl cells in uvi4 undergo one extra round of endo-reduplication. The uvi4 mutation also promoted the progression of endo-reduplication during leaf development. The UVI4 gene is expressed mainly in actively dividing cells. In the leaves of P(UVI4)::GUS plants, the GUS signal disappeared in basipetal fashion as the leaf developed. The total leaf blade area was not different between uvi4 and the wild-type through leaf development, while the average cell area in the adaxial epidermis was considerably larger in uvi4, suggesting that the uvi4 leaves have fewer but larger epidermal cells. These results suggest that UVI4 is necessary for the maintenance of the mitotic state, and the loss of UVI4 function stimulated endo-reduplication. Tetraploid Arabidopsis was hyper-resistant to UV-B compared to diploid Arabidopsis, suggesting that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4 mutant.

  11. Fitness cost of chromosomal drug resistance-conferring mutations.

    PubMed

    Sander, Peter; Springer, Burkhard; Prammananan, Therdsak; Sturmfels, Antje; Kappler, Martin; Pletschette, Michel; Böttger, Erik C

    2002-05-01

    To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

  12. Mutations in durum wheat SBEII genes conferring increased amylose and resistant starch affect grain yield components, semolina and pasta quality and fermentation responses in rats

    USDA-ARS?s Scientific Manuscript database

    Increased amylose in wheat (Triticum spp.) starch is associated with increased resistant starch, a fermentable dietary fiber. Fermentation of resistant starch in the large intestine produces short-chain fatty acids that provide human health benefits. Since wheat foods are an important component of t...

  13. Multidrug resistance conferred by novel DNA polymerase mutations in human cytomegalovirus isolates.

    PubMed

    Scott, Gillian M; Weinberg, Adriana; Rawlinson, William D; Chou, Sunwen

    2007-01-01

    The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.

  14. Filaggrin Mutations That Confer Risk of Atopic Dermatitis Confer Greater Risk for Eczema Herpeticum

    PubMed Central

    Gao, Pei-Song; Rafaels, Nicholas M; Hand, Tracey; Murray, Tanda; Boguniewicz, Mark; Hata, Tissa; Schneider, Lynda; Hanifin, Jon M; Gallo, Richard L; Gao, Li; Beaty, Terri H; Beck, Lisa A; Barnes, Kathleen C; Leung, Donald YM

    2015-01-01

    Background Loss-of-function null mutations R501X and 2282del4 in the skin barrier gene, filaggrin (FLG), represent the most replicated genetic risk factors for atopic dermatitis (AD). Associations have not been reported in African ancestry populations. Eczema herpeticum (ADEH) is a rare but serious complication of AD resulting from disseminated cutaneous HSV infections. Objective We aimed to determine whether FLG polymorphisms contribute to ADEH susceptibility. Methods Two common loss-of-function mutations plus nine FLG single nucleotide polymorphisms (SNPs) were genotyped in 278 European American AD patients, of whom 112 had ADEH, and 157non-atopic controls. Replication was performed on 339 African Americans. Results Significant associations were observed for both the R501X and 2282del4 mutations and AD among European Americans (P=1.46×10−5,3.87×10−5, respectively), but the frequency of the R501X mutation was three times higher (25.% vs 9%) for ADEH compared to AD without EH (odds ratio [OR]=3.4 (1.7–6.8), P=0.0002). Associations with ADEH were stronger with the combined null mutations (OR=10.1 (4.7–22.1), P=1.99×10−11). Associations with the R501X mutation were replicated in the African American population; the null mutation was absent among healthy African Americans, but present among AD (3.2%, P=0.035) and common among ADEH (9.4%; P=0.0049) patients. However, the 2282del4 mutation was absent among African American ADEH patients and rare (<1%) among healthy individuals. Conclusion The R501X mutation in the gene encoding filaggrin, one of the strongest genetic predictors of AD, confers an even greater risk for ADEH in both European and African ancestry populations, suggesting a role for defective skin barrier in this devastating condition. Clinical Implications The Filaggrin (FLG) R501X Mutation, a major risk factor for atopic dermatitis, confers a greater risk of the severe, HSV-associated complication, eczema herpeticum in diverse ethnic groups

  15. Selection of mutations for increased protein stability.

    PubMed

    van den Burg, Bertus; Eijsink, Vincent G H

    2002-08-01

    There are many ways to select mutations that increase the stability of proteins, including rational design, functional screening of randomly generated mutant libraries, and comparison of naturally occurring homologous proteins. The protein engineer's toolbox is expanding and the number of successful examples of engineered protein stability is increasing. Still, the selection of thermostable mutations is not a standard process. Selection is complicated by lack of knowledge of the process that leads to thermal inactivation and by the fact that proteins employ a large variety of structural tricks to achieve stability.

  16. Ribosomal Mutations in Arcanobacterium pyogenes Confer a Unique Spectrum of Macrolide Resistance

    PubMed Central

    Jost, B. Helen; Trinh, Hien T.; Songer, J. Glenn; Billington, Stephen J.

    2004-01-01

    Four macrolide-resistant Arcanobacterium pyogenes isolates contained A2058T, A2058G, or C2611G (Escherichia coli numbering) mutations in their 23S rRNA genes. While these mutations conferred resistance to erythromycin, oleandomycin, and spiramycin, they did not confer resistance to tylosin. PMID:14982799

  17. Mutations Located outside the Integrase Gene Can Confer Resistance to HIV-1 Integrase Strand Transfer Inhibitors.

    PubMed

    Malet, Isabelle; Subra, Frédéric; Charpentier, Charlotte; Collin, Gilles; Descamps, Diane; Calvez, Vincent; Marcelin, Anne-Geneviève; Delelis, Olivier

    2017-09-26

    Resistance to the integrase strand transfer inhibitors raltegravir and elvitegravir is often due to well-identified mutations in the integrase gene. However, the situation is less clear for patients who fail dolutegravir treatment. Furthermore, most in vitro experiments to select resistance to dolutegravir have resulted in few mutations of the integrase gene. We performed an in vitro dolutegravir resistance selection experiment by using a breakthrough method. First, MT4 cells were infected with human immunodeficiency virus type 1 (HIV-1) Lai. After integration into the host cell genome, cells were washed to remove unbound virus and 500 nM dolutegravir was added to the cell medium. This high concentration of the drug was maintained throughout selection. At day 80, we detected a virus highly resistant to dolutegravir, raltegravir, and elvitegravir that remained susceptible to zidovudine. Sequencing of the virus showed no mutations in the integrase gene but highlighted the emergence of five mutations, all located in the nef region, of which four were clustered in the 3' polypurine tract (PPT). Mutations selected in vitro by dolutegravir, located outside the integrase gene, can confer a high level of resistance to all integrase inhibitors. Thus, HIV-1 can use an alternative mechanism to develop resistance to integrase inhibitors by selecting mutations in the 3' PPT region. Further studies are required to determine to what extent these mutations may explain virological failure during integrase inhibitor therapy.IMPORTANCE Integrase strand transfer inhibitors (INSTIs) are increasingly used both as first-line drugs and in rescue therapy because of their low toxicity and high efficacy in both treatment-naive and treatment-experienced patients. Until now, resistance mutations selected by INSTI exposure have either been described in patients or selected in vitro and involve the integrase gene. Most mutations selected by raltegravir, elvitegravir, or dolutegravir exposure

  18. Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria.

    PubMed

    Kinga Modrzynska, Katarzyna; Creasey, Alison; Loewe, Laurence; Cezard, Timothee; Trindade Borges, Sofia; Martinelli, Axel; Rodrigues, Louise; Cravo, Pedro; Blaxter, Mark; Carter, Richard; Hunt, Paul

    2012-03-21

    Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions. Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter. Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to

  19. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

    PubMed Central

    Straimer, Judith; Gnädig, Nina F.; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D.; Urnov, Fyodor D.; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M.; Ménard, Didier; Fidock, David A.

    2015-01-01

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  20. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila

    PubMed Central

    Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-01-01

    ABSTRACT Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila. Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings. PMID:28069647

  1. Ribosomal Mutations Conferring Macrolide Resistance in Legionella pneumophila.

    PubMed

    Descours, Ghislaine; Ginevra, Christophe; Jacotin, Nathalie; Forey, Françoise; Chastang, Joëlle; Kay, Elisabeth; Etienne, Jerome; Lina, Gérard; Doublet, Patricia; Jarraud, Sophie

    2017-03-01

    Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.

  2. Mutations in the Pneumocystis jirovecii DHPS gene confer cross-resistance to sulfa drugs.

    PubMed

    Iliades, Peter; Meshnick, Steven R; Macreadie, Ian G

    2005-02-01

    Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP) and results in a high degree of mortality in immunocompromised individuals. The drug of choice for PCP is typically sulfamethoxazole (SMX) or dapsone in conjunction with trimethoprim. Drug treatment failure and sulfa drug resistance have been implicated epidemiologically with point mutations in dihydropteroate synthase (DHPS) of P. jirovecii. P. jirovecii cannot be cultured in vitro; however, heterologous complementation of the P. jirovecii trifunctional folic acid synthesis (PjFAS) genes with an E. coli DHPS-disrupted strain was recently achieved. This enabled the evaluation of SMX resistance conferred by DHPS mutations. In this study, we sought to determine whether DHPS mutations conferred sulfa drug cross-resistance to 15 commonly available sulfa drugs. It was established that the presence of amino acid substitutions (T(517)A or P(519)S) in the DHPS domain of PjFAS led to cross-resistance against most sulfa drugs evaluated. The presence of both mutations led to increased sulfa drug resistance, suggesting cooperativity and the incremental evolution of sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine [SCP] and sulfamethoxypyridazine [SMP]) that had a higher inhibitory potential than SMX were identified. In addition, SCP, SMP, and sulfadiazine (SDZ) were found to be capable of inhibiting the clinically observed drug-resistant mutants. We propose that SCP, SMP, and SDZ should be considered for clinical evaluation against PCP or for future development of novel sulfa drug compounds.

  3. Mutations in gidB Confer Low-Level Streptomycin Resistance in Mycobacterium tuberculosis▿†

    PubMed Central

    Wong, Sharon Y.; Lee, Jong Seok; Kwak, Hyun Kyung; Via, Laura E.; Boshoff, Helena I. M.; Barry, Clifton E.

    2011-01-01

    The global threat posed by drug-resistant strains of Mycobacterium tuberculosis demands a greater understanding of the genetic basis and molecular mechanisms that govern how such strains develop resistance against various antituberculous drugs. In this report, we examine a new genetic basis for resistance to one of the oldest and most widely used second-line drugs employed in tuberculosis therapy, streptomycin (SM). This marker for SM resistance was first discovered on the basis of genomic data obtained from drug-resistant M. tuberculosis strains collected in Japan, wherein an association was observed between SM resistance and a mutation in gidB, a putative 16S rRNA methyltransferase. By evaluating an isogenic ΔgidB mutant strain constructed from strain H37Rv, we demonstrate the causal role of gidB in conferring a low-level SM-resistant phenotype in M. tuberculosis with a 16-fold increase in the MIC over the parent strain. Among clinical isolates, the modest increase in SM resistance conferred by a gidB mutation leads to an MIC distribution of gidB mutation-containing strains that spans the recommended SM breakpoint concentration currently used in drug susceptibility testing protocols. As such, some gidB mutation-containing isolates are found to be SM sensitive, while others are SM resistant. On the basis of a pharmacodynamic analysis and Monte Carlo simulation, those isolates that are found to be SM sensitive should still respond favorably to SM treatment, while nearly half of those found to be SM resistant will likely respond poorly. This report provides the first microbiological evidence for the contribution of gidB in streptomycin resistance and examines the clinical implications of mutations in the gidB gene. PMID:21444711

  4. Molecular survey of turfgrass species for mutations conferring resistance to ACCase inhibiting herbicides

    USDA-ARS?s Scientific Manuscript database

    The control of grassy weeds in turfgrass is often problematic due to lack of herbicide selectivity. Seven different naturally occurring mutation sites have been reported to confer resistance to Acetyl coenzyme A carboxylase inhibiting herbicides. One or more of these mutation sites may hold potentia...

  5. Bactobolin Resistance Is Conferred by Mutations in the L2 Ribosomal Protein

    PubMed Central

    Chandler, Josephine R.; Truong, Thao T.; Silva, Patricia M.; Seyedsayamdost, Mohammad R.; Carr, Gavin; Radey, Matthew; Jacobs, Michael A.; Sims, Elizabeth H.; Clardy, Jon; Greenberg, E. Peter

    2012-01-01

    ABSTRACT Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). PMID:23249812

  6. Increased transversions in a novel mutator colon cancer cell line.

    PubMed

    Eshleman, J R; Donover, P S; Littman, S J; Swinler, S E; Li, G M; Lutterbaugh, J D; Willson, J K; Modrich, P; Sedwick, W D; Markowitz, S D; Veigl, M L

    1998-03-05

    We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

  7. Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma.

    PubMed

    Jiang, Xiaomo; Hao, Huai-Xiang; Growney, Joseph D; Woolfenden, Steve; Bottiglio, Cindy; Ng, Nicholas; Lu, Bo; Hsieh, Mindy H; Bagdasarian, Linda; Meyer, Ronald; Smith, Timothy R; Avello, Monika; Charlat, Olga; Xie, Yang; Porter, Jeffery A; Pan, Shifeng; Liu, Jun; McLaughlin, Margaret E; Cong, Feng

    2013-07-30

    A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells. LGK974 inhibited proliferation and induced differentiation of RNF43-mutant pancreatic adenocarcinoma xenograft models. Our data suggest that mutational inactivation of RNF43 in pancreatic adenocarcinoma confers Wnt dependency, and the presence of RNF43 mutations could be used as a predictive biomarker for patient selection supporting the clinical development of Wnt inhibitors in subtypes of cancer.

  8. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

    PubMed

    Prudhomme, M; Méjean, V; Martin, B; Claverys, J P

    1991-11-01

    DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.

  9. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Emergence of a dhfr mutation conferring high-level drug resistance in Plasmodium falciparum populations from southwest Uganda.

    PubMed

    Lynch, Caroline; Pearce, Richard; Pota, Hirva; Cox, Jonathan; Abeku, Tarekegn A; Rwakimari, John; Naidoo, Inbarani; Tibenderana, James; Roper, Cally

    2008-06-01

    The S108N, C59R, and N51I mutations in the Plasmodium falciparum gene that encodes dihydrofolate reductase, dhfr, confer resistance to pyrimethamine and are common in Africa. However, the I164L mutation, which confers high-level resistance, is rarely seen. We found a 14% prevalence of the I164L mutation among a sample of 51 patients with malaria in Kabale District in southwest Uganda in 2005 and a 4% prevalence among 72 patients with malaria in the neighboring district of Rukungiri during the same year. Surveillance at 6 sites across Uganda during 2002-2004 reported a single case of infection involving an I164L mutant, also in the southwest, suggesting that this is a regional hot spot. The spatial clustering and increasing prevalence of the I164L mutation is indicative of local transmission of the mutant. Targeted surveillance is needed to confirm the extent of the spread of the I164L mutation and to monitor the impact of I164L on the efficacy of antifolates for intermittent preventive treatment of pregnant women and/or infants with falciparum malaria.

  11. A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

    PubMed

    Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

    2015-09-01

    Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs.

  12. Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer

    PubMed Central

    Tavtigian, Sean V.; Oefner, Peter J.; Babikyan, Davit; Hartmann, Anne; Healey, Sue; Le Calvez-Kelm, Florence; Lesueur, Fabienne; Byrnes, Graham B.; Chuang, Shu-Chun; Forey, Nathalie; Feuchtinger, Corinna; Gioia, Lydie; Hall, Janet; Hashibe, Mia; Herte, Barbara; McKay-Chopin, Sandrine; Thomas, Alun; Vallée, Maxime P.; Voegele, Catherine; Webb, Penelope M.; Whiteman, David C.; Sangrajrang, Suleeporn; Hopper, John L.; Southey, Melissa C.; Andrulis, Irene L.; John, Esther M.; Chenevix-Trench, Georgia

    2009-01-01

    The susceptibility gene for ataxia telangiectasia, ATM, is also an intermediate-risk breast-cancer-susceptibility gene. However, the spectrum and frequency distribution of ATM mutations that confer increased risk of breast cancer have been controversial. To assess the contribution of rare variants in this gene to risk of breast cancer, we pooled data from seven published ATM case-control mutation-screening studies, including a total of 1544 breast cancer cases and 1224 controls, with data from our own mutation screening of an additional 987 breast cancer cases and 1021 controls. Using an in silico missense-substitution analysis that provides a ranking of missense substitutions from evolutionarily most likely to least likely, we carried out analyses of protein-truncating variants, splice-junction variants, and rare missense variants. We found marginal evidence that the combination of ATM protein-truncating and splice-junction variants contribute to breast cancer risk. There was stronger evidence that a subset of rare, evolutionarily unlikely missense substitutions confer increased risk. On the basis of subset analyses, we hypothesize that rare missense substitutions falling in and around the FAT, kinase, and FATC domains of the protein may be disproportionately responsible for that risk and that a subset of these may confer higher risk than do protein-truncating variants. We conclude that a comparison between the graded distributions of missense substitutions in cases versus controls can complement analyses of truncating variants and help identify susceptibility genes and that this approach will aid interpretation of the data emerging from new sequencing technologies. PMID:19781682

  13. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  14. Mitochondrial DNA Mutations in Mutator Mice Confer Respiration Defects and B-Cell Lymphoma Development

    PubMed Central

    Mito, Takayuki; Kikkawa, Yoshiaki; Shimizu, Akinori; Hashizume, Osamu; Katada, Shun; Imanishi, Hirotake; Ota, Azusa; Kato, Yukina; Nakada, Kazuto; Hayashi, Jun-Ichi

    2013-01-01

    Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ0) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan. PMID:23418460

  15. Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

    PubMed

    Mito, Takayuki; Kikkawa, Yoshiaki; Shimizu, Akinori; Hashizume, Osamu; Katada, Shun; Imanishi, Hirotake; Ota, Azusa; Kato, Yukina; Nakada, Kazuto; Hayashi, Jun-Ichi

    2013-01-01

    Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0)) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

  16. Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice

    PubMed Central

    Sanchez, Jamila R.; Reddick, Traci L.; Perez, Marissa; Centonze, Victoria E.; Mitra, Sankar; Izumi, Tadahide; McMahan, C. Alex; Walter, Christi A.

    2015-01-01

    Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells. PMID:26201249

  17. Novel mutations in ribosomal proteins L4 and L22 that confer erythromycin resistance in Escherichia coli.

    PubMed

    Zaman, Sephorah; Fitzpatrick, Megan; Lindahl, Lasse; Zengel, Janice

    2007-11-01

    L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated 'tentacles' that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance.

  18. Novel mutations in ribosomal proteins L4 and L22 that confer erythromycin resistance in Escherichia coli

    PubMed Central

    Zaman, Sephorah; Fitzpatrick, Megan; Lindahl, Lasse; Zengel, Janice

    2007-01-01

    L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated ‘tentacles’ that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. Mutations in the tentacles of L4 and L22 confer macrolide resistance in a variety of pathogenic and non-pathogenic bacteria. In Escherichia coli, a Lys-to-Glu mutation in L4 and a three-amino-acid deletion in the L22 had been reported. To learn more about the roles of the tentacles in ribosome assembly and function, we isolated additional erythromycin-resistant E. coli mutants. Eight new mutations mapped in L4, all within the tentacle. Two new mutations were identified in L22; one mapped outside the tentacle. Insertion mutations were found in both genes. All of the mutants grew slower than the parent, and they all showed reduced in vivo rates of peptide-chain elongation and increased levels of precursor 23S rRNA. Large insertions in L4 and L22 resulted in very slow growth and accumulation of abnormal ribosomal subunits. Our results highlight the important role of L4 and L22 in ribosome function and assembly, and indicate that a variety of changes in these proteins can mediate macrolide resistance. PMID:17956547

  19. Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

    PubMed Central

    Almeida, Deepak; Ioerger, Thomas; Tyagi, Sandeep; Li, Si-Yang; Mdluli, Khisimuzi; Andries, Koen; Grosset, Jacques; Sacchettini, Jim

    2016-01-01

    The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene. PMID:27185800

  20. Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis.

    PubMed

    Almeida, Deepak; Ioerger, Thomas; Tyagi, Sandeep; Li, Si-Yang; Mdluli, Khisimuzi; Andries, Koen; Grosset, Jacques; Sacchettini, Jim; Nuermberger, Eric

    2016-08-01

    The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

  1. Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation

    PubMed Central

    Charmandari, E.; Guan, R.; Zhang, M.; Silveira, L. G.; Fan, Q. R.; Chrousos, G. P.; Sertedaki, A. C.; Latronico, A. C.

    2016-01-01

    We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated. PMID:26554443

  2. Germline mutation in the RAD51B gene confers predisposition to breast cancer

    PubMed Central

    2013-01-01

    Background Most currently known breast cancer predisposition genes play a role in DNA repair by homologous recombination. Recent studies conducted on RAD51 paralogs, involved in the same DNA repair pathway, have identified rare germline mutations conferring breast and/or ovarian cancer predisposition in the RAD51C, RAD51D and XRCC2 genes. The present study analysed the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) to estimate their contribution to breast and ovarian cancer predisposition. Methods The study was conducted on 142 unrelated patients with breast and/or ovarian cancer either with early onset or with a breast/ovarian cancer family history. Patients were referred to a French family cancer clinic and had been previously tested negative for a BRCA1/2 mutation. Coding sequences of the five genes were analysed by EMMA (Enhanced Mismatch Mutation Analysis). Detected variants were characterized by Sanger sequencing analysis. Results Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys. No RAD51D and XRCC2 gene mutations were detected. These mutations and variants were detected in families with both breast and ovarian cancers, except for the RAD51B c.475C > T/p.Arg159Cys variant that occurred in a family with 3 breast cancer cases. Conclusions This study identified the first RAD51B mutation in a breast and ovarian cancer family and is the first report of XRCC3 mutation analysis in breast and ovarian cancer. It confirms that RAD51 paralog mutations confer breast and ovarian cancer predisposition and are rare events. In view of the low frequency of RAD51 paralog mutations, international collaboration of family cancer clinics will be required to more accurately estimate their penetrance and establish clinical guidelines in carrier individuals. PMID

  3. Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells

    PubMed Central

    Mertz, Tony M.; Baranovskiy, Andrey G.; Wang, Jing; Tahirov, Tahir H.; Shcherbakova, Polina V.

    2017-01-01

    Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→TA transversions and GC→AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors. PMID:28368425

  4. Association of TERT Promoter Mutation, But Not BRAF Mutation, With Increased Mortality in PTC

    PubMed Central

    George, Jonathan R.; Henderson, Ying C.; Williams, Michelle D.; Roberts, Dianna B.; Hei, Hu; Lai, Stephen Y.

    2015-01-01

    Context: Papillary thyroid carcinoma (PTC) carrying the BRAF mutation has been reported to be associated with high recurrence and potentially increased mortality. PTC carrying the TERT promoter mutation has been associated with older age, recurrence, and aggressive disease. Objective: The objective of this study was to determine the association of BRAF and TERT promoter gene alterations with recurrence and survival in a high-risk population. Design: Genomic DNA was analyzed for the BRAF mutation from 256 persistent/recurrent PTC (p/rPTC; 202 new, 54 previously reported) and for the TERT promoter mutation and polymorphism (242 p/rPTC). Two-tailed Fisher exact tests or the Pearson χ2 test were performed for the associations between mutations and other variables. Overall and disease-free survivals were compared by log rank tests on Kaplan-Meier plots and by Cox regression analysis. TERT promoter constructs were tested in PTC cell lines to determine their activities in these cells. Results: BRAF V600E mutation was identified in 235 of 256 (91.8%), TERT promoter mutation at −124 was detected in 77 of 242 (31.8%), and TERT promoter polymorphism at −245 was found in 113 of 242 (46.7%) p/rPTC patients. A significant difference in survival was found in p/rPTC patients with the TERT promoter mutation, which also displayed increased activity in vitro as compared to the nonmutated promoter sequence. No association was noted between the BRAF mutation or TERT promoter polymorphism and recurrence or survival. A drawback of our study could be the limited number of patients with nonmutated BRAF (21 of 256 [8.2%]). Conclusions: Mutation in the TERT promoter, but not in BRAF, was associated with decreased survival in 19 (24.7%) p/rPTC patients who died of disease and in 38 (49.4%) p/rPTC patients who died at last contact. The presence or absence of the BRAF mutation and TERT promoter polymorphism, however, was not significantly correlated with survival. PMID:26461266

  5. Association of TERT Promoter Mutation, But Not BRAF Mutation, With Increased Mortality in PTC.

    PubMed

    George, Jonathan R; Henderson, Ying C; Williams, Michelle D; Roberts, Dianna B; Hei, Hu; Lai, Stephen Y; Clayman, Gary L

    2015-12-01

    Papillary thyroid carcinoma (PTC) carrying the BRAF mutation has been reported to be associated with high recurrence and potentially increased mortality. PTC carrying the TERT promoter mutation has been associated with older age, recurrence, and aggressive disease. The objective of this study was to determine the association of BRAF and TERT promoter gene alterations with recurrence and survival in a high-risk population. Genomic DNA was analyzed for the BRAF mutation from 256 persistent/recurrent PTC (p/rPTC; 202 new, 54 previously reported) and for the TERT promoter mutation and polymorphism (242 p/rPTC). Two-tailed Fisher exact tests or the Pearson χ(2) test were performed for the associations between mutations and other variables. Overall and disease-free survivals were compared by log rank tests on Kaplan-Meier plots and by Cox regression analysis. TERT promoter constructs were tested in PTC cell lines to determine their activities in these cells. BRAF V600E mutation was identified in 235 of 256 (91.8%), TERT promoter mutation at -124 was detected in 77 of 242 (31.8%), and TERT promoter polymorphism at -245 was found in 113 of 242 (46.7%) p/rPTC patients. A significant difference in survival was found in p/rPTC patients with the TERT promoter mutation, which also displayed increased activity in vitro as compared to the nonmutated promoter sequence. No association was noted between the BRAF mutation or TERT promoter polymorphism and recurrence or survival. A drawback of our study could be the limited number of patients with nonmutated BRAF (21 of 256 [8.2%]). Mutation in the TERT promoter, but not in BRAF, was associated with decreased survival in 19 (24.7%) p/rPTC patients who died of disease and in 38 (49.4%) p/rPTC patients who died at last contact. The presence or absence of the BRAF mutation and TERT promoter polymorphism, however, was not significantly correlated with survival.

  6. Inhibition of FAAH confers increased stem cell migration via PPARα

    PubMed Central

    Wollank, Yvonne; Ramer, Robert; Ivanov, Igor; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2015-01-01

    Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors. PMID:26263913

  7. Inhibition of FAAH confers increased stem cell migration via PPARα.

    PubMed

    Wollank, Yvonne; Ramer, Robert; Ivanov, Igor; Salamon, Achim; Peters, Kirsten; Hinz, Burkhard

    2015-10-01

    Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors.

  8. Mutations in the aph(2")-Ic Gene Are Responsible for Increased Levels of Aminoglycoside Resistance

    PubMed Central

    Lee, Hae Kyung; Vakulenko, Sergei B.; Clewell, Don B.; Lerner, Stephen A.; Chow, Joseph W.

    2002-01-01

    Random PCR mutagenesis of the enterococcal aph(2")-Ic gene followed by selection for mutant enzymes that confer enhanced levels of aminoglycoside resistance resulted in mutants of APH(2")-Ic with His-258-Leu and Phe-108-Leu substitutions, all of which conferred rises in the MICs of several aminoglycosides. The mutated residues are located outside conserved regions of aminoglycoside phosphotransferases. PMID:12234853

  9. Novel K540N mutation in Plasmodium falciparum dihydropteroate synthetase confers a lower level of sulfa drug resistance than does a K540E mutation.

    PubMed

    Lumb, Vanshika; Sharma, Yagya D

    2011-05-01

    Sulfadoxine (SDX) and sulfamethoxazole (SMX) each inhibit the Plasmodium falciparum dihydropteroate synthetase (PfDHPS), and certain point mutations in this enzyme yield the drug-resistant parasite. Using a simple Escherichia coli model system, we describe here the effect of the recently reported novel K540N mutation in PfDHPS on the level of SDX/SMX resistance. The survival rate of the transformed E. coli (DHPS-deficient strain) under different SDX or SMX concentrations revealed that the K540N mutation confers a lower level of drug resistance than its contemporary K540E mutation. Further, SMX was more effective than SDX in the E. coli system.

  10. Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates.

    PubMed

    Moon, Dong Chan; Seol, Sung Yong; Gurung, Mamata; Jin, Jong Sook; Choi, Chul Hee; Kim, Jungmin; Lee, Yoo Chul; Cho, Dong Taek; Lee, Je Chul

    2010-01-01

    Mutations in DNA gyrase and topoisomerase IV genes are the main mechanisms of resistance to quinolones. In this study, we determined mutations in gyrA, gyrB, parC and parE among 57 ciprofloxacin-resistant Escherichia coli isolates from a South Korean hospital and analysed the relationship between the minimal inhibitory concentrations (MICs) of fluoroquinolones and mutations in the topoisomerase IV gene. All ciprofloxacin-resistant E. coli isolates carried double mutations in gyrA and at least a single mutation in parC; some isolates also carried a single mutation in parE. The most common mutations were S83L and D87N in gyrA, S80I in parC and S458A in parE, which accounted for 25% of isolates. Single mutations in parE at L445I, S458P and S458W were identified for the first time. Double mutations in parC and a combination of single mutations in parC and parE significantly increased the MIC values of fluoroquinolones. In vitro induction of resistance to ciprofloxacin showed that double mutations in gyrA were a prerequisite to conferring a resistant phenotype to fluoroquinolones, and an additional mutation in the topoisomerase IV gene increased the MIC values of ciprofloxacin. In conclusion, emergence of a new mutation in parC and parE and its accumulation induces high levels of resistance to fluoroquinolones in E. coli.

  11. The Distribution of Fitness Costs of Resistance-Conferring Mutations Is a Key Determinant for the Future Burden of Drug-Resistant Tuberculosis: A Model-Based Analysis.

    PubMed

    Knight, Gwenan M; Colijn, Caroline; Shrestha, Sourya; Fofana, Mariam; Cobelens, Frank; White, Richard G; Dowdy, David W; Cohen, Ted

    2015-10-15

    Drug resistance poses a serious challenge for the control of tuberculosis in many settings. It is well established that the expected future trend in resistance depends on the reproductive fitness of drug-resistant Mycobacterium tuberculosis. However, the variability in fitness between strains with different resistance-conferring mutations has been largely ignored when making these predictions. We developed a novel approach for incorporating the variable fitness costs of drug resistance-conferring mutations and for tracking this distribution of fitness costs over time within a transmission model. We used this approach to describe the effects of realistic fitness cost distributions on the future prevalence of drug-resistant tuberculosis. The shape of the distribution of fitness costs was a strong predictor of the long-term prevalence of resistance. While, as expected, lower average fitness costs of drug resistance-conferring mutations were associated with more severe epidemics of drug-resistant tuberculosis, fitness distributions with greater variance also led to higher levels of drug resistance. For example, compared to simulations in which the fitness cost of resistance was fixed, introducing a realistic amount of variance resulted in a 40% increase in prevalence of drug-resistant tuberculosis after 20 years. The differences in the fitness costs associated with drug resistance-conferring mutations are a key determinant of the future burden of drug-resistant tuberculosis. Future studies that can better establish the range of fitness costs associated with drug resistance-conferring mutations will improve projections and thus facilitate better public health planning efforts. © 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.

  12. The Distribution of Fitness Costs of Resistance-Conferring Mutations Is a Key Determinant for the Future Burden of Drug-Resistant Tuberculosis: A Model-Based Analysis

    PubMed Central

    Knight, Gwenan M.; Colijn, Caroline; Shrestha, Sourya; Fofana, Mariam; Cobelens, Frank; White, Richard G.; Dowdy, David W.; Cohen, Ted

    2015-01-01

    Background. Drug resistance poses a serious challenge for the control of tuberculosis in many settings. It is well established that the expected future trend in resistance depends on the reproductive fitness of drug-resistant Mycobacterium tuberculosis. However, the variability in fitness between strains with different resistance-conferring mutations has been largely ignored when making these predictions. Methods. We developed a novel approach for incorporating the variable fitness costs of drug resistance-conferring mutations and for tracking this distribution of fitness costs over time within a transmission model. We used this approach to describe the effects of realistic fitness cost distributions on the future prevalence of drug-resistant tuberculosis. Results. The shape of the distribution of fitness costs was a strong predictor of the long-term prevalence of resistance. While, as expected, lower average fitness costs of drug resistance–conferring mutations were associated with more severe epidemics of drug-resistant tuberculosis, fitness distributions with greater variance also led to higher levels of drug resistance. For example, compared to simulations in which the fitness cost of resistance was fixed, introducing a realistic amount of variance resulted in a 40% increase in prevalence of drug-resistant tuberculosis after 20 years. Conclusions. The differences in the fitness costs associated with drug resistance–conferring mutations are a key determinant of the future burden of drug-resistant tuberculosis. Future studies that can better establish the range of fitness costs associated with drug resistance–conferring mutations will improve projections and thus facilitate better public health planning efforts. PMID:26409276

  13. Ordered accumulation of mutations conferring resistance to sulfadoxine-pyrimethamine in the Plasmodium falciparum parasite.

    PubMed

    Mita, Toshihiro; Ohashi, Jun; Venkatesan, Meera; Marma, Aung Swi Prue; Nakamura, Masatoshi; Plowe, Christopher V; Tanabe, Kazuyuki

    2014-01-01

    Monitoring the prevalence of drug resistant Plasmodium falciparum is essential for effective malaria control. Resistance to pyrimethamine and sulfadoxine increases as mutations accumulate in the parasite genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), respectively. Although parasites are exposed to these antifolate drugs simultaneously, it remains virtually unknown whether dhfr and dhps mutations accumulate along interrelated paths. We investigated the order of step-wise accumulation in dhfr and dhps by cumulative analyses using binomial tests in 575 P. falciparum isolates obtained from 7 countries in Asia and Melanesia. An initial step in the accumulation of mutations preferentially occurred in dhfr (2 mutations), followed by 1 mutation in dhps. In a subsequent step, mutations were estimated separately for 5 dhfr/dhps-resistant lineages identified using 12 microsatellites flanking dhfr and dhps. Among these lineages, we found 3 major mutational paths, each of which follows a unique stepwise trajectory to produce the most highly resistant form with 4 mutations in dhfr and 3 in dhps. The ordered accumulation of mutations in dhfr and dhps elucidated here will assist in predicting the status and progression of antifolate resistance in malaria-endemic regions where antifolate drugs are used for intermittent preventive treatment.

  14. FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

    PubMed Central

    Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

    2014-01-01

    Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

  15. Mutation in the Bimd Gene of Aspergillus Nidulans Confers a Conditional Mitotic Block and Sensitivity to DNA Damaging Agents

    PubMed Central

    Denison, S. H.; Kafer, E.; May, G. S.

    1993-01-01

    Mutation in the bimD gene of Aspergillus nidulans results in a mitotic block in anaphase characterized by a defective mitosis. Mutation in bimD also confers, at temperatures permissive for the mitotic arrest phenotype, an increased sensitivity to DNA damaging agents, including methyl methanesulfonate and ultraviolet light. In order to better understand the relationship between DNA damage and mitotic progression, we cloned the bimD gene from Aspergillus. A cosmid containing the bimD gene was identified among pools of cosmids by cotransformation with the nutritional selective pyrG gene of a strain carrying the recessive, temperature-sensitive lethal bimD6 mutation. The bimD gene encodes a predicted polypeptide of 166,000 daltons in mass and contains amino acid sequence motifs similar to those found in some DNA-binding transcription factors. These sequences include a basic domain followed by a leucine zipper, which together are called a bZIP motif, and a carboxyl-terminal domain enriched in acidic amino acids. Overexpression of the wild-type bimD protein resulted in an arrest of the nuclear division cycle that was reversible and determined to be in either the G(1) or S phase of the cell cycle. Our data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis. PMID:8375649

  16. Is Increased Low-dose somatic Radiosensitivity Associated with Increased Transgenerational Germline Mutation

    SciTech Connect

    Brenner, David J.

    2008-10-02

    Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm+/–) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm+/– males did not significantly differ from that in wild-type BALB/c mice. Acute gamma-ray exposure did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm+/– and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analyzed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

  17. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP

    SciTech Connect

    Yun, C.H.; Mengwasser, K.E.; Toms, A.V.; Woo, M.S.; Greulich, H.; Wong, K.K.; Meyerson, M.; Eck, M.J.

    2008-07-15

    Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the 'gatekeeper' residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a 'generic' resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

  18. Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance

    PubMed Central

    Wei, Xiaolei; Chen, Peiying; Gao, Rongsui; Li, Yeqi; Zhang, Anxue; Liu, Feifei

    2016-01-01

    ABSTRACT The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance. PMID:27799210

  19. Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance.

    PubMed

    Wei, Xiaolei; Chen, Peiying; Gao, Rongsui; Li, Yeqi; Zhang, Anxue; Liu, Feifei; Lu, Ling

    2017-01-01

    The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance. Copyright © 2016 American Society for Microbiology.

  20. Increasing Role of Titin Mutations in Neuromuscular Disorders

    PubMed Central

    Savarese, Marco; Sarparanta, Jaakko; Vihola, Anna; Udd, Bjarne; Hackman, Peter

    2016-01-01

    The TTN gene with 363 coding exons encodes titin, a giant muscle protein spanning from the Z-disk to the M-band within the sarcomere. Mutations in the TTN gene have been associated with different genetic disorders, including hypertrophic and dilated cardiomyopathy and several skeletal muscle diseases. Before the introduction of next generation sequencing (NGS) methods, the molecular analysis of TTN has been laborious, expensive and not widely used, resulting in a limited number of mutations identified. Recent studies however, based on the use of NGS strategies, give evidence of an increasing number of rare and unique TTN variants. The interpretation of these rare variants of uncertain significance (VOUS) represents a challenge for clinicians and researchers. The main aim of this review is to describe the wide spectrum of muscle diseases caused by TTN mutations so far determined, summarizing the molecular findings as well as the clinical data, and to highlight the importance of joint efforts to respond to the challenges arising from the use of NGS. An international collaboration through a clinical and research consortium and the development of a single accessible database listing variants in the TTN gene, identified by high throughput approaches, may be the key to a better assessment of titinopathies and to systematic genotype– phenotype correlation studies. PMID:27854229

  1. Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model

    PubMed Central

    Li, Li; Piloto, Obdulio; Nguyen, Ho Bao; Greenberg, Kathleen; Takamiya, Kogo; Racke, Frederick; Huso, David; Small, Donald

    2008-01-01

    Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)–spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia. PMID:18245664

  2. Renal FMD may not confer a familial hypertensive risk nor is it caused by ACTA2 mutations.

    PubMed

    Marks, Stephen D; Gullett, Ambrose M; Brennan, Eileen; Tullus, Kjell; Jaureguiberry, Graciana; Klootwijk, Enriko; Stanescu, Horia C; Kleta, Robert; Woolf, Adrian S

    2011-10-01

    Renal fibromuscular dysplasia (FMD) can cause hypertension, and previous reports suggest that FMD is familial. We hypothesized that, in families containing an individual with proven FMD, relatives of index cases would have an increased risk of hypertension. ACTA2 mutations cause a spectrum of extra-renal arteriopathy, leading to our second hypothesis that mutations are implicated in FMD. The blood pressure of first-degree relatives was measured using standard devices and, when indicated, with 24-h ambulatory monitoring. Leucocyte DNA was obtained from FMD index cases and ACTA2 sequenced. Thirteen unrelated index cases, aged 2-32 (median 15) years, were recruited. Blood pressure was assessed in 40 first-degree relatives, comprising 22 parents aged 28-58 (median 44) years and 18 siblings aged 3-30 (median 13) years. Hypertension was evident in six (27%) parents but in none of the eight adult siblings. Of the ten screened siblings aged less than 18 years, one teenager was pre-hypertensive (90th-95th centile), the remainder being normotensive. No ACTA2 mutations were found in 13 index cases. Hypertension was evident in 20% of all assessed adult first-degree relatives and is therefore not increased relative to 25% of the adult population. Although hypertensive parents did not undergo angiography to assign FMD status, this observation, together with the lack of hypertension in 18 siblings, indicates that FMD is unlikely to confer an excess hypertension risk in first-degree relatives up to middle-age. Furthermore, in our cohort, FMD was not caused by ACTA2 mutations.

  3. A mutation in yeast topoisomerase II that confers hypersensitivity to multiple classes of topoisomerase II poisons.

    PubMed

    Dong, J; Walker, J; Nitiss, J L

    2000-03-17

    A mutation was constructed in the CAP homology domain of yeast topoisomerase II that resulted in hypersensitivity to the intercalating agent N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and the fluoroquinolone 6, 8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxyli c acid, but not to etoposide. This mutation, which changes threonine at position 744 to proline, also confers hypersensitivity to anti-bacterial fluoroquinolones. The purified T744P mutant protein had wild type enzymatic activity in the absence of drugs, and no alteration in drug-independent DNA cleavage. Enhanced DNA cleavage in the presence of N-[4-(9-acridinylamino)-3-methoxy-phenyl]methanesulfonamide and fluoroquinolones was observed, in agreement with the results observed in vivo. DNA cleavage was also seen in the presence of norfloxacin and oxolinic acid, two quinolones that are inactive against eukaryotic topoisomerase II. The hypersensitivity was not associated with heat-stable covalent complexes, as was seen in another drug-hypersensitive mutant. Molecular modeling suggests that the mutation in the CAP homology domain may displace amino acids that play important roles in catalysis by topoisomerase II and may explain the drug-hypersensitive phenotype.

  4. Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations

    PubMed Central

    Menozzi, Philippe; Shi, Ming An; Lougarre, Andrée; Tang, Zhen Hua; Fournier, Didier

    2004-01-01

    Background Organophosphate and carbamate insecticides irreversibly inhibit acetylcholinesterase causing death of insects. Resistance-modified acetylcholinesterases(AChEs) have been described in many insect species and sequencing of their genes allowed several point mutations to be described. However, their relative frequency and their cartography had not yet been addressed. Results To analyze the most frequent mutations providing insecticide resistance in Drosophila melanogaster acetylcholinesterase, the Ace gene was cloned and sequenced in several strains harvested from different parts of the world. Sequence comparison revealed four widespread mutations, I161V, G265A, F330Y and G368A. We confirm here that mutations are found either isolated or in combination in the same protein and we show that most natural populations are heterogeneous, composed of a mixture of different alleles. In vitro expression of mutated proteins showed that combining mutations in the same protein has two consequences: it increases resistance level and provides a wide spectrum of resistance. Conclusion The presence of several alleles in natural populations, offering various resistance to carbamate and organophosphate compounds will complicate the establishment of resistance management programs. PMID:15018651

  5. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    PubMed Central

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  6. G118R and F121Y mutations identified in patients failing raltegravir treatment confer dolutegravir resistance.

    PubMed

    Munir, Soundasse; Thierry, Eloise; Malet, Isabelle; Subra, Frédéric; Calvez, Vincent; Marcelin, Anne-Geneviève; Deprez, Eric; Delelis, Olivier

    2015-03-01

    Strand transfer inhibitors (raltegravir, elvitegravir and dolutegravir) are now commonly used to inhibit HIV-1 integration. To date, three main pathways conferring raltegravir/elvitegravir resistance, involving residues Y143, Q148 and N155, have been described. However, no pathway has been clearly described for dolutegravir resistance. The aim of this study was to characterize the susceptibility of two mutations, F121Y and G118R, originally described in patients failing raltegravir-containing regimens, to dolutegravir and raltegravir, and then to compare the resistance of these mutations with that of other well-known mutations involved in raltegravir resistance. Both the F121Y and G118R mutations were introduced by site-directed mutagenesis into the pNL4.3 backbone and studied in cell-based and in vitro assays. The effects of the mutations were characterized at the different steps of infection by quantitative PCR. Results obtained with in vitro and ex vivo assays consistently showed that both mutations impaired the catalytic properties of integrase, especially at the integration step. Moreover, both mutations conferred an intermediate level of resistance to dolutegravir. Interestingly, the F121Y mutation, but not the G118R mutation, displayed differential resistance to raltegravir and dolutegravir. Indeed, the F121Y mutation was more resistant to raltegravir than to dolutegravir. Mutations at G118 and F121, which have been described in patients failing raltegravir-containing regimens, must be included in drug-resistance-testing algorithms. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. JAK-2 V617F mutation increases heparanase procoagulant activity.

    PubMed

    Kogan, Inna; Chap, Dafna; Hoffman, Ron; Axelman, Elena; Brenner, Benjamin; Nadir, Yona

    2016-01-01

    Patients with polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are at increased risk of arterial and venous thrombosis. In patients with ET a positive correlation was observed between JAK-2 V617F mutation, that facilitates erythropoietin receptor signalling, and thrombotic events, although the mechanism involved is not clear. We previously demonstrated that heparanase protein forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF) which leads to increased factor Xa production and subsequent activation of the coagulation system. The present study was aimed to evaluate heparanase procoagulant activity in myeloproliferative neoplasms. Forty bone marrow biopsies of patients with ET, PV, PMF and chronic myelogenous leukaemia (CML) were immunostained to heparanase, TF and TF pathway inhibitor (TFPI). Erythropoietin receptor positive cell lines U87 human glioma and MCF-7 human breast carcinoma were studied. Heparanase and TFPI staining were more prominent in ET, PV and PMF compared to CML. The strongest staining was in JAK-2 positive ET biopsies. Heparanase level and procoagulant activity were higher in U87 cells transfected to over express JAK-2 V617F mutation compared to control and the effect was reversed using JAK-2 inhibitors (Ruxolitinib, VZ3) and hydroxyurea, although the latter drug did not inhibit JAK-2 phosphorylation. Erythropoietin increased while JAK-2 inhibitors decreased the heparanase level and procoagulant activity in U87 and MCF-7 parental cells. In conclusion, JAK-2 is involved in heparanase up-regulation via the erythropoietin receptor. The present findings may potentially point to a new mechanism of thrombosis in JAK-2 positive ET patients.

  8. A single gene mutation that increases maize seed weight

    SciTech Connect

    Giroux, M.J.; Shaw, J.; Hannah, L.C. |

    1996-06-11

    The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3 feet to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts. 20 refs., 5 figs., 5 tabs.

  9. Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance

    PubMed Central

    LaMonte, Gregory; Lim, Michelle Yi-Xiu; Wree, Melanie; Reimer, Christin; Nachon, Marie; Corey, Victoria; Gedeck, Peter; Plouffe, David; Du, Alan; Figueroa, Nelissa; Yeung, Bryan; Winzeler, Elizabeth A.

    2016-01-01

    ABSTRACT Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites. PMID:27381290

  10. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor

    PubMed Central

    Yao, Jia-Long; Dong, Yi-Hu; Morris, Bret A. M.

    2001-01-01

    Fruit development in higher plants normally requires pollination and fertilization to stimulate cell division of specific floral tissues. In some cases, parthenocarpic fruit development proceeds without either pollination or fertilization. Parthenocarpic fruit without seed has higher commercial value than seeded fruit. Several apple (Malus domestica) mutants (Rae Ime, Spencer Seedless and Wellington Bloomless) are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruit. Through genetics, a single recessive gene has been identified to control this trait in apple. Flower phenotypes of these apple mutants are strikingly similar to those of the Arabidopsis mutant pistillata (pi), which produces flowers where petals are transformed to sepals and stamens to carpels. In this study, we have cloned the apple PI homolog (MdPI) that shows 64% amino acid sequence identity and closely conserved intron positions and mRNA expression patterns to the Arabidopsis PI. We have identified that in the apetalous mutants MdPI has been mutated by a retrotransposon insertion in intron 4 in the case of Rae Ime and in intron 6 in the case of Spencer Seedless and Wellington Bloomless. The insertion apparently abolishes the normal expression of the MdPI gene. We conclude that the loss of function mutation in the MdPI MADS-box transcription factor confers parthenocarpic fruit development in these apple varieties and demonstrates another function for the MADS- box gene family. The knowledge generated here could be used to produce parthenocarpic fruit cultivars through genetic engineering. PMID:11158635

  11. Frequency of spontaneous mutations that confer antibiotic resistance in Chlamydia spp.

    PubMed

    Binet, Rachel; Maurelli, Anthony T

    2005-07-01

    Mutations in rRNA genes (rrn) that confer resistance to ribosomal inhibitors are typically recessive or weakly codominant and have been mostly reported for clinical strains of pathogens possessing only one or two rrn operons, such as Helicobacter pylori and Mycobacterium spp. An analysis of the genome sequences of several members of the Chlamydiaceae revealed that these obligate intracellular bacteria harbor only one or two sets of rRNA genes. To study the contribution of rRNA mutations to the emergence of drug resistance in the Chlamydiaceae, we used the sensitivities of Chlamydia trachomatis L2 (two rrn operons) and Chlamydophila psittaci 6BC (one rrn operon) to the aminoglycoside spectinomycin as a model. Confluent cell monolayers were infected in a plaque assay with about 10(8) wild-type infectious particles and then treated with the antibiotic. After a 2-week incubation time, plaques formed by spontaneous spectinomycin-resistant (Spc(r)) mutants appeared with a frequency of 5 x 10(-5) for C. psittaci 6BC. No Spc(r) mutants were isolated for C. trachomatis L2, although the frequencies of rifampin resistance were in the same range for both strains (i.e., 10(-7)). The risk of emergence of Chlamydia strains resistant to tetracyclines and macrolides, the ribosomal drugs currently used to treat chlamydial infections, is discussed.

  12. The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids.

    PubMed

    Smith, T J; Lee, S H; Ingles, P J; Knipple, D C; Soderlund, D M

    1997-10-01

    Voltage-sensitive sodium channels encoded by a full-length cDNA corresponding to the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes either alone or in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1 cRNA alone produced very small (50-150 nA) sodium currents, whereas the combination of Vssc1 and tipE cRNAs produced robust (0.5-3 microA), rapidly inactivating sodium currents. The pyrethroid insecticide cismethrin prolonged the sodium current carried by Vssc1/tipE sodium channels during a depolarizing pulse and induced a tail current after repolarization. The Vssc1 cDNA was specifically mutated to substitute phenylalanine for leucine at position 1014 of the inferred amino acid sequence (L1014F), a polymorphism shown previously to be associated with the kdr (knockdown resistance) trait of the house fly. The L1014F substitution reduced the sensitivity of expressed house fly sodium channels to cismethrin at least 10-fold and increased the rate of decay of pyrethroid-induced sodium tail currents. These results demonstrate that the resistance-associated L1014F mutation confers a reduction in the sensitivity of house fly sodium channels to pyrethroids that is sufficient to account for the kdr resistance trait.

  13. Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster.

    PubMed

    Perry, Trent; Heckel, David G; McKenzie, John A; Batterham, Philip

    2008-05-01

    Resistance to insecticides by modification of their molecular targets is a serious problem in chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods. The spectrum of possible resistance-conferring mutations of this receptor is poorly understood. Prediction of resistance is complicated by the existence of multiple genes encoding the different subunits of this essential component of neurotransmission. We focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram was performed on hybrids carrying a deficiency for this chromosomal region. Two complementation groups were defined for the four strains isolated. Molecular characterisation of the mutations found lesions in two nAChR subunit genes, Dalpha1 (encoding an alpha-type subunit) and Dbeta2 (beta-type). Mutations conferring resistance in beta-type receptors have not previously been reported, but we found several lesions in the Dbeta2 sequence, including locations distant from the predicted neonicotinoid-binding site. This study illustrates that mutations in a single-receptor subunit can confer nitenpyram resistance. Moreover, some of the mutations may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than affecting binding affinities of neonicotinoids to the channel.

  14. ON pathway mutations increase susceptibility to form-deprivation myopia

    PubMed Central

    Chakraborty, Ranjay; Park, Han na; Hanif, Adam M.; Sidhu, Curran; Iuvone, P. Michael; Pardue, Machelle T.

    2015-01-01

    The ON pathway mutation in nob mice is associated with altered refractive development, and an increased susceptibility to form-deprivation (FD) myopia. In this study, we used mGluR6−/− mice, another ON pathway mutant, to determine whether the nob phenotype was due to the Nyx mutation or abnormal ON pathway transmission. Refractive development under a normal visual environment for mGluR6−/− and age-matched wild-type (WT) mice was measured every 2 weeks from 4 to 16 weeks of age. The response to monocular FD from 4 weeks of age was measured weekly in a separate cohort of mice. Refraction and ocular biometry were obtained using a photorefractor and optical coherence tomography. Retinas were harvested at 16 weeks, and analyzed for dopamine (DA) and DOPAC using high-performance liquid chromatography. Under normal conditions, mGluR6−/− mice were significantly more myopic than their WT controls (refraction at 12 weeks; WT: 9.40 ± 0.16 D, mGluR6−/−: 6.91 ± 0.38 D). Similar to nob mice, two weeks of FD resulted in a significant myopic shift of −5.57 ± 0.72 D in mGluR6−/− mice compared to −1.66 ± 0.19 D in WT animals. No significant axial length changes were observed with either normal or FD visual conditions. At 16 weeks, mGluR6−/− retinas showed significantly lower DOPAC levels (111.2 ± 33.0 pg/mg) compared to their WT counterparts (197.5 ± 11.2 pg/mg). Retinal DA levels were similar between the different genotypes. Our results indicate that reduced retinal DA metabolism/turnover may be associated with increased susceptibility to myopia in mice with ON pathway defect mutations. PMID:26072023

  15. RET mutation and increased angiogenesis in medullary thyroid carcinomas.

    PubMed

    Verrienti, Antonella; Tallini, Giovanni; Colato, Chiara; Boichard, Amélie; Checquolo, Saula; Pecce, Valeria; Sponziello, Marialuisa; Rosignolo, Francesca; de Biase, Dario; Rhoden, Kerry; Casadei, Gian Piero; Russo, Diego; Visani, Michela; Acquaviva, Giorgia; Ferdeghini, Marco; Filetti, Sebastiano; Durante, Cosimo

    2016-08-01

    Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs. RET mutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somatic RET mutations (RETmut group) and 34 with wild-type RET (RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared with RETwt tumors, RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels in RETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis in RETmut MTCs may be more intense and complete than that found in RETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density, RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors.

  16. A germ-line-selective advantage rather than an increased mutation rate can explain some unexpectedly common human disease mutations.

    PubMed

    Choi, Soo-Kyung; Yoon, Song-Ro; Calabrese, Peter; Arnheim, Norman

    2008-07-22

    Two nucleotide substitutions in the human FGFR2 gene (C755G or C758G) are responsible for virtually all sporadic cases of Apert syndrome. This condition is 100-1,000 times more common than genomic mutation frequency data predict. Here, we report on the C758G de novo Apert syndrome mutation. Using data on older donors, we show that spontaneous mutations are not uniformly distributed throughout normal testes. Instead, we find foci where C758G mutation frequencies are 3-4 orders of magnitude greater than the remaining tissue. We conclude this nucleotide site is not a mutation hot spot even after accounting for possible Luria-Delbruck "mutation jackpots." An alternative explanation for such foci involving positive selection acting on adult self-renewing Ap spermatogonia experiencing the rare mutation could not be rejected. Further, the two youngest individuals studied (19 and 23 years old) had lower mutation frequencies and smaller foci at both mutation sites compared with the older individuals. This implies that the mutation frequency of foci increases as adults age, and thus selection could explain the paternal age effect for Apert syndrome and other genetic conditions. Our results, now including the analysis of two mutations in the same set of testes, suggest that positive selection can increase the relative frequency of premeiotic germ cells carrying such mutations, although individuals who inherit them have reduced fitness. In addition, we compared the anatomical distribution of C758G mutation foci with both new and old data on the C755G mutation in the same testis and found their positions were not correlated with one another.

  17. Migraine Mutations Increase Stroke Vulnerability by Facilitating Ischemic Depolarizations

    PubMed Central

    Eikermann-Haerter, Katharina; Lee, Jeong Hyun; Yuzawa, Izumi; Liu, Christina H.; Zhou, Zhipeng; Shin, Hwa Kyoung; Zheng, Yi; Qin, Tao; Kurth, Tobias; Waeber, Christian; Ferrari, Michel D.; van den Maagdenberg, Arn M. J. M.; Moskowitz, Michael A.; Ayata, Cenk

    2012-01-01

    Background Migraine is an independent risk factor for stroke. Mechanisms underlying this association are unclear. Familial hemiplegic migraine (FHM), a migraine subtype that also carries an increased stroke risk, is a useful model for common migraine phenotypes because of shared aura and headache features, trigger factors, and underlying glutamatergic mechanisms. Methods and Results Here, we show that FHM type 1 (FHM1) mutations in CaV2.1 voltage-gated Ca2+ channels render the brain more vulnerable to ischemic stroke. Compared to wild-type, two FHM1 mutant mouse strains developed earlier onset of anoxic depolarization and more frequent peri-infarct depolarizations, associated with rapid expansion of infarct core on diffusion-weighted MRI and larger perfusion deficits on laser speckle flowmetry. Cerebral blood flow required for tissue survival was higher in the mutants, leading to infarction with milder ischemia. As a result, mutants developed larger infarcts and worse neurological outcomes after stroke, which were selectively attenuated by a glutamate receptor antagonist. Conclusions We propose that enhanced susceptibility to ischemic depolarizations akin to spreading depression predisposes migraineurs to infarction during mild ischemic events, thereby increasing the stroke risk. PMID:22144569

  18. Mex3c mutation reduces adiposity and increases energy expenditure.

    PubMed

    Jiao, Yan; George, Sunil K; Zhao, Qingguo; Hulver, Matthew W; Hutson, Susan M; Bishop, Colin E; Lu, Baisong

    2012-11-01

    The function of MEX3C, the mammalian homolog of Caenorhabditis elegans RNA-binding protein muscle excess 3 (MEX-3), was unknown until our recent report that MEX3C is necessary for normal postnatal growth and enhances the expression of local bone Igf1 expression. Here we report the pivotal role of Mex3c in energy balance regulation. Mex3c mutation caused leanness in both heterozygous and homozygous transgenic mice, as well as a more beneficial blood glucose and lipid profile in homozygous transgenic mice, in both sexes. Although transgenic mice showed normal food intake and fecal lipid excretion, they had increased energy expenditure independent of physical activity. Mutant mice had normal body temperature, Ucp1 expression in brown adipose tissue, and muscle and liver fatty acid oxidation. Mex3c is expressed in neurons and is detectable in the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. Mex3c was not detected in NPY or POMC neurons but was detected in leptin-responsive neurons in the ventromedial nucleus. Mex3c and Leptin double mutant mice were growth retarded and obese and had blood profiles similar to those of ob/ob mice but showed none of the steatosis observed in ob/ob mice. Our data show that Mex3c is involved in energy balance regulation.

  19. Sdt97: A Point Mutation in the 5' Untranslated Region Confers Semidwarfism in Rice.

    PubMed

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-06-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5' untranslated region of Sdt97 qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5' untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice.

  20. Sdt97: A Point Mutation in the 5′ Untranslated Region Confers Semidwarfism in Rice

    PubMed Central

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-01-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5′ untranslated region of Sdt97. qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5′ untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice. PMID:27172200

  1. Nuclear Mutation Increases Streptomycin and Spectinomycin Sensitivity in Chlamydomonas

    PubMed Central

    Lee, Robert W.; Sapp, Jan A.

    1978-01-01

    A spontaneously arising nuclear mutation, ss-1, has been identified in Chlamydomonas reinhardtii that decreases both streptomycin and spectinomycin resistance levels about 10-fold after its introduction into all wild-type, streptomycin-resistant and spectinomycin-resistant strains examined. The mutations for resistance map to nuclear and uniparentally inherited (chloroplast) loci. In contrast, no modification of erythromycin resistance was detected after introducing ss-1 into wild-type strains or into strains carrying nuclear or uniparentally inherited erythromycin-resistance mutations. We suggest that ss-1 affects the small subunit of the chloroplast ribosome because others have shown that streptomycin and spectinomycin resistance in C. reinhardtii are associated with this subunit, whereas erythromycin resistance is associated with the large subunit. ss-1 shows no linkage with the nuclear locus for streptomycin resistance. PMID:148390

  2. Increasing the imaging depth through computational scattering correction (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Koberstein-Schwarz, Benno; Omlor, Lars; Schmitt-Manderbach, Tobias; Mappes, Timo; Ntziachristos, Vasilis

    2016-03-01

    Imaging depth is one of the most prominent limitations in light microscopy. The depth in which we are still able to resolve biological structures is limited by the scattering of light within the sample. We have developed an algorithm to compensate for the influence of scattering. The potential of algorithm is demonstrated on a 3D image stack of a zebrafish embryo captured with a selective plane illumination microscope (SPIM). With our algorithm we were able shift the point in depth, where scattering starts to blur the imaging and effect the image quality by around 30 µm. For the reconstruction the algorithm only uses information from within the image stack. Therefore the algorithm can be applied on the image data from every SPIM system without further hardware adaption. Also there is no need for multiple scans from different views to perform the reconstruction. The underlying model estimates the recorded image as a convolution between the distribution of fluorophores and a point spread function, which describes the blur due to scattering. Our algorithm performs a space-variant blind deconvolution on the image. To account for the increasing amount of scattering in deeper tissue, we introduce a new regularizer which models the increasing width of the point spread function in order to improve the image quality in the depth of the sample. Since the assumptions the algorithm is based on are not limited to SPIM images the algorithm should also be able to work on other imaging techniques which provide a 3D image volume.

  3. A point mutation in influenza B neuraminidase confers resistance to peramivir and loss of slow binding.

    PubMed

    Baum, Ellen Z; Wagaman, Pamela C; Ly, Linh; Turchi, Ignatius; Le, Jianhua; Bucher, Doris; Bush, Karen

    2003-06-01

    The influenza neuraminidase (NA) inhibitors peramivir, oseltamivir, and zanamivir are potent inhibitors of NAs from both influenza A and B strains. In general, these inhibitors are slow, tight binders of NA, exhibiting time-dependent inhibition. A mutant of influenza virus B/Yamagata/16/88 which was resistant to peramivir was generated by passage of the virus in tissue culture, in the presence of increasing concentrations (0.1-120 microM over 15 passages) of the compound. Whereas the wild type (WT) virus was inhibited by peramivir with an EC(50) value of 0.10 microM, virus isolated at passages 3 and 15 displayed EC(50) values of 10 and >50 microM, respectively. Passage 3 virus contained 3 hemagglutinin (HA) mutations, but no NA mutation. Passage 15 (P15R) virus contained an additional 3 HA mutations, plus the NA mutation His273Tyr. The mechanism of inhibition of WT and P15R NA by peramivir was examined in enzyme assays. The WT and P15R NAs displayed IC(50) values of 8.4+/-0.4 and 127+/-16 nM, respectively, for peramivir. Peramivir inhibited the WT enzyme in a time-dependent fashion, with a K(i) value of 0.066+/-0.002nM. In contrast, the P15R enzyme did not display the property of slow binding and was inhibited competitively with a K(i) value of 4.69+/-0.44nM. Molecular modeling suggested that His273 was relatively distant from peramivir (>5A) in the NA active site, but that Tyr273 introduced a repulsive interaction between the enzyme and inhibitor, which may have been responsible for peramivir resistance.

  4. Mutation of environmental mycobacteria to resist silver nanoparticles also confers resistance to a common antibiotic.

    PubMed

    Larimer, Curtis; Islam, Mohammad Shyful; Ojha, Anil; Nettleship, Ian

    2014-08-01

    Non-tuberculous mycobacteria are a threat to human health, gaining entry to the body through contaminated water systems, where they form persistent biofilms despite extensive attempts at disinfection. Silver is a natural antibacterial agent and in nanoparticle form activity is increased by a high surface area. Silver nanoparticles (AgNPs) have been used as alternative disinfectants in circulating water systems, washing machines and even clothing. However, nanoparticles, like any other antibiotic that has a pervasive durable presence, carry the risk of creating a resistant population. In this study Mycobacterium smegmatis strain mc(2)155 was cultured in AgNP enriched agar such that only a small population survived. Surviving cultures were isolated and re-exposed to AgNPs and AgNO3 and resistance to silver was compared to a negative control. After only a single exposure, mutant M. smegmatis populations were resistant to AgNPs and AgNO3. Further, the silver resistant mutants were exposed to antibiotics to determine if general resistance had been conferred. The minimum inhibitory concentration of isoniazid was four times higher for silver resistant mutants than for strain mc(2)155. However, core resistance was not conferred to other toxic metal ions. The mutants had lower resistance to CuSO4 and ZnSO4 than the mc(2)155 strain.

  5. Gestational mutations in radiation carcinogenesis

    NASA Astrophysics Data System (ADS)

    Meza, R.; Luebeck, G.; Moolgavkar, S.

    Mutations in critical genes during gestation could increase substantially the risk of cancer. We examine the consequences of such mutations using the Luebeck-Moolgavkar model for colorectal cancer and the Lea-Coulson modification of the Luria-Delbruck model for the accumulation of mutations during gestation. When gestational mutation rates are high, such mutations make a significant contribution to cancer risk even for adult tumors. Furthermore, gestational mutations ocurring at distinct times during emryonic developmemt lead to substantially different numbers of mutated cells at birth, with early mutations leading to a large number (jackpots) of mutated cells at birth and mutation occurring late leading to only a few mutated cells. Thus gestational mutations could confer considerable heterogeneity of the risk of cancer. If the fetus is exposed to an environmental mutagen, such as ionizing radiation, the gestational mutation rate would be expected to increase. We examine the consequences of such exposures during gestation on the subsequent development of cancer.

  6. A mutation in Escherichia coli DNA gyrase conferring quinolone resistance results in sensitivity to drugs targeting eukaryotic topoisomerase II.

    PubMed

    Gruger, Thomas; Nitiss, John L; Maxwell, Anthony; Zechiedrich, E Lynn; Heisig, Peter; Seeber, Siegfried; Pommier, Yves; Strumberg, Dirk

    2004-12-01

    Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (Gyr(S83W)) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of Gyr(S83W), and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for Gyr(S83W)-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the Gyr(S83W) mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.

  7. Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).

    PubMed

    Ranade, Sonali Sachin; Ganea, Laura-Stefana; Razzak, Abdur M; García Gil, M R

    2015-01-01

    Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134).

  8. Increasing Participation in Learning. Symposium 19. [Concurrent Symposium Session at AHRD Annual Conference, 2000.

    ERIC Educational Resources Information Center

    2000

    This document contains three papers from a symposium on increasing participation in learning that was conducted as part of a conference on human resource development (HRD). "Factors Influencing Employee Participation in Training: An Empirical Investigation" (Reid A. Bates) reports on a mediated model of employee participation in training…

  9. Using Student Conferences to Increase Participation in the Classroom: A Case Study

    ERIC Educational Resources Information Center

    Arenas, M. G.; Castillo, P. A.; de Vega, F. F.; Merelo, J. J.

    2012-01-01

    This paper describes the use of a student conference as a novel experience aimed at motivating students enrolled in various computer architecture courses, such as Microprocessor Systems. The goal was to increase student engagement, to decrease failure rates, and to introduce students to the world of research. This multidisciplinary experience…

  10. Increasing Job Satisfaction. Symposium 22. [Concurrent Symposium Session at AHRD Annual Conference, 2000.

    ERIC Educational Resources Information Center

    2000

    This document contains three papers from a symposium on increasing job satisfaction that was conducted as part of a conference on human resource development (HRD). "A Systematic Model of Job Design by Examining the Organizational Factors Affecting Satisfaction" (Zhichao Cheng, Danyang Yang, Fenglou Liu) reports on a project in which…

  11. Increased mitochondrial mutation frequency after an island colonization: positive selection or accumulation of slightly deleterious mutations?

    PubMed

    Hardouin, Emilie A; Tautz, Diethard

    2013-04-23

    Island colonizations are excellent models for studying early processes of evolution. We found in a previous study on mice that had colonized the sub-Antarctic Kerguelen Archipelago about 200 years ago that they were derived from a single founder lineage and that this showed an unexpectedly large number of new mutations in the mitochondrial D-loop. To assess whether positive selection has played a role in the emergence of these variants, we have obtained 16 full mitochondrial genome sequences from these mice. For comparison, we have compiled 57 mitochondrial genome sequences from laboratory inbred lines that became established about 100 years ago, also starting from a single founder lineage. We find that the island mice and the laboratory lines show very similar mutation frequencies and patterns. None of the patterns in the Kerguelen mice provides evidence for positive selection. We conclude that nearly neutral evolutionary processes that assume the presence of slightly deleterious variants can fully explain the patterns. This supports the notion of time-dependency of molecular evolution and provides a new calibration point. Based on the observed mutation frequency, we calculate an average evolutionary rate of 0.23 substitutions per site per Myr for the earliest time frame of divergence, which is about six times higher than the long-term rate of 0.037 substitutions per site per Myr.

  12. Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas.

    PubMed

    Castro-Vega, Luis Jaime; Buffet, Alexandre; De Cubas, Aguirre A; Cascón, Alberto; Menara, Mélanie; Khalifa, Emmanuel; Amar, Laurence; Azriel, Sharona; Bourdeau, Isabelle; Chabre, Olivier; Currás-Freixes, Maria; Franco-Vidal, Valérie; Guillaud-Bataille, Marine; Simian, Christophe; Morin, Aurélie; Letón, Rocío; Gómez-Graña, Alvaro; Pollard, Patrick J; Rustin, Pierre; Robledo, Mercedes; Favier, Judith; Gimenez-Roqueplo, Anne-Paule

    2014-05-01

    Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an 'SDH-like' molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2-succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P = 0.007) and multiple tumors (P = 0.02) were significantly more frequent in patients with FH mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior.

  13. Genetic instability and increased mutational load: which diagnostic tool best direct patients with cancer to immunotherapy?

    PubMed

    Palmieri, Giuseppe; Colombino, Maria; Cossu, Antonio; Marchetti, Antonio; Botti, Gerardo; Ascierto, Paolo A

    2017-01-21

    The occurrence of high rates of somatic mutations in cancer is believed to correspond to increased frequency of neo-epitope formation and tumor immunogenicity. Thus, classification of patients with cancer according to degree a somatic hyper-mutational status could be proposed as a predictive biomarker of responsiveness to immunotherapy with immune checkpoint inhibitors. Here, we discuss the suitable and reliable tests easily adoptable in clinical practice to assess somatic mutational status in patients with advanced cancer.

  14. Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae

    PubMed Central

    Kaya, Alaattin; Lobanov, Alexei V.; Gerashchenko, Maxim V.; Koren, Amnon; Fomenko, Dmitri E.; Koc, Ahmet; Gladyshev, Vadim N.

    2014-01-01

    Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. PMID:25173844

  15. Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India.

    PubMed

    Kaur, Simerpreet; Rana, Vibhuti; Singh, Pooja; Trivedi, Garima; Anand, Shashi; Kaur, Amanpreet; Gupta, Pawan; Jain, Amita; Sharma, Charu

    2016-01-01

    Twenty-nine Kanamycin resistant clinical isolates of Mycobacterium tuberculosis from Northern India were screened to evaluate genetic mutations in rrs gene, eis gene with its promoter, and whiB7 gene along with its 5'UTR. 14 strains (~48.0%) collectively exhibited mutations in rrs, eis or whiB7 target regions. While the highest frequency of mutations was found in rrs gene, eis and whiB7 loci displayed novel mutations. The novel mutations displayed by eis and whiB7 loci were found to be associated specifically with the Kanamycin resistance as none of the twenty nine Kanamycin sensitive strains harbor them. The inclusion of novel mutations of eis and whiB7 loci will be useful in improving the specificity of future diagnostics.

  16. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

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

  17. A novel insertion mutation on exon 20 of epidermal growth factor receptor, conferring resistance to erlotinib.

    PubMed

    Khan, Nawazish A; Mirshahidi, Saied; Mirshahidi, Hamid R

    2014-05-01

    The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein tyrosine kinase receptor. The small-molecule tyrosine kinase receptor inhibitors (TKIs) are in clinical use to treat non-small cell lung cancer with EGFR mutations. Variable tumor responses to erlotinib and gefitinib have been observed. The response to these TKIs varies by the type of EGFR mutations found in the tumor. The deletion on exon 19 and the L858R substitution on exon 21 constitute the most frequent mutations and are known to show good response to TKIs. However, mutations on exon 20 are less common and seem to respond poorly to TKIs. In clinical settings, the reported response of exon 20 mutations to reversible TKIs (both gefitinib and erlotinib) remains inconstant. The type of coexisting mutation seems to affect the response of these insertions to TKIs. We herein present a case of disease progression despite the use of erlotinib in a female patient who had a novel insertion mutation on exon 20. Our patient was a never-smoker and was identified to have a Pro772_His773insGlnCysPro mutation on exon 20. She had previously been treated with cisplatin and gemcitabine and then with carboplatin and pemetrexed. She was treated with erlotinib upon intolerance to second-line chemotherapy and did not respond. Our patient had a novel insertion mutation on exon 20, which was found to be resistant to erlotinib.

  18. Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors

    PubMed Central

    Baker, Theresa; Nerle, Sujata; Pritchard, Justin; Zhao, Boyang; Rivera, Victor M.

    2015-01-01

    Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i. PMID:26360609

  19. Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides

    PubMed Central

    Lee, Hyejin; Rustgi, Sachin; Kumar, Neeraj; Burke, Ian; Yenish, Joseph P.; Gill, Kulvinder S.; von Wettstein, Diter; Ullrich, Steven E.

    2011-01-01

    Induced mutagenesis can be an effective way to increase variability in self-pollinated crops for a wide variety of agronomically important traits. Crop resistance to a given herbicide can be of practical value to control weeds with efficient chemical use. In some crops (for example, wheat, maize, and canola), resistance to imidazolinone herbicides (IMIs) has been introduced through mutation breeding and is extensively used commercially. However, this production system imposes plant-back restrictions on rotational crops because of herbicide residuals in the soil. In the case of barley, a preferred rotational crop after wheat, a period of 9–18 mo is required. Thus, introduction of barley varieties showing resistance to IMIs will provide greater flexibility as a rotational crop. The objective of the research reported was to identify resistance in barley for IMIs through induced mutagenesis. To achieve this objective, a sodium azide-treated M2/M3 population of barley cultivar Bob was screened for resistance against acetohydroxy acid synthase (AHAS)-inhibiting herbicides. The phenotypic screening allowed identification of a mutant line showing resistance against IMIs. Molecular analysis identified a single-point mutation leading to a serine 653 to asparagine amino acid substitution in the herbicide-binding site of the barley AHAS gene. The transcription pattern of the AHAS gene in the mutant (Ser653Asn) and WT has been analyzed, and greater than fourfold difference in transcript abundance was observed. Phenotypic characteristics of the mutant line are promising and provide the base for the release of IMI-resistant barley cultivar(s). PMID:21551103

  20. Screening for streptomycin resistance conferring mutations in Mycobacterium tuberculosis isolates from Iran.

    PubMed

    Rezaei, Faranak; Haeili, Mehri; Imani Fooladi, Abbasali; Azari Garmjan, Gholam Ali; Feizabadi, Mohammad Mehdi

    2017-02-01

    Point mutations in the rpsL and rrs genes can lead to development of streptomycin (STR) resistance in Mycobacterium tuberculosis. The aims of this study were to determine the frequency of mutations in STR resistant M. tuberculosis isolates in Iran and to analyze the possible relationship between bacterial genotype and STR resistance. Twenty-three M. tuberculosis samples comprising 9 multidrug-resistant (MDR) and 14 non-MDR isolates, recovered from TB patients in four regions: Tehran (n = 14), Isfahan (n = 2), Zahedan (n = 2), and Khorasan (n = 5), were analysed. Mutational profiling was performed by sequencing of the rrs and rpsL genes and spoligotyping method was used for genotyping. Nineteen isolates were resistant to STR, among them 7 exhibited mutations in the rpsL gene and 7 had mutations in the rrs gene. The remaining 5 STR resistant as well as all susceptible isolates lacked any mutation in both genes. Beijing genotype was associated with both MDR and STR resistance in which all mutations occurred at codon 43 of the rpsL gene. There was an association between mutations in the rpsL and rrs genes and STR resistance. We also found a correlation between Beijing genotype and STR resistance.

  1. Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

    PubMed Central

    Melançon, P; Tapprich, W E; Brakier-Gingras, L

    1992-01-01

    Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes. PMID:1281147

  2. Subclonal mutations in SETBP1 confer a poor prognosis in juvenile myelomonocytic leukemia

    PubMed Central

    Troup, Camille B.; Gelston, Laura C.; Haliburton, John; Chow, Eric D.; Yu, Kristie B.; Akutagawa, Jon; Taylor-Weiner, Amaro N.; Liu, Y. Lucy; Wang, Yong-Dong; Beckman, Kyle; Emanuel, Peter D.; Braun, Benjamin S.; Abate, Adam; Gerbing, Robert B.; Alonzo, Todd A.; Loh, Mignon L.

    2015-01-01

    Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Children’s Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006). PMID:25395418

  3. A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509.

    PubMed

    Joseph, James D; Lu, Nhin; Qian, Jing; Sensintaffar, John; Shao, Gang; Brigham, Dan; Moon, Michael; Maneval, Edna Chow; Chen, Isan; Darimont, Beatrice; Hager, Jeffrey H

    2013-09-01

    Despite the impressive clinical activity of the second-generation antiandrogens enzalutamide and ARN-509 in patients with prostate cancer, acquired resistance invariably emerges. To identify the molecular mechanisms underlying acquired resistance, we developed and characterized cell lines resistant to ARN-509 and enzalutamide. In a subset of cell lines, ARN-509 and enzalutamide exhibit agonist activity due to a missense mutation (F876L) in the ligand-binding domain of the androgen receptor (AR). AR F876L is sufficient to confer resistance to ARN-509 and enzalutamide in in vitro and in vivo models of castration-resistant prostate cancer (CRPC). Importantly, the AR F876L mutant is detected in plasma DNA from ARN-509-treated patients with progressive CRPC. Thus, selective outgrowth of AR F876L is a clinically relevant mechanism of second-generation antiandrogen resistance that can potentially be targeted with next-generation antiandrogens. A missense mutation in the ligand-binding domain of the androgen receptor F876L confers resistance to the second-generation antiandrogens enzalutamide and ARN-509 in preclinical models of AR function and prostate cancer and is detected in plasma DNA from ARN-509-treated patients with progressive disease. These results chart a new path for the discovery and development of next-generation antiandrogens that could be coupled with a blood-based companion diagnostic to guide treatment decisions. ©2013 AACR.

  4. Increasing Faculty Attendance at Emergency Medicine Resident Conferences: Does CME Credit Make a Difference?

    PubMed Central

    Lefebvre, Cedric W.; Hiestand, Brian; Bond, Michael C.; Fox, Sean M.; Char, Doug; Weber, Drew S.; Glenn, David; Patterson, Leigh A.; Manthey, David E.

    2013-01-01

    Background Faculty involvement in resident teaching events is beneficial to resident education, yet evidence about the factors that promote faculty attendance at resident didactic conferences is limited. Objective To determine whether offering continuing medical education (CME) credits would result in an increase in faculty attendance at weekly emergency medicine conferences and whether faculty would report the availability of CME credit as a motivating factor. Methods Our prospective, multi-site, observational study of 5 emergency medicine residency programs collected information on the number of faculty members present at CME and non-CME lectures for 9 months and collected information from faculty on factors influencing decisions to attend resident educational events and from residents on factors influencing their learning experience. Results Lectures offering CME credit on average were attended by 5 additional faculty members per hour, compared with conferences that did not offer CME credit (95% confidence interval [CI], 3.9–6.1; P < .001). Faculty reported their desire to “participate in resident education” was the most influential factor prompting them to attend lectures, followed by “explore current trends in emergency medicine” and the lecture's “specific topic.” Faculty also reported that “clinical/administrative duties” and “family responsibilities” negatively affected their ability to attend. Residents reported that the most important positive factor influencing their conference experience was “lectures given by faculty.” Conclusions Although faculty reported that CME credit was not an important factor in their decision to attend resident conferences, offering CME credit resulted in significant increases in faculty attendance. Residents reported that “lectures given by faculty” and “faculty attendance” positively affected their learning experience. PMID:24404225

  5. Increasing Faculty Attendance at Emergency Medicine Resident Conferences: Does CME Credit Make a Difference?

    PubMed

    Lefebvre, Cedric W; Hiestand, Brian; Bond, Michael C; Fox, Sean M; Char, Doug; Weber, Drew S; Glenn, David; Patterson, Leigh A; Manthey, David E

    2013-03-01

    Faculty involvement in resident teaching events is beneficial to resident education, yet evidence about the factors that promote faculty attendance at resident didactic conferences is limited. To determine whether offering continuing medical education (CME) credits would result in an increase in faculty attendance at weekly emergency medicine conferences and whether faculty would report the availability of CME credit as a motivating factor. Our prospective, multi-site, observational study of 5 emergency medicine residency programs collected information on the number of faculty members present at CME and non-CME lectures for 9 months and collected information from faculty on factors influencing decisions to attend resident educational events and from residents on factors influencing their learning experience. Lectures offering CME credit on average were attended by 5 additional faculty members per hour, compared with conferences that did not offer CME credit (95% confidence interval [CI], 3.9-6.1; P < .001). Faculty reported their desire to "participate in resident education" was the most influential factor prompting them to attend lectures, followed by "explore current trends in emergency medicine" and the lecture's "specific topic." Faculty also reported that "clinical/administrative duties" and "family responsibilities" negatively affected their ability to attend. Residents reported that the most important positive factor influencing their conference experience was "lectures given by faculty." Although faculty reported that CME credit was not an important factor in their decision to attend resident conferences, offering CME credit resulted in significant increases in faculty attendance. Residents reported that "lectures given by faculty" and "faculty attendance" positively affected their learning experience.

  6. Antagonistic coevolution with parasites increases the cost of host deleterious mutations

    PubMed Central

    Buckling, Angus; Wei, Yan; Massey, Ruth C; Brockhurst, Michael A; Hochberg, Michael E

    2005-01-01

    The fitness consequences of deleterious mutations are sometimes greater when individuals are parasitized, hence parasites may result in the more rapid purging of deleterious mutations from host populations. The significance of host deleterious mutations when hosts and parasites antagonistically coevolve (reciprocal evolution of host resistance and parasite infectivity) has not previously been experimentally investigated. We addressed this by coevolving the bacterium Pseudomonas fluorescens and a parasitic bacteriophage in laboratory microcosms, using bacteria with high and low mutation loads. Directional coevolution between bacterial resistance and phage infectivity occurred in all populations. Bacterial population fitness, as measured by competition experiments with ancestral genotypes in the absence of phage, declined with time spent coevolving. However, this decline was significantly more rapid in bacteria with high mutation loads, suggesting the cost of bacterial resistance to phage was greater in the presence of deleterious mutations (synergistic epistasis). As such, resistance to phage was more costly to evolve in the presence of a high mutation load. Consistent with these data, bacteria with high mutation loads underwent less rapid directional coevolution with their phage populations, and showed lower levels of resistance to their coevolving phage populations. These data suggest that coevolution with parasites increases the rate at which deleterious mutations are purged from host populations. PMID:16519233

  7. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

    PubMed

    Zabriskie, Matthew S; Eide, Christopher A; Tantravahi, Srinivas K; Vellore, Nadeem A; Estrada, Johanna; Nicolini, Franck E; Khoury, Hanna J; Larson, Richard A; Konopleva, Marina; Cortes, Jorge E; Kantarjian, Hagop; Jabbour, Elias J; Kornblau, Steven M; Lipton, Jeffrey H; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J; Press, Richard D; Chuah, Charles; Goldberg, Stuart L; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R; Heaton, William L; Eiring, Anna M; Pomicter, Anthony D; Khorashad, Jamshid S; Kelley, Todd W; Baron, Riccardo; Druker, Brian J; Deininger, Michael W; O'Hare, Thomas

    2014-09-08

    Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

  8. KIT mutations confer a distinct gene expression signature in core binding factor leukaemia.

    PubMed

    Lück, Sonja C; Russ, Annika C; Du, Juan; Gaidzik, Verena; Schlenk, Richard F; Pollack, Jonathan R; Döhner, Konstanze; Döhner, Hartmut; Bullinger, Lars

    2010-03-01

    Core binding factor (CBF) leukaemias, characterized by either inv(16)(p13.1q22) or t(8;21)(q22;q22), constitute acute myeloid leukaemia (AML) subgroups with favourable prognosis. However, 40-50% of patients relapse, emphasizing the need for risk-adapted treatment approaches. In this regard, studying secondary genetic aberrations, such as mutations of the KIT gene, is of great interest, particularly as they can be targeted by receptor tyrosine kinase inhibitors (TKI). However, so far little is known about the biology underlying KIT-mutated CBF leukaemias. We analysed gene expression profiles of 83 CBF AML cases with known KIT mutation status in order to gain novel insights in KIT-mutated CBF pathogenesis. KIT-mutated cases were characterized by deregulation of genes belonging to the NFkB signalling complex suggesting impaired control of apoptosis. Notably, a subgroup of KIT wildtype cases was also characterized by the KIT mutation signature due to yet unknown aberrations. Our data suggest that this CBF leukaemia subgroup might profit from TKI therapy, however, the relevance of the KIT mutation-associated signature remains to be validated prior to clinical implementation. Nevertheless, the existence of such a signature supports the notion of relevant biological differences in CBF leukaemia and might serve as diagnostic tool in the future.

  9. Somatostatin analogues increase AIP expression in somatotropinomas, irrespective of Gsp mutations.

    PubMed

    Jaffrain-Rea, Marie-Lise; Rotondi, Sandra; Turchi, Annarita; Occhi, Gianluca; Barlier, Anne; Peverelli, Erika; Rostomyan, Lilya; Defilles, Céline; Angelini, Mariolina; Oliva, Maria-Antonietta; Ceccato, Filippo; Maiorani, Orlando; Daly, Adrian F; Esposito, Vincenzo; Buttarelli, Francesca; Figarella-Branger, Dominique; Giangaspero, Felice; Spada, Anna; Scaroni, Carla; Alesse, Edoardo; Beckers, Albert

    2013-10-01

    Germline aryl hydrocarbon receptor interacting protein (AIP) gene mutations confer a predisposition to pituitary adenoma (PA), predominantly GH-secreting (GH-PA). As recent data suggest a role for AIP in the pathogenesis of sporadic GH-PA and their response to somatostatin analogues (SSA), the expression of AIP and its partner, aryl hydrocarbon receptor (AHR), was determined by semiquantitative immunohistochemistry scoring in 62 sporadic GH-PA (37 treated with SSA preoperatively). The influence of Gsp status was studied in a subset of tumours (n=39, 14 Gsp(+)) and six GH-PA were available for primary cultures. AIP and AHR were detected in most cases, with a positive correlation between AIP and cytoplasmic AHR (P=0.012). Low AIP expression was significantly more frequent in untreated vs SSA-treated tumours (44.0 vs 20.5%, P=0.016). AHR expression or localisation did not differ between the two groups. Similarly, in vitro octreotide induced a median twofold increase in AIP expression (range 1.2-13.9, P=0.027) in GH-PA. In SSA-treated tumours, the AIP score was significantly higher in the presence of preoperative IGF1 decrease or tumour shrinkage (P=0.008 and P=0.014 respectively). In untreated tumours, low AIP expression was significantly associated with invasiveness (P=0.028) and suprasellar extension (P=0.019). The only effect of Gsp status was a significantly lower nuclear AHR score in Gsp(+) vs Gsp(-) tumours (P=0.025), irrespective of SSA. In conclusion, AIP is involved in the aggressiveness of sporadic GH-PA, regardless of Gsp status, and AIP up-regulation in SSA-treated tumours is associated with a better preoperative response, with no clear role for AHR.

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

    PubMed

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

    2017-05-23

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

  11. Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

    PubMed Central

    Zhang, Haonan; Wu, Shuwen; Yang, Yihua; Tabashnik, Bruce E.; Wu, Yidong

    2012-01-01

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins. PMID:23285292

  12. Non-recessive Bt toxin resistance conferred by an intracellular cadherin mutation in field-selected populations of cotton bollworm.

    PubMed

    Zhang, Haonan; Wu, Shuwen; Yang, Yihua; Tabashnik, Bruce E; Wu, Yidong

    2012-01-01

    Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

  13. Human Slack potassium channel mutations increase positive cooperativity between individual channels

    PubMed Central

    Barcia, Giulia; Quraishi, Imran H.; Martin, Hilary C.; Blair, Edward; Taylor, Jenny C.; Dulac, Olivier; Colleaux, Laurence

    2015-01-01

    Summary Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation or voltage-dependence. We examined nine different mutations of the KCNT1 (Slack) Na+-activated K+ channel that give rise to three distinct forms of epilepsy. All produced many fold-increases in current amplitude over that of the wild type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gives rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of the mutations is to alter channel-channel interactions. PMID:25482562

  14. Human slack potassium channel mutations increase positive cooperativity between individual channels.

    PubMed

    Kim, Grace E; Kronengold, Jack; Barcia, Giulia; Quraishi, Imran H; Martin, Hilary C; Blair, Edward; Taylor, Jenny C; Dulac, Olivier; Colleaux, Laurence; Nabbout, Rima; Kaczmarek, Leonard K

    2014-12-11

    Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na(+)-activated K(+) channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions.

  15. Acquired resistance to dasatinib in lung cancer cell lines conferred by DDR2 gatekeeper mutation and NF1 loss.

    PubMed

    Beauchamp, Ellen M; Woods, Brittany A; Dulak, Austin M; Tan, Li; Xu, Chunxiao; Gray, Nathanael S; Bass, Adam J; Wong, Kwok-kin; Meyerson, Matthew; Hammerman, Peter S

    2014-02-01

    The treatment of non-small cell lung cancer has evolved dramatically over the past decade with the adoption of widespread use of effective targeted therapies in patients with distinct molecular alterations. In lung squamous cell carcinoma (lung SqCC), recent studies have suggested that DDR2 mutations are a biomarker for therapeutic response to dasatinib and clinical trials are underway testing this hypothesis. Although targeted therapeutics are typically quite effective as initial therapy for patients with lung cancer, nearly all patients develop resistance with long-term exposure to targeted drugs. Here, we use DDR2-dependent lung cancer cell lines to model acquired resistance to dasatinib therapy. We perform targeted exome sequencing to identify two distinct mechanisms of acquired resistance: acquisition of the T654I gatekeeper mutation in DDR2 and loss of NF1. We show that NF1 loss activates a bypass pathway, which confers ERK dependency downstream of RAS activation. These results indicate that acquired resistance to dasatinib can occur via both second-site mutations in DDR2 and by activation of bypass pathways. These data may help to anticipate mechanisms of resistance that may be identified in upcoming clinical trials of anti-DDR2 therapy in lung cancer and suggest strategies to overcome resistance.

  16. Molecular and phenotypic characterization of Als1 and Als2 mutations conferring tolerance to acetolactate synthase herbicides in soybean

    PubMed Central

    Walter, Kay L; Strachan, Stephen D; Ferry, Nancy M; Albert, Henrik H; Castle, Linda A; Sebastian, Scott A

    2014-01-01

    BACKGROUND Sulfonylurea (SU) herbicides are effective because they inhibit acetolactate synthase (ALS), a key enzyme in branched-chain amino acid synthesis required for plant growth. A soybean line known as W4-4 was developed through rounds of seed mutagenesis and was demonstrated to have a high degree of ALS-based resistance to both post-emergence and pre-emergence applications of a variety of SU herbicides. This report describes the molecular and phenotypic characterization of the Als1 and Als2 mutations that confer herbicide resistance to SUs and other ALS inhibitors. RESULTS The mutations are shown to occur in two different ALS genes that reside on different chromosomes: Als1 (P178S) on chromosome 4 and Als2 (W560L) on chromosome 6 (P197S and W574L in Arabidopsis thaliana). CONCLUSION Although the Als1 and Als2 genes are unlinked, the combination of these two mutations is synergistic for improved tolerance of soybeans to ALS-inhibiting herbicides. © 2014 DuPont Pioneer. Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24425499

  17. Hair keratin mutations in tooth enamel increase dental decay risk.

    PubMed

    Duverger, Olivier; Ohara, Takahiro; Shaffer, John R; Donahue, Danielle; Zerfas, Patricia; Dullnig, Andrew; Crecelius, Christopher; Beniash, Elia; Marazita, Mary L; Morasso, Maria I

    2014-12-01

    Tooth enamel is the hardest substance in the human body and has a unique combination of hardness and fracture toughness that protects teeth from dental caries, the most common chronic disease worldwide. In addition to a high mineral content, tooth enamel comprises organic material that is important for mechanical performance and influences the initiation and progression of caries; however, the protein composition of tooth enamel has not been fully characterized. Here, we determined that epithelial hair keratins, which are crucial for maintaining the integrity of the sheaths that support the hair shaft, are expressed in the enamel organ and are essential organic components of mature enamel. Using genetic and intraoral examination data from 386 children and 706 adults, we found that individuals harboring known hair disorder-associated polymorphisms in the gene encoding keratin 75 (KRT75), KRT75(A161T) and KRT75(E337K), are prone to increased dental caries. Analysis of teeth from individuals carrying the KRT75(A161T) variant revealed an altered enamel structure and a marked reduction of enamel hardness, suggesting that a functional keratin network is required for the mechanical stability of tooth enamel. Taken together, our results identify a genetic locus that influences enamel structure and establish a connection between hair disorders and susceptibility to dental caries.

  18. Hair keratin mutations in tooth enamel increase dental decay risk

    PubMed Central

    Duverger, Olivier; Ohara, Takahiro; Shaffer, John R.; Donahue, Danielle; Zerfas, Patricia; Dullnig, Andrew; Crecelius, Christopher; Beniash, Elia; Marazita, Mary L.; Morasso, Maria I.

    2014-01-01

    Tooth enamel is the hardest substance in the human body and has a unique combination of hardness and fracture toughness that protects teeth from dental caries, the most common chronic disease worldwide. In addition to a high mineral content, tooth enamel comprises organic material that is important for mechanical performance and influences the initiation and progression of caries; however, the protein composition of tooth enamel has not been fully characterized. Here, we determined that epithelial hair keratins, which are crucial for maintaining the integrity of the sheaths that support the hair shaft, are expressed in the enamel organ and are essential organic components of mature enamel. Using genetic and intraoral examination data from 386 children and 706 adults, we found that individuals harboring known hair disorder–associated polymorphisms in the gene encoding keratin 75 (KRT75), KRT75A161T and KRT75E337K, are prone to increased dental caries. Analysis of teeth from individuals carrying the KRT75A161T variant revealed an altered enamel structure and a marked reduction of enamel hardness, suggesting that a functional keratin network is required for the mechanical stability of tooth enamel. Taken together, our results identify a genetic locus that influences enamel structure and establish a connection between hair disorders and susceptibility to dental caries. PMID:25347471

  19. Autism Linked to Increased Oncogene Mutations but Decreased Cancer Rate

    PubMed Central

    Zimmerman, M. Bridget; Mahajan, Vinit B.; Bassuk, Alexander G.

    2016-01-01

    Autism spectrum disorder (ASD) is one phenotypic aspect of many monogenic, hereditary cancer syndromes. Pleiotropic effects of cancer genes on the autism phenotype could lead to repurposing of oncology medications to treat this increasingly prevalent neurodevelopmental condition for which there is currently no treatment. To explore this hypothesis we sought to discover whether autistic patients more often have rare coding, single-nucleotide variants within tumor suppressor and oncogenes and whether autistic patients are more often diagnosed with neoplasms. Exome-sequencing data from the ARRA Autism Sequencing Collaboration was compared to that of a control cohort from the Exome Variant Server database revealing that rare, coding variants within oncogenes were enriched for in the ARRA ASD cohort (p<1.0x10-8). In contrast, variants were not significantly enriched in tumor suppressor genes. Phenotypically, children and adults with ASD exhibited a protective effect against cancer, with a frequency of 1.3% vs. 3.9% (p<0.001), but the protective effect decreased with age. The odds ratio of neoplasm for those with ASD relative to controls was 0.06 (95% CI: 0.02, 0.19; p<0.0001) in the 0 to 14 age group; 0.35 (95% CI: 0.14, 0.87; p = 0.024) in the 15 to 29 age group; 0.41 (95% CI: 0.15, 1.17; p = 0.095) in the 30 to 54 age group; and 0.49 (95% CI: 0.14, 1.74; p = 0.267) in those 55 and older. Both males and females demonstrated the protective effect. These findings suggest that defects in cellular proliferation, and potentially senescence, might influence both autism and neoplasm, and already approved drugs targeting oncogenic pathways might also have therapeutic value for treating autism. PMID:26934580

  20. Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1☆

    PubMed Central

    Sugi, Tatsuki; Kobayashi, Kyousuke; Takemae, Hitoshi; Gong, Haiyan; Ishiwa, Akiko; Murakoshi, Fumi; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-01-01

    Toxoplasma gondii is an important food and waterborne pathogen that causes severe disease in immunocompromised patients. Bumped kinase inhibitors (BKIs) have an antiparasitic effect on T. gondii tachyzoite growth by targeting T. gondii calmodulin-domain protein kinase 1 (TgCDPK1). To identify mutations that confer resistance to BKIs, chemical mutagenesis was performed, followed by selection in media containing either 250 or 1000 nM 1NM-PP1. Whole-genome sequence analysis of resistant clones revealed single nucleotide mutations in T. gondii mitogen-activated protein kinase 1 (TgMAPK1) at amino acids 162 (L162Q) and 171 (I171N). Plasmid constructs having the TgMAPK1 L162Q mutant sequence successfully replaced native TgMAPK1 genome locus in the presence of 1000 nM 1NM-PP1. The inhibitory effect of 1NM-PP1 on cell division observed in the parent clone was decreased in 1NM-PP1-resistant clones; however, effects on parasite invasion and calcium-induced egress were similar in both parent and resistant clones. A plasmid construct expressing the full length TgMAPK1 splicing isoform with L162Q mutation successfully complemented TgMAPK1 function in the pressure of 250 nM 1NM-PP1 in plaque assay. 1NM-PP1-resistant clones showed resistance to other BKIs (3MB-PP1 and 3BrB-PP1) with different levels. Here we identify TgMAPK1 as a novel target for 1NM-PP1 activity. This inhibitory effect is mediated through inhibition of tachyzoite cell division, and can be overcome through mutations at multiple residues in TgMAPK1. PMID:24533298

  1. Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

    PubMed Central

    Jacobs, Lauren; Maria, Ann; Villano, Danylo; Gaddam, Pragna; Wu, Gang; McGee, Rose B.; Quinn, Emily; Inaba, Hiroto; Hartford, Christine; Pui, Ching-hon; Pappo, Alberto; Edmonson, Michael; Zhang, Michael Y.; Stepensky, Polina; Steinherz, Peter; Schrader, Kasmintan; Lincoln, Anne; Bussel, James; Lipkin, Steve M.; Goldgur, Yehuda; Harit, Mira; Stadler, Zsofia K.; Mullighan, Charles; Weintraub, Michael; Shimamura, Akiko; Zhang, Jinghui; Downing, James R.; Nichols, Kim E.; Offit, Kenneth

    2015-01-01

    Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition. PMID:26102509

  2. Genetic Interactions among Chlamydomonas Reinhardtii Mutations That Confer Resistance to anti-Microtubule Herbicides

    PubMed Central

    James, S. W.; Lefebvre, P. A.

    1992-01-01

    We previously described two types of genetic interactions among recessive mutations in the APM1 and APM2 loci of Chlamydomonas reinhardtii that may reflect a physical association of the gene products or their involvement in a common structure/process: (1) allele-specific synthetic lethality, and (2) unlinked noncomplementation, or dominant enhancement. To further investigate these interactions, we isolated revertants in which the heat sensitivity caused by the apm2-1 mutation is lost. The heat-insensitive revertants were either fully or partially suppressed for the drug-resistance caused by the apm2-1 allele. In recombination tests the revertants behaved as if the suppressing mutation mapped within the APM2 locus; the partial suppressors of apm2-1 herbicide resistance failed to complement apm2-1, leading to the conclusion that they were likely to be intragenic pseudorevertants. The apm2-1 partial suppressor mutations reversed apm1(-)apm2-1 synthetic lethality in an allele-specific manner with respect both to apm1(-) alleles and apm2-1 suppressor mutations. Those apm1(-) apm2-1(rev) strains that regained viability also regained heat sensitivity characteristic of the original apm2-1 mutation, even though the apm2-1 suppressor strains were fully heat-insensitive. The Hs(+) phenotypes of apm2-1 partial suppressors were also reversed by treatment with the microtubule-stabilizing agent deuterium oxide (D(2)O). In addition to the above interactions, we observed interallelic complementation and phenotypic enhancement of temperature conditionality among apm1(-) alleles. Evidence of a role for the products of the two genes in microtubule-based processes was obtained from studying flagellar assembly in apm1(-) and apm2(-) mutants. PMID:1311696

  3. Independent origins of loss-of-function mutations conferring oxamniquine resistance in a Brazilian schistosome population.

    PubMed

    Chevalier, Frédéric D; Le Clec'h, Winka; Eng, Nina; Rugel, Anastasia R; Assis, Rafael Ramiro de; Oliveira, Guilherme; Holloway, Stephen P; Cao, Xiaohang; Hart, P John; LoVerde, Philip T; Anderson, Timothy J C

    2016-06-01

    Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles.

  4. Determination of the primary target of a quinolone drug and the effect of quinolone resistance-conferring mutations by measuring quinolone sensitivity based on its mode of action.

    PubMed

    Pfeiffer, Emily S; Hiasa, Hiroshi

    2007-09-01

    We used an assay to measure quinolone sensitivity as a shift in the position of the cleavage-religation equilibrium. This assay was found to be useful in identifying the primary target of a quinolone drug and assessing the effect of quinolone resistance-conferring mutations.

  5. Increased cancer risk of heterozygotes with NBS1 germline mutations in Poland.

    PubMed

    Steffen, Jan; Varon, Raymonda; Mosor, Maria; Maneva, Galina; Maurer, Martin; Stumm, Markus; Nowakowska, Dorota; Rubach, Maryna; Kosakowska, Ewa; Ruka, Włodzimierz; Nowecki, Zbigniew; Rutkowski, Piotr; Demkow, Tomasz; Sadowska, Małgorzata; Bidziński, Mariusz; Gawrychowski, Krzysztof; Sperling, Karl

    2004-08-10

    It has been suggested based on familial data that Nijmegen breakage syndrome (NBS) heterozygotes have an increased risk of malignant tumors. We found 15 carriers of the 657del5 mutation and 8 carriers of the R215W molecular variant of the NBS1 gene among 1,289 consecutive patients from Central Poland with various cancers and only 10 and 4 such carriers, respectively, in 1,620 controls from this region. Most of the 657del5 mutation carriers were found among patients with melanoma (4/105), non-Hodgkin lymphoma (2/42) and breast cancer (4/224) and of the 234 patients with colorectal carcinoma 3 carried the 657del5 mutation and 3 others the R215W molecular variant. The frequencies of 657del5 mutation carriers among patients with melanoma and non-Hodgkin lymphoma and of R215W carriers in patients with colorectal cancer were significantly higher than in controls (p < 0.01, < 0.05 and < 0.05 respectively). The pooled frequencies of 657del5 and R215W mutations in all cancer patients were also significantly higher than in controls (p < 0.05). Two carriers of the 657del5 mutation had second primary tumors. Malignant tumors among parents and siblings of 657del5 mutation carriers (14/77) were twice more frequent than in population controls. Three carriers of this mutation (2 probands with melanoma) reported melanoma in relatives. These results suggest strongly that NBS1 heterozygosity may be associated with elevated risk of some cancers. Larger studies are needed to evaluate the impact of the high frequency of germline NBS1 mutations on the cancer burden in the Slav populations. Copyright 2004 Wiley-Liss, Inc.

  6. The Trojan Female Technique for pest control: a candidate mitochondrial mutation confers low male fertility across diverse nuclear backgrounds in Drosophila melanogaster.

    PubMed

    Dowling, Damian K; Tompkins, Daniel M; Gemmell, Neil J

    2015-10-01

    Pest species represent a major ongoing threat to global biodiversity. Effective management approaches are required that regulate pest numbers, while minimizing collateral damage to nontarget species. The Trojan Female Technique (TFT) was recently proposed as a prospective approach to biological pest control. The TFT draws on the evolutionary hypothesis that maternally inherited mitochondrial genomes are prone to the accumulation of male, but not female, harming mutations. These mutations could be harnessed to provide trans-generational fertility-based control of pest species. A candidate TFT mutation was recently described in the fruit fly, Drosophila melanogaster, which confers male-only sterility in the specific isogenic nuclear background in which it is maintained. However, applicability of the TFT relies on mitochondrial mutations whose male-sterilizing effects are general across nuclear genomic contexts. We test this assumption, expressing the candidate TFT-mutation bearing haplotype alongside a range of nuclear backgrounds and comparing its fertility in males, relative to that of control haplotypes. We document consistently lower fertility for males harbouring the TFT mutation, in both competitive and noncompetitive mating contexts, across all nuclear backgrounds screened. This indicates that TFT mutations conferring reduced male fertility can segregate within populations and could be harnessed to facilitate this novel form of pest control.

  7. The Trojan Female Technique for pest control: a candidate mitochondrial mutation confers low male fertility across diverse nuclear backgrounds in Drosophila melanogaster

    PubMed Central

    Dowling, Damian K; Tompkins, Daniel M; Gemmell, Neil J

    2015-01-01

    Pest species represent a major ongoing threat to global biodiversity. Effective management approaches are required that regulate pest numbers, while minimizing collateral damage to nontarget species. The Trojan Female Technique (TFT) was recently proposed as a prospective approach to biological pest control. The TFT draws on the evolutionary hypothesis that maternally inherited mitochondrial genomes are prone to the accumulation of male, but not female, harming mutations. These mutations could be harnessed to provide trans-generational fertility-based control of pest species. A candidate TFT mutation was recently described in the fruit fly, Drosophila melanogaster, which confers male-only sterility in the specific isogenic nuclear background in which it is maintained. However, applicability of the TFT relies on mitochondrial mutations whose male-sterilizing effects are general across nuclear genomic contexts. We test this assumption, expressing the candidate TFT-mutation bearing haplotype alongside a range of nuclear backgrounds and comparing its fertility in males, relative to that of control haplotypes. We document consistently lower fertility for males harbouring the TFT mutation, in both competitive and noncompetitive mating contexts, across all nuclear backgrounds screened. This indicates that TFT mutations conferring reduced male fertility can segregate within populations and could be harnessed to facilitate this novel form of pest control. PMID:26495040

  8. The incidence of p53 mutations increases with progression of head and neck cancer.

    PubMed

    Boyle, J O; Hakim, J; Koch, W; van der Riet, P; Hruban, R H; Roa, R A; Correo, R; Eby, Y J; Ruppert, J M; Sidransky, D

    1993-10-01

    To establish a genetic model of the progression of head and neck squamous carcinoma we have defined the incidence and timing of p53 mutations in this type of cancer. We sequenced the conserved regions of the p53 gene in 102 head and neck squamous carcinoma lesions. These included 65 primary invasive carcinomas and 37 noninvasive archival specimens consisting of 13 severe dysplasias and 24 carcinoma in situ lesions. The incidence of p53 mutations in noninvasive lesions was 19% (7/37) and increased to 43% (28/65) in invasive carcinomas. These data suggest that p53 mutations can precede invasion in primary head and neck cancer. Furthermore, the spectrum of codon hotspots is similar to that seen in squamous carcinoma of the lung and 64% of mutations are at G nucleotides, implicating carcinogens from tobacco smoke in the etiology of head and neck squamous carcinoma.

  9. A mutation in Caenorhabditis elegans that increases recombination frequency more than threefold.

    PubMed

    Rose, A M; Baillie, D L

    1979-10-18

    In higher organisms the rate of recombination between genetic loci is presumably responsive to selective pressure. Recently, selective pressures and mutational events that influence recombination have been reviewed. Mutational sites and chromosomal rearrangements that enhance or suppress recombination frequency in specific regions are known, but general mechanisms that enhance recombination have not yet been discovered. We describe here the isolation and characterisation of a strain of the hermaphroditic nematode, Caenorhabditis elegans, that has a recombination frequency at least threefold higher than that found in the wild type. In this strain, rec-1, the number of reciprocal recombination events between linked loci is increased. This is true for all pairs of linked loci studies so far. The high recombination strain behaves as if it carries a classical recessive mutation, although a second mutation exists which can alter the recessive behaviour of rec-1.

  10. The A395T mutation in ERG11 gene confers fluconazole resistance in Candida tropicalis causing candidemia.

    PubMed

    Tan, Jingwen; Zhang, Jinqing; Chen, Wei; Sun, Yi; Wan, Zhe; Li, Ruoyu; Liu, Wei

    2015-04-01

    The mechanism of fluconazole resistance in Candida tropicalis is still unclear. Recently, we isolated a fluconazole-resistant strain of C. tropicalis from the blood specimen of a patient with candidemia in China. In vitro antifungal susceptibility of the isolate was determined by using CLSI M27-A3 and E-test methods. The sequence of ERG11 gene was then analyzed, and the three-dimensional model of Erg11p encoded by ERG11 gene was also investigated. The sequencing of ERG11 gene revealed the mutation of A395T in this fluconazole-resistant isolate of C. tropicalis, resulting in the Y132F substitution in Erg11p. Sequence alignment and three-dimensional model comparison of Erg11ps showed high similarity between fluconazole-susceptible isolates of C. tropicalis and Candida albicans. The comparison of the three-dimensional models of Erg11ps demonstrated that the position of the Y132F substitution in this isolate of C. tropicalis is identical to the isolate of C. albicans with fluconazole resistance resulting from Y132F substitution in Erg11p. Hence, we ascertain that the Y132F substitution of Erg11p caused by A395T mutation in ERG11 gene confers the fluconazole resistance in C. tropicalis.

  11. Free-energy computations identify the mutations required to confer trans-sialidase activity into Trypanosoma rangeli sialidase.

    PubMed

    Pierdominici-Sottile, Gustavo; Palma, Juliana; Roitberg, Adrian E

    2014-03-01

    Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans-sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH-33). They share 70% of sequence identity and their crystallographic Cα RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl-glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans-sialidase activity through appropriate point mutations. Recently, we calculated the free-energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA5mut), which has faint trans-sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA5mut that should create a mutant (TrSA10mut ) with high trans-sialidase activity.

  12. Independent mutations in the Rdl locus confer dieldrin resistance to Anopheles gambiae and An. arabiensis.

    PubMed

    Du, W; Awolola, T S; Howell, P; Koekemoer, L L; Brooke, B D; Benedict, M Q; Coetzee, M; Zheng, L

    2005-04-01

    Substitutions of a conserved alanine residue in the Rdl locus coding for a gamma-aminobutyric acid (GABA) receptor subunit with serine or glycine confer resistance to dieldrin in various insect species. Here, we show that alanine to glycine substitution in the Rdl locus of the malaria vector, Anopheles gambiae, is genetically linked to resistance to dieldrin. An alanine to serine substitution developed independently in a dieldrin resistant strain of An. arabiensis. An allele-specific polymerase chain reaction (PCR) assay was able to differentiate dieldrin resistant and susceptible mosquitoes.

  13. Extended life-span conferred by cotransporter gene mutations in Drosophila.

    PubMed

    Rogina, B; Reenan, R A; Nilsen, S P; Helfand, S L

    2000-12-15

    Aging is genetically determined and environmentally modulated. In a study of longevity in the adult fruit fly, Drosophila melanogaster, we found that five independent P-element insertional mutations in a single gene resulted in a near doubling of the average adult life-span without a decline in fertility or physical activity. Sequence analysis revealed that the product of this gene, named Indy (for I'm not dead yet), is most closely related to a mammalian sodium dicarboxylate cotransporter-a membrane protein that transports Krebs cycle intermediates. Indy was most abundantly expressed in the fat body, midgut, and oenocytes: the principal sites of intermediary metabolism in the fly. Excision of the P element resulted in a reversion to normal life-span. These mutations may create a metabolic state that mimics caloric restriction, which has been shown to extend life-span.

  14. Increased in vivo amyloid-β42 production, exchange, and loss in presenilin mutation carriers.

    PubMed

    Potter, Rachel; Patterson, Bruce W; Elbert, Donald L; Ovod, Vitaliy; Kasten, Tom; Sigurdson, Wendy; Mawuenyega, Kwasi; Blazey, Tyler; Goate, Alison; Chott, Robert; Yarasheski, Kevin E; Holtzman, David M; Morris, John C; Benzinger, Tammie L S; Bateman, Randall J

    2013-06-12

    Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-β (Aβ) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aβ42 compared to Aβ40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers. We used compartmental modeling of stable isotope labeling kinetic (SILK) studies in human carriers of PSEN mutations and related noncarriers to evaluate the pathophysiological effects of PSEN1 and PSEN2 mutations on the production and turnover of Aβ isoforms. We compared these findings by mutation status and amount of fibrillar amyloid deposition as measured by positron emission tomography (PET) using the amyloid tracer Pittsburgh compound B (PIB). CNS Aβ42 to Aβ40 production rates were 24% higher in mutation carriers compared to noncarriers, and this was independent of fibrillar amyloid deposits quantified by PET PIB imaging. The fractional turnover rate of soluble Aβ42 relative to Aβ40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aβ42 into plaques, leading to reduced recovery of Aβ42 in cerebrospinal fluid (CSF). Reversible exchange of Aβ42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aβ42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aβ42 concentrations in the CSF.

  15. Global Dissemination of a Single Mutation Conferring White Pericarp in Rice

    PubMed Central

    Sweeney, Megan T; Thomson, Michael J; Cho, Yong Gu; Park, Yong Jin; Williamson, Scott H; Bustamante, Carlos D; McCouch, Susan R

    2007-01-01

    Here we report that the change from the red seeds of wild rice to the white seeds of cultivated rice (Oryza sativa) resulted from the strong selective sweep of a single mutation, a frame-shift deletion within the Rc gene that is found in 97.9% of white rice varieties today. A second mutation, also within Rc, is present in less than 3% of white accessions surveyed. Haplotype analysis revealed that the predominant mutation originated in the japonica subspecies and crossed both geographic and sterility barriers to move into the indica subspecies. A little less than one Mb of japonica DNA hitchhiked with the rc allele into most indica varieties, suggesting that other linked domestication alleles may have been transferred from japonica to indica along with white pericarp color. Our finding provides evidence of active cultural exchange among ancient farmers over the course of rice domestication coupled with very strong, positive selection for a single white allele in both subspecies of O. sativa. PMID:17696613

  16. Mutation of Rv2887, a marR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

    PubMed Central

    Winglee, Kathryn; Lun, Shichun; Pieroni, Marco; Kozikowski, Alan

    2015-01-01

    Drug resistance is a major problem in Mycobacterium tuberculosis control, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity against M. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independent M. tuberculosis mutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations in Rv2887 were common to all three MP-III-71-resistant mutants, and we confirmed the role of Rv2887 as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified in Escherichia coli to negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation of Rv2887 abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations of Rv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance of M. tuberculosis Rv2887 mutants may involve efflux pump upregulation and also drug methylation. PMID:26303802

  17. Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum.

    PubMed

    Leßmeier, Lennart; Wendisch, Volker F

    2015-10-16

    Methanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes. The methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by experimental evolution in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1. The methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.

  18. Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

    PubMed

    Roditakis, Emmanouil; Steinbach, Denise; Moritz, Gerald; Vasakis, Emmanouil; Stavrakaki, Marianna; Ilias, Aris; García-Vidal, Lidia; Martínez-Aguirre, María Del Rosario; Bielza, Pablo; Morou, Evangelia; Silva, Jefferson E; Silva, Wellington M; Siqueira, Ηerbert A A; Iqbal, Sofia; Troczka, Bartlomiej J; Williamson, Martin S; Bass, Chris; Tsagkarakou, Anastasia; Vontas, John; Nauen, Ralf

    2017-01-01

    Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance

  19. A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance.

    PubMed

    Sharma, Paresh; Rayavara, Kempaiah; Ito, Daisuke; Basore, Katherine; Desai, Sanjay A

    2015-06-01

    Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted α-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.

  20. Lamivudine/Adefovir Treatment Increases the Rate of Spontaneous Mutation of Hepatitis B Virus in Patients

    PubMed Central

    Pereira-Gómez, Marianoel; Bou, Juan-Vicente; Andreu, Iván; Sanjuán, Rafael

    2016-01-01

    The high levels of genetic diversity shown by hepatitis B virus (HBV) are commonly attributed to the low fidelity of its polymerase. However, the rate of spontaneous mutation of human HBV in vivo is currently unknown. Here, based on the evolutionary principle that the population frequency of lethal mutations equals the rate at which they are produced, we have estimated the mutation rate of HBV in vivo by scoring premature stop codons in 621 publicly available, full-length, molecular clone sequences derived from patients. This yielded an estimate of 8.7 × 10−5 spontaneous mutations per nucleotide per cell infection in untreated patients, which should be taken as an upper limit estimate because PCR errors and/or lack of effective lethality may inflate observed mutation frequencies. We found that, in patients undergoing lamivudine/adefovir treatment, the HBV mutation rate was elevated by more than sixfold, revealing a mutagenic effect of this treatment. Genome-wide analysis of single-nucleotide polymorphisms indicated that lamivudine/adefovir treatment increases the fraction of A/T-to-G/C base substitutions, consistent with recent work showing similar effects of lamivudine in cellular DNA. Based on these data, the rate at which HBV produces new genetic variants in treated patients is similar to or even higher than in RNA viruses. PMID:27649318

  1. Disruption of Xpg increases spontaneous mutation frequency, particularly A:T to C:G transversion.

    PubMed

    Shiomi, N; Hayashi, E; Sasanuma, S; Mita, K; Shiomi, T

    2001-12-19

    Cells isolated from Xpg (the mouse counterpart of XPG)-disrupted mice underwent premature senescence and showed early onset of immortalization, suggesting that Xpg might be involved in genetic stability. Recent studies showed that human XPG, in addition to its function in the nucleotide excision repair (NER), was involved in the repair of oxidative base damages such as thymine glycol (Tg) and 8-oxo-guanine (8-oxoG), and this may explain the genetic instability observed in Xpg-deficient cells. To clarify this point, we determined spontaneous mutation frequencies and the type of spontaneous base substitution mutations in cells obtained from normal and Xpg-deficient mice using the supF shuttle vector (pNY200) for mutation assay. The spontaneous mutation frequency of the supF gene in pNY200 propagated in the Xpg-deficient cells was about three times higher than that in normal cells, indicating the importance of Xpg in reducing the frequency of spontaneous mutations. The frequency of spontaneous base substitution mutations at A:T sites, particularly that of the A:T to C:G transversion, increased markedly in the Xpg-deficient cells.

  2. GATA1 mutations in patients with down syndrome and acute megakaryoblastic leukaemia do not always confer a good prognosis.

    PubMed

    Ariffin, Hany; Garcia, Jaime Castillo; Daud, Siti Sarah; Ibrahim, Kamariah; Aizah, Nik; Ong, Gek-Bee; Chong, Lee-Ai; Mohamad, Zulqarnain

    2009-07-01

    Children with Down syndrome and acute megakaryoblastic leukemia (DS-AMKL) have been shown to have increased sensitivity to cytarabine based chemotherapy. The excellent prognosis in patients with DS-AMKL may be due to mutations in the GATA1 gene leading to reduced expression of the enzyme cytidine deaminase. This leads to a decreased ability to convert cytarabine into its inactive metabolite, resulting in high intracellular concentration of this cytotoxic agent. We report two cases of DS-AMKL with GATA1 mutations who had poor outcome. These patients had high expression levels of cytidine deaminase mRNA transcripts. We speculate that other factors can affect overall outcome in patients with DS-AMKL irrespective of the presence of GATA1 mutations.

  3. RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer.

    PubMed

    Yaeger, Rona; Cowell, Elizabeth; Chou, Joanne F; Gewirtz, Alexandra N; Borsu, Laetitia; Vakiani, Efsevia; Solit, David B; Rosen, Neal; Capanu, Marinela; Ladanyi, Marc; Kemeny, Nancy

    2015-04-15

    RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P < .01). RAS mutant mCRC exhibited a significantly higher cumulative incidence of lung, bone, and brain metastasis and on multivariate analysis was an independent predictor of involvement of these sites (HR, 1.5, 1.6, and 3.7, respectively). PIK3CA mutations occurred in 10% of the 786 cases genotyped, did not predict for worse survival, and did not exhibit a site-specific pattern of metastatic spread. The metastatic potential of CRC varies with the presence of RAS mutation. RAS mutation is associated with worse OS and increased incidence of lung, bone, and brain metastasis. An understanding of this site-specific pattern of spread may help to inform physicians' assessment of symptoms in patients with mCRC. © 2014 American Cancer Society.

  4. Mitochondrial mutations contribute to HIF1α accumulation via increased reactive oxygen species and upregulated PDK2 in head and neck squamous cell carcinoma

    PubMed Central

    Sun, Wenyue; Zhou, Shaoyu; Chang, Steven S.; McFate, Thomas; Verma, Ajay; Califano, Joseph A.

    2008-01-01

    Purpose Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1α accumulation and increased cell growth. The purpose of present study was to examine the pathways relating these alterations. Experimental Design Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1α, pyruvate dehydrogenease (PDH), phospho-PDH, and pyruvate dehydrogenease kinase (PDK) 2, together with ROS generation, were compared between the mutant and wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2, and mitochondrial targeted catalase, were evaluated on the ND2 mutant transfectants. Results We determined that ND2 mutant downregulated PDH expression via upregulated PDK2, with an increase in phospho-PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1α accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1α expression, and introduction of mitochondrial targeted catalase decreased mitochondrial mutation mediated PDK2 and HIF1α expression and suppressed cell growth. Conclusions Our findings suggest that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, upregulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1α stabilization. This may provide insight into a potential mechanism by which mitochondrial mutations contribute to HNSCC development. PMID:19147752

  5. LEOPARD syndrome-associated SHP2 mutation confers leanness and protection from diet-induced obesity

    PubMed Central

    Tajan, Mylène; Batut, Aurélie; Cadoudal, Thomas; Deleruyelle, Simon; Le Gonidec, Sophie; Saint Laurent, Céline; Vomscheid, Maëlle; Wanecq, Estelle; Tréguer, Karine; De Rocca Serra-Nédélec, Audrey; Vinel, Claire; Marques, Marie-Adeline; Pozzo, Joffrey; Kunduzova, Oksana; Salles, Jean-Pierre; Tauber, Maithé; Raynal, Patrick; Cavé, Hélène; Edouard, Thomas; Valet, Philippe; Yart, Armelle

    2014-01-01

    LEOPARD syndrome (multiple Lentigines, Electrocardiographic conduction abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth, sensorineural Deafness; LS), also called Noonan syndrome with multiple lentigines (NSML), is a rare autosomal dominant disorder associating various developmental defects, notably cardiopathies, dysmorphism, and short stature. It is mainly caused by mutations of the PTPN11 gene that catalytically inactivate the tyrosine phosphatase SHP2 (Src-homology 2 domain-containing phosphatase 2). Besides its pleiotropic roles during development, SHP2 plays key functions in energetic metabolism regulation. However, the metabolic outcomes of LS mutations have never been examined. Therefore, we performed an extensive metabolic exploration of an original LS mouse model, expressing the T468M mutation of SHP2, frequently borne by LS patients. Our results reveal that, besides expected symptoms, LS animals display a strong reduction of adiposity and resistance to diet-induced obesity, associated with overall better metabolic profile. We provide evidence that LS mutant expression impairs adipogenesis, triggers energy expenditure, and enhances insulin signaling, three features that can contribute to the lean phenotype of LS mice. Interestingly, chronic treatment of LS mice with low doses of MEK inhibitor, but not rapamycin, resulted in weight and adiposity gains. Importantly, preliminary data in a French cohort of LS patients suggests that most of them have lower-than-average body mass index, associated, for tested patients, with reduced adiposity. Altogether, these findings unravel previously unidentified characteristics for LS, which could represent a metabolic benefit for patients, but may also participate to the development or worsening of some traits of the disease. Beyond LS, they also highlight a protective role of SHP2 global LS-mimicking modulation toward the development of obesity and associated disorders

  6. Functional EGFR germline polymorphisms may confer risk for EGFR somatic mutations in non-small cell lung cancer, with a predominant effect on exon 19 microdeletions

    PubMed Central

    Liu, Wanqing; He, Lijun; Ramírez, Jacqueline; Krishnaswamy, Soundararajan; Kanteti, Rajani; Wang, Yi-Ching; Salgia, Ravi; Ratain, Mark J

    2011-01-01

    Somatic mutations in the EGFR tyrosine kinase (TK) domain play a critical role in the development and treatment of non-small cell lung cancer (NSCLC). Strong genetic influence on susceptibility to these mutations has been suggested. To identify the genetic factors conferring risk for the EGFR TK mutations in NSCLC, a case-control study was conducted in 141 Taiwanese NSCLC patients by focusing on three functional polymorphisms in the EGFR gene [-216G/T, intron 1(CA)n and R497K]. Allelic imbalance (AI) of the EGFR -216G/T polymorphism was also tested in the heterozygous patients as well as in the NCI-60 cancer cell lines to further verify its function. We found that the frequencies of the alleles -216T and CA-19 are significantly higher in the patients with any mutation (p=0.032 and 0.01, respectively), in particular in those with exon 19 microdeletions (p=0.006 and 0.033, respectively), but not in the patients with L858R mutation. The -216T allele is favored to be amplified in both tumor DNA of lung cancer patients and cancer cell lines. We conclude that the local haplotype structures across the EGFR gene may favor the development of cellular malignancies and thus significantly confer risk to the occurrence of EGFR mutations in NSCLC, particularly the exon 19 microdeletions. PMID:21292812

  7. Genetic Correlations Greatly Increase Mutational Robustness and Can Both Reduce and Enhance Evolvability

    PubMed Central

    Greenbury, Sam F.; Schaper, Steffen; Ahnert, Sebastian E.; Louis, Ard A.

    2016-01-01

    Mutational neighbourhoods in genotype-phenotype (GP) maps are widely believed to be more likely to share characteristics than expected from random chance. Such genetic correlations should strongly influence evolutionary dynamics. We explore and quantify these intuitions by comparing three GP maps—a model for RNA secondary structure, the HP model for protein tertiary structure, and the Polyomino model for protein quaternary structure—to a simple random null model that maintains the number of genotypes mapping to each phenotype, but assigns genotypes randomly. The mutational neighbourhood of a genotype in these GP maps is much more likely to contain genotypes mapping to the same phenotype than in the random null model. Such neutral correlations can be quantified by the robustness to mutations, which can be many orders of magnitude larger than that of the null model, and crucially, above the critical threshold for the formation of large neutral networks of mutationally connected genotypes which enhance the capacity for the exploration of phenotypic novelty. Thus neutral correlations increase evolvability. We also study non-neutral correlations: Compared to the null model, i) If a particular (non-neutral) phenotype is found once in the 1-mutation neighbourhood of a genotype, then the chance of finding that phenotype multiple times in this neighbourhood is larger than expected; ii) If two genotypes are connected by a single neutral mutation, then their respective non-neutral 1-mutation neighbourhoods are more likely to be similar; iii) If a genotype maps to a folding or self-assembling phenotype, then its non-neutral neighbours are less likely to be a potentially deleterious non-folding or non-assembling phenotype. Non-neutral correlations of type i) and ii) reduce the rate at which new phenotypes can be found by neutral exploration, and so may diminish evolvability, while non-neutral correlations of type iii) may instead facilitate evolutionary exploration and so

  8. Functional and computational assessment of missense variants in the ataxia-telangiectasia mutated (ATM) gene: mutations with increased cancer risk.

    PubMed

    Mitui, M; Nahas, S A; Du, L T; Yang, Z; Lai, C H; Nakamura, K; Arroyo, S; Scott, S; Purayidom, A; Concannon, P; Lavin, M; Gatti, R A

    2009-01-01

    The functional consequences of missense variants are often difficult to predict. This becomes especially relevant when DNA sequence changes are used to determine a diagnosis or prognosis. To analyze the consequences of 12 missense variants in patients with mild forms of ataxia-telangiectasia (A-T), we employed site-directed mutagenesis of ataxia-telangiectasia mutated (ATM) cDNA followed by stable transfections into a single A-T cell line to isolate the effects of each allele on the cellular phenotype. After induction of the transfected cells with CdCl2, we monitored for successful ATM transcription and subsequently assessed: 1) intracellular ATM protein levels; 2) ionizing radiation (IR)-induced ATM kinase activity; and 3) cellular radiosensitivity. We then calculated SIFT and PolyPhen scores for the missense changes. Nine variants produced little or no correction of the A-T cellular phenotype and were interpreted to be ATM mutations; SIFT/PolyPhen scores supported this. Three variants corrected the cellular phenotype, suggesting that they represented benign variants or polymorphisms. SIFT and PolyPhen scores supported the functional analyses for one of these variants (c.1709T>C); the other two were predicted to be "not tolerated" (c.6188G>A and c.6325T>G) and were classified as "operationally neutral." Genotype/phenotype relationships were compared: three deleterious missense variants were associated with an increased risk of cancer (c.6679C>T, c.7271T>G, and c.8494C>T). In situ mutagenesis represents an effective experimental approach for distinguishing deleterious missense mutations from benign or operationally neutral missense variants. Copyright 2008 Wiley-Liss, Inc.

  9. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism.

    PubMed

    Grassian, Alexandra R; Parker, Seth J; Davidson, Shawn M; Divakaruni, Ajit S; Green, Courtney R; Zhang, Xiamei; Slocum, Kelly L; Pu, Minying; Lin, Fallon; Vickers, Chad; Joud-Caldwell, Carol; Chung, Franklin; Yin, Hong; Handly, Erika D; Straub, Christopher; Growney, Joseph D; Vander Heiden, Matthew G; Murphy, Anne N; Pagliarini, Raymond; Metallo, Christian M

    2014-06-15

    Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation. ©2014 American Association for Cancer Research.

  10. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses.

    PubMed

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F X; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A; Roffler, Stefan

    2016-09-07

    DNA (class 2) transposons are mobile genetic elements which move within their 'host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind.

  11. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

    PubMed Central

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  12. IDH1 Mutations Alter Citric Acid Cycle Metabolism and Increase Dependence on Oxidative Mitochondrial Metabolism

    PubMed Central

    Grassian, Alexandra R.; Parker, Seth J.; Davidson, Shawn M.; Divakarun, Ajit S.; Green, Courtney R.; Zhang, Xiamei; Slocum, Kelly L.; Pu, Minying; Lin, Fallon; Vickers, Chad; Joud-Caldwell, Carol; Chung, Franklin; Yin, Hong; Handly, Erika D.; Straub, Christopher; Growney, Joseph D.; Vander Heiden, Matthew G.; Murphy, Anne N.; Pagliarini, Raymond; Metallo, Christian M.

    2016-01-01

    Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed 13C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation. PMID:24755473

  13. The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

    PubMed Central

    Julias, J G; Kim, T; Arnold, G; Pathak, V K

    1997-01-01

    It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

  14. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    PubMed Central

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W.; Amin, Hesham M.; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D.; Rosen, Steven T.; Pham, Lan V.; Ngo, Vu N.

    2016-01-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL. PMID:27713153

  15. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

    PubMed

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W; Amin, Hesham M; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D; Rosen, Steven T; Pham, Lan V; Ngo, Vu N

    2016-11-08

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5-10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

  16. In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.

    PubMed

    Logan, Angela; Shabalina, Irina G; Prime, Tracy A; Rogatti, Sebastian; Kalinovich, Anastasia V; Hartley, Richard C; Budd, Ralph C; Cannon, Barbara; Murphy, Michael P

    2014-08-01

    In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria-targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro-apoptotic and pro-inflammatory redox signaling pathways. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  17. Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: catalase, peroxidase, and INH-NADH adduct formation activities.

    PubMed

    Cade, Christine E; Dlouhy, Adrienne C; Medzihradszky, Katalin F; Salas-Castillo, Saida Patricia; Ghiladi, Reza A

    2010-03-01

    Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD(+)/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H(2)O(2)), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct.

  18. Isoniazid-resistance conferring mutations in Mycobacterium tuberculosis KatG: Catalase, peroxidase, and INH-NADH adduct formation activities

    PubMed Central

    Cade, Christine E; Dlouhy, Adrienne C; Medzihradszky, Katalin F; Salas-Castillo, Saida Patricia; Ghiladi, Reza A

    2010-01-01

    Mycobacterium tuberculosis catalase-peroxidase (KatG) is a bifunctional hemoprotein that has been shown to activate isoniazid (INH), a pro-drug that is integral to frontline antituberculosis treatments. The activated species, presumed to be an isonicotinoyl radical, couples to NAD+/NADH forming an isoniazid-NADH adduct that ultimately confers anti-tubercular activity. To better understand the mechanisms of isoniazid activation as well as the origins of KatG-derived INH-resistance, we have compared the catalytic properties (including the ability to form the INH-NADH adduct) of the wild-type enzyme to 23 KatG mutants which have been associated with isoniazid resistance in clinical M. tuberculosis isolates. Neither catalase nor peroxidase activities, the two inherent enzymatic functions of KatG, were found to correlate with isoniazid resistance. Furthermore, catalase function was lost in mutants which lacked the Met-Tyr-Trp crosslink, the biogenic cofactor in KatG which has been previously shown to be integral to this activity. The presence or absence of the crosslink itself, however, was also found to not correlate with INH resistance. The KatG resistance-conferring mutants were then assayed for their ability to generate the INH-NADH adduct in the presence of peroxide (t-BuOOH and H2O2), superoxide, and no exogenous oxidant (air-only background control). The results demonstrate that residue location plays a critical role in determining INH-resistance mechanisms associated with INH activation; however, different mutations at the same location can produce vastly different reactivities that are oxidant-specific. Furthermore, the data can be interpreted to suggest the presence of a second mechanism of INH-resistance that is not correlated with the formation of the INH-NADH adduct. PMID:20054829

  19. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  20. Frameshift Mutation Confers Function as Virulence Factor to Leucine-Rich Repeat Protein from Acidovorax avenae

    PubMed Central

    Kondo, Machiko; Hirai, Hiroyuki; Furukawa, Takehito; Yoshida, Yuki; Suzuki, Aika; Kawaguchi, Takemasa; Che, Fang-Sik

    2017-01-01

    Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (KΔLrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (NΔLrp) did not interfere with infection of finger millet. In addition, NΔLrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation. PMID:28101092

  1. Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4.

    PubMed

    Dai, Xiangpeng; Gan, Wenjian; Li, Xiaoning; Wang, Shangqian; Zhang, Wei; Huang, Ling; Liu, Shengwu; Zhong, Qing; Guo, Jianping; Zhang, Jinfang; Chen, Ting; Shimizu, Kouhei; Beca, Francisco; Blattner, Mirjam; Vasudevan, Divya; Buckley, Dennis L; Qi, Jun; Buser, Lorenz; Liu, Pengda; Inuzuka, Hiroyuki; Beck, Andrew H; Wang, Liewei; Wild, Peter J; Garraway, Levi A; Rubin, Mark A; Barbieri, Christopher E; Wong, Kwok-Kin; Muthuswamy, Senthil K; Huang, Jiaoti; Chen, Yu; Bradner, James E; Wei, Wenyi

    2017-09-01

    The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3(SPOP) earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

  2. Mechanisms by which a CACNA1H mutation in epilepsy patients increases seizure susceptibility

    PubMed Central

    Eckle, Veit-Simon; Shcheglovitov, Aleksandr; Vitko, Iuliia; Dey, Deblina; Yap, Chan Choo; Winckler, Bettina; Perez-Reyes, Edward

    2014-01-01

    T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca2+ channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I–II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca2+-regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Cav3.2 T-type Ca2+ channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression. PMID:24277868

  3. Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin

    PubMed Central

    Frohloff, Frank; Fichtner, Lars; Jablonowski, Daniel; Breunig, Karin D.; Schaffrath, Raffael

    2001-01-01

    Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G1 cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with Elongator. Intriguingly, strains lacking non-Elongator HATs (gcn5Δ, hat1Δ, hpa3Δ and sas3Δ) or non-Elongator transcription elongation factors TFIIS (dst1Δ) and Spt4p (spt4Δ) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin. PMID:11296232

  4. Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells

    PubMed Central

    Spender, Lindsay C.; Ferguson, G. John; Liu, Sijia; Cui, Chao; Girotti, Maria Romina; Sibbet, Gary; Higgs, Ellen B.; Shuttleworth, Morven K.; Hamilton, Tom; Lorigan, Paul; Weller, Michael; Vincent, David F.; Sansom, Owen J.; Frame, Margaret; Dijke, Peter ten; Marais, Richard; Inman, Gareth J.

    2016-01-01

    Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance. PMID:27835901

  5. Fast growth increases the selective advantage of a mutation arising recurrently during evolution under metal limitation.

    PubMed

    Chou, Hsin-Hung; Berthet, Julia; Marx, Christopher J

    2009-09-01

    Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B(12), a cobalt-containing cofactor, to sustain two vitamin B(12)-dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate-dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences.

  6. Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

    PubMed Central

    Chou, Hsin-Hung; Berthet, Julia; Marx, Christopher J.

    2009-01-01

    Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences. PMID:19763169

  7. A single locus confers tolerance to continuous light and allows substantial yield increase in tomato.

    PubMed

    Velez-Ramirez, Aaron I; van Ieperen, Wim; Vreugdenhil, Dick; van Poppel, Pieter M J A; Heuvelink, Ep; Millenaar, Frank F

    2014-08-05

    An important constraint for plant biomass production is the natural day length. Artificial light allows for longer photoperiods, but tomato plants develop a detrimental leaf injury when grown under continuous light--a still poorly understood phenomenon discovered in the 1920s. Here, we report a dominant locus on chromosome 7 of wild tomato species that confers continuous light tolerance. Genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III light harvesting chlorophyll a/b binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In Arabidopsis thaliana, this protein is thought to have a regulatory role balancing light harvesting by photosystems I and II. Introgressing the tolerance into modern tomato hybrid lines, results in up to 20% yield increase, showing that limitations for crop productivity, caused by the adaptation of plants to the terrestrial 24-h day/night cycle, can be overcome.

  8. A novel mutation, D404N, in the connection subdomain of reverse transcriptase of HIV-1 CRF08_BC subtype confers cross-resistance to NNRTIs

    PubMed Central

    Zhang, Xiao-Min; Wu, Hao; Zhang, Qiwei; Lau, Terrence Chi-Kong; Chu, Hin; Chen, Zhi-Wei; Jin, Dong-Yan; Zheng, Bo-Jian

    2015-01-01

    Objectives Growing evidence suggests that mutations in the connection domain of the HIV-1 reverse transcriptase (RT) can contribute to viral resistance to RT inhibitors. This work was designed to determine the effects of a novel mutation, D404N, in the connection subdomain of RT of HIV-1 CRF08_BC subtype on drug resistance, viral replication capacity (RC) and RT activity. Methods Mutation D404N, alone or together with the other reported mutations, was introduced into an HIV-1 CRF08_BC subtype infectious clone by site-directed mutagenesis. Viral susceptibility to nine RT inhibitors, viral RC and the DNA polymerase activity of viral RT of the constructed virus mutants were investigated. A modelling study using the server SWISS-MODEL was conducted to explore the possible structure-related drug resistance mechanism of the mutation D404N. Results Single mutations D404N and H221Y conferred low-level resistance to nevirapine, efavirenz, rilpivirine and zidovudine. Double mutations Y181C/D404N and Y181C/H221Y significantly reduced susceptibility to NNRTIs. The most pronounced resistance to NNRTIs was observed with the triple mutation Y181C/D404N/H221Y. Virus containing D404N as the only mutation displayed ∼50% RC compared with the WT virus. The modelling study suggested that the D404N mutation might abolish the hydrogen bonds between residues 404 and K30 in p51 or K431 in p66, leading to impaired RT subunit structure and enhanced drug resistance. Conclusions These results indicate that D404N is a novel NNRTI-associated mutation in the HIV-1 subtype CRF08_BC and provides information valuable for the monitoring of clinical RTI resistance. PMID:25637519

  9. Increased frequency of MEFV gene mutations in patients with primary dysmenorrhea.

    PubMed

    Erten, Sukran; Altunoglu, A; Keskin, H L; Ceylan, G G; Yazıcı, A; Dalgaci, A F; Uyanık, G; Avsar, A F

    2013-09-01

    Familial Mediterranean fever (FMF) is an autoinflammatory disease characterized by recurrent attacks of fever and polyserositis and an autosomal recessive inheritance mode. Up to 15 % of FMF patients are reported to experience perimenstrual attacks. Primary dysmenorrhea could be an incomplete abdominal attack, or patients with dysmenorrhea may have increased frequency of MEFV gene mutation carriage. Therefore, we aimed to evaluate the frequency of MEFV gene mutations in patients with dysmenorrhea. Eighty-four patients with primary dysmenorrhea attending consecutively to our gynecology department and 73 healthy female controls selected from hospital staff were included in the study, and MEFV gene mutations were analyzed. The prevalence of total allelic variants was significantly increased in dysmenorrhea patients (p = 0.015); analysis of individual variant rates revealed a significant increase in the frequency of MEFV gene mutations in dysmenorrhea patients compared with the control group (p = 0.036). Gynecologists and primary care physicians must be aware of FMF in the differential diagnosis of dysmenorrhea.

  10. Increased use of Twitter at a medical conference: a report and a review of the educational opportunities.

    PubMed

    McKendrick, Douglas R A; Cumming, Grant P; Lee, Amanda J

    2012-12-11

    Most consider Twitter as a tool purely for social networking. However, it has been used extensively as a tool for online discussion at nonmedical and medical conferences, and the academic benefits of this tool have been reported. Most anesthetists still have yet to adopt this new educational tool. There is only one previously published report of the use of Twitter by anesthetists at an anesthetic conference. This paper extends that work. We report the uptake and growth in the use of Twitter, a microblogging tool, at an anesthetic conference and review the potential use of Twitter as an educational tool for anesthetists. A unique Twitter hashtag (#WSM12) was created and promoted by the organizers of the Winter Scientific Meeting held by The Association of Anaesthetists of Great Britain and Ireland (AAGBI) in London in January 2012. Twitter activity was compared with Twitter activity previously reported for the AAGBI Annual Conference (September 2011 in Edinburgh). All tweets posted were categorized according to the person making the tweet and the purpose for which they were being used. The categories were determined from a literature review. A total of 227 tweets were posted under the #WSM12 hashtag representing a 530% increase over the previously reported anesthetic conference. Sixteen people joined the Twitter stream by using this hashtag (300% increase). Excellent agreement (κ = 0.924) was seen in the classification of tweets across the 11 categories. Delegates primarily tweeted to create and disseminate notes and learning points (55%), describe which session was attended, undertake discussions, encourage speakers, and for social reasons. In addition, the conference organizers, trade exhibitors, speakers, and anesthetists who did not attend the conference all contributed to the Twitter stream. The combined total number of followers of those who actively tweeted represented a potential audience of 3603 people. This report demonstrates an increase in uptake and

  11. BRCA1 and BRCA2 mutations in ovarian cancer patients from China: ethnic-related mutations in BRCA1 associated with an increased risk of ovarian cancer.

    PubMed

    Shi, Tingyan; Wang, Pan; Xie, Caixia; Yin, Sheng; Shi, Di; Wei, Congchong; Tang, Wenbin; Jiang, Rong; Cheng, Xi; Wei, Qingyi; Wang, Qing; Zang, Rongyu

    2017-05-01

    BRCA1/2 are cancer predisposition genes involved in hereditary breast and ovarian cancer (HBOC). Mutation carriers display an increased sensitivity to inhibitors of poly(ADP-ribose) polymerase (PARP). Despite a number of small-size hospital-based studies being previously reported, there is not yet, to our knowledge, precise data of BRCA1/2 mutations among Chinese ovarian cancer patients. We performed a multicenter cohort study including 916 unselected consecutive epithelial ovarian cancer (EOC) patients from eastern China to screen for BRCA1/2 mutations using the next-generation sequencing approach. A total of 153 EOC patients were found to carry pathogenic germline mutations in BRCA1/2, accounting for an overall mutation incidence of 16.7% with the predominance in BRCA1 (13.1%) compared with BRCA2 (3.9%). We identified 53 novel pathogenic mutations, among which the c.283_286delCTTG and the c.4573C > T of BRCA1 were both found in two unrelated patients. More importantly, the most common mutation found in this study, c.5470_5477del8 was most likely to be Chinese population-related without an apparent founder origin. This hot-spot mutation was presumably associated with an increased risk of ovarian cancer. Taken together, germline BRCA1/2 mutations were common in Chinese EOC patients with distinct mutational spectrum compared to Western populations. Our study contributes to the current understanding of BRCA1/2 mutation prevalence worldwide. We recommend BRCA1/2 genetic testing to all Chinese women diagnosed with EOC to identify HBOC families, to provide genetic counseling and clinical management for at-risk relatives. Mutation carriers may also benefit from PARP-targeted therapies. © 2017 UICC.

  12. Mutations in alternative carbon utilization pathways in Candida albicans attenuate virulence and confer pleiotropic phenotypes.

    PubMed

    Ramírez, Melissa A; Lorenz, Michael C

    2007-02-01

    The interaction between Candida albicans and cells of the innate immune system is a key determinant of disease progression. Transcriptional profiling has revealed that C. albicans has a complex response to phagocytosis, much of which is similar to carbon starvation. This suggests that nutrient limitation is a significant stress in vivo, and we have shown that glyoxylate cycle mutants are less virulent in mice. To examine whether other aspects of carbon metabolism are important in vivo during an infection, we have constructed strains lacking FOX2 and FBP1, which encode key components of fatty acid beta-oxidation and gluconeogenesis, respectively. As expected, fox2Delta mutants failed to utilize several fatty acids as carbon sources. Surprisingly, however, these mutants also failed to grow in the presence of several other carbon sources, whose assimilation is independent of beta-oxidation, including ethanol and citric acid. Mutants lacking the glyoxylate enzyme ICL1 also had more severe carbon utilization phenotypes than were expected. These results suggest that the regulation of alternative carbon metabolism in C. albicans is significantly different from that in other fungi. In vivo, fox2Delta mutants show a moderate but significant reduction in virulence in a mouse model of disseminated candidiasis, while disruption of the glyoxylate cycle or gluconeogenesis confers a severe attenuation in this model. These data indicate that C. albicans often encounters carbon-poor conditions during growth in the host and that the ability to efficiently utilize multiple nonfermentable carbon sources is a virulence determinant. Consistent with this in vivo requirement, C. albicans uniquely regulates carbon metabolism in a more integrated manner than in Saccharomyces cerevisiae, such that defects in one part of the machinery have wider impacts than expected. These aspects of alternative carbon metabolism may then be useful as targets for therapeutic intervention.

  13. Mutations of the TATA-binding protein confer enhanced tolerance to hyperosmotic stress in Saccharomyces cerevisiae.

    PubMed

    Kim, Na-Rae; Yang, Jungwoo; Kwon, Hyeji; An, Jieun; Choi, Wonja; Kim, Wankee

    2013-09-01

    Previously, it was shown that overexpression of either of two SPT15 mutant alleles, SPT15-M2 and SPT15-M3, which encode mutant TATA-binding proteins, confer enhanced ethanol tolerance in Saccharomyces cerevisiae. In this study, we demonstrated that strains overexpressing SPT15-M2 or SPT15-M3 were tolerant to hyperosmotic stress caused by high concentrations of glucose, salt, and sorbitol. The enhanced tolerance to high glucose concentrations in particular improved ethanol production from very high gravity (VHG) ethanol fermentations. The strains displayed constitutive and sustained activation of Hog1, a central kinase in the high osmolarity glycerol (HOG) signal transduction pathway of S. cerevisiae. However, the cell growth defect known to be caused by constitutive and sustained activation of Hog1 was not observed. We also found that reactive oxygen species (ROS) were accumulated to a less extent upon exposure to high glucose concentration in our osmotolerant strains. We identified six new genes (GPH1, HSP12, AIM17, SSA4, USV1, and IGD1), the individual deletion of which renders cells sensitive to 50 % glucose. In spite of the presence of multiple copies of stress response element in their promoters, it was apparent that those genes were not controlled at the transcriptional level by the HOG pathway under the high glucose conditions. Combined with previously published results, overexpression of SPT15-M2 or SPT15-M3 clearly provides a basis for improved tolerance to ethanol and osmotic stress, which enables construction of strains of any genetic background that need enhanced tolerance to high concentrations of ethanol and glucose, promoting the feasibility for VHG ethanol fermentation.

  14. Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

    PubMed Central

    Sexton, Nicole R.; Smith, Everett Clinton; Blanc, Hervé; Vignuzzi, Marco; Peersen, Olve B.

    2016-01-01

    ABSTRACT Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity

  15. Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice.

    PubMed

    Morgan, Daniel J; Davis, Brian J; Kearn, Chris S; Marcus, David; Cook, Alex J; Wager-Miller, Jim; Straiker, Alex; Myoga, Michael H; Karduck, Jeffrey; Leishman, Emma; Sim-Selley, Laura J; Czyzyk, Traci A; Bradshaw, Heather B; Selley, Dana E; Mackie, Ken

    2014-04-09

    For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

  16. A Mutation in a Saccharomyces Cerevisiae Gene (Rad3) Required for Nucleotide Excision Repair and Transcription Increases the Efficiency of Mismatch Correction

    PubMed Central

    Yang, Y.; Johnson, A. L.; Johnston, L. H.; Siede, W.; Friedberg, E. C.; Ramachandran, K.; Kunz, B. A.

    1996-01-01

    RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase δ was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency. PMID:8889512

  17. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    PubMed Central

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  18. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    PubMed

    Tay, Wee Tek; Mahon, Rod J; Heckel, David G; Walsh, Thomas K; Downes, Sharon; James, William J; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K; Gordon, Karl H J

    2015-11-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  19. Origin and dissemination of the pollen-part mutated SC haplotype which confers self-compatibility in apricot (Prunus armeniaca).

    PubMed

    Halász, Júlia; Pedryc, Andrzej; Hegedus, Attila

    2007-01-01

    In China, its centre of origin, apricot (Prunus armeniaca) is self-incompatible. However, most European cultivars are self-compatible. In most cases, self-compatibility is a result of a loss-of-function mutation within the pollen gene (SFB) in the SC haplotype. Controlled pollinations performed in this work revealed that the cross 'Ceglédi óriás' (S8S9)x'Ceglédi arany' (SCS9) set well, as expected, but the reciprocal cross did not. Apricot S8, S9 and SC haplotypes were analysed using a multilevel approach including fruit set evaluation, pollen tube growth analysis, RNase activity assays, polymerase chain reaction (PCR) analysis and DNA sequencing of the S-RNase and SFB alleles. SFB8 was revealed to be the first known progenitor allele of a naturally occurring self-compatibility allele in Prunus, and consequently SC=The first intron of SC-RNase is a phase one intron, indicating its more recent evolutionary origin compared with the second intron. Sequence analysis of different cultivars revealed that more single nucleotide polymorphisms accumulated in SC-RNase than in SFBC. New methods were designed to allow high-throughput analysis of S genotypes of apricot cultivars and selections. S-RNase sequence data from various sources helped to elucidate the putative origin and dissemination of self-compatibility in apricot conferred by the SC haplotype.

  20. A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B☆

    PubMed Central

    Robinson, Jerid W.; Dickey, Deborah M.; Miura, Kohji; Michigami, Toshimi; Ozono, Keiichi; Potter, Lincoln R.

    2015-01-01

    C-type natriuretic peptide (CNP) increases long bone growth by stimulating guanylyl cyclase (GC)-B/NPR-B/NPR2. Recently, a Val to Met missense mutation at position 883 in the catalytic domain of GC-B was identified in humans with increased blood cGMP levels that cause abnormally long bones. Here, we determined how this mutation activates GC-B. In the absence of CNP, cGMP levels in cells expressing V883M-GC-B were increased more than 20 fold compared to cells expressing wild-type (WT)-GC-B, and the addition of CNP only further increased cGMP levels 2-fold. In the absence of CNP, maximal enzymatic activity (Vmax) of V883M-GC-B was increased 15-fold compared to WT-GC-B but the affinity of the enzymes for substrate as revealed by the Michaelis constant (Km) was unaffected. Surprisingly, CNP decreased the Km of V883M-GC-B 10-fold in a concentration dependent manner without increasing Vmax. Unlike the WT enzyme the Km reduction of V883M-GC-B did not require ATP. Unexpectedly, V883M-GC-B, but not WT-GC-B, failed to inactivate with time. Phosphorylation elevated but was not required for the activity increase associated with the mutation because the Val to Met substitution also activated a GC-B mutant lacking all known phosphorylation sites. We conclude that the V883M mutation increases maximal velocity in the absence of CNP, eliminates the requirement for ATP in the CNP-dependent Km reduction, and disrupts the normal inactivation process. PMID:23827346

  1. An Arabidopsis soil-salinity-tolerance mutation confers ethylene-mediated enhancement of sodium/potassium homeostasis.

    PubMed

    Jiang, Caifu; Belfield, Eric J; Cao, Yi; Smith, J Andrew C; Harberd, Nicholas P

    2013-09-01

    High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ethylene overproducer1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ethylene resistant1-constitutive triple response1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of respiratory burst oxidase homolog F (RBOHF)-dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated high-affinity K(+) TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation.

  2. An Arabidopsis Soil-Salinity–Tolerance Mutation Confers Ethylene-Mediated Enhancement of Sodium/Potassium Homeostasis[W

    PubMed Central

    Jiang, Caifu; Belfield, Eric J.; Cao, Yi; Smith, J. Andrew C.; Harberd, Nicholas P.

    2013-01-01

    High soil Na concentrations damage plants by increasing cellular Na accumulation and K loss. Excess soil Na stimulates ethylene-induced soil-salinity tolerance, the mechanism of which we here define via characterization of an Arabidopsis thaliana mutant displaying transpiration-dependent soil-salinity tolerance. This phenotype is conferred by a loss-of-function allele of ETHYLENE OVERPRODUCER1 (ETO1; mutant alleles of which cause increased production of ethylene). We show that lack of ETO1 function confers soil-salinity tolerance through improved shoot Na/K homeostasis, effected via the ETHYLENE RESISTANT1–CONSTITUTIVE TRIPLE RESPONSE1 ethylene signaling pathway. Under transpiring conditions, lack of ETO1 function reduces root Na influx and both stelar and xylem sap Na concentrations, thereby restricting root-to-shoot delivery of Na. These effects are associated with increased accumulation of RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF)–dependent reactive oxygen species in the root stele. Additionally, lack of ETO1 function leads to significant enhancement of tissue K status by an RBOHF-independent mechanism associated with elevated HIGH-AFFINITY K+ TRANSPORTER5 transcript levels. We conclude that ethylene promotes soil-salinity tolerance via improved Na/K homeostasis mediated by RBOHF-dependent regulation of Na accumulation and RBOHF-independent regulation of K accumulation. PMID:24064768

  3. Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

    PubMed Central

    Effner, Emily E.; Iglesias-Sánchez, Maria José; Golden, Susan S.

    2016-01-01

    In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongatus PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678–16683, 2012, http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide of S. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains. PMID:26921432

  4. Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL

    PubMed Central

    Zhang, Yixi; Meng, Xiangkun; Yang, Yuanxue; Li, Hong; Wang, Xin; Yang, Baojun; Zhang, Jianhua; Li, Chunrui; Millar, Neil S.; Liu, Zewen

    2016-01-01

    Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance. PMID:27557781

  5. Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL.

    PubMed

    Zhang, Yixi; Meng, Xiangkun; Yang, Yuanxue; Li, Hong; Wang, Xin; Yang, Baojun; Zhang, Jianhua; Li, Chunrui; Millar, Neil S; Liu, Zewen

    2016-08-25

    Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance.

  6. Women in Nontraditional Jobs: A Conference Guide. Increasing Job Options for Women.

    ERIC Educational Resources Information Center

    Women's Bureau (DOL), Washington, DC.

    Designed to help organizations interested in expanding job options for women to plan and hold a community-based conference on nontraditional jobs, this guide outlines basic steps in planning, provides information about successful programs, and makes suggestions about how to deal with the mechanics of a conference. Following an introduction which…

  7. Dual E627K and D701N mutations in the PB2 protein of A(H7N9) influenza virus increased its virulence in mammalian models

    PubMed Central

    Zhu, Wenfei; Li, Long; Yan, Zhigang; Gan, Tanhuan; Li, Lifeng; Chen, Rirong; Chen, Ruidong; Zheng, Zuoyi; Hong, Wenshan; Wang, Jia; Smith, David K.; Guan, Yi; Zhu, Huachen; Shu, Yuelong

    2015-01-01

    The ongoing avian H7N9 influenza outbreaks in China have caused significant human fatal cases and the virus is becoming established in poultry. Mutations with potential to increase mammalian adaptation have occurred in the polymerase basic protein 2 (PB2) and other viral genes. Here we found that dual 627K and 701N mutations could readily occur during transmission of the virus among ferrets via direct physical contact, and these mutations conferred higher polymerase activity and improved viral replication in mammalian cells, and enhanced virulence in mice. Special attention needs to be paid to patients with such mutations, as these may serve as an indicator of higher virus replication and increased pathogenicity. PMID:26391278

  8. Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect

    PubMed Central

    Zhang, Dandan; Gong, Lingling; He, Fei; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E.; Wu, Kongming

    2016-01-01

    Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control. PMID:26872031

  9. Resistance to Bacillus thuringiensis Mediated by an ABC Transporter Mutation Increases Susceptibility to Toxins from Other Bacteria in an Invasive Insect.

    PubMed

    Xiao, Yutao; Liu, Kaiyu; Zhang, Dandan; Gong, Lingling; He, Fei; Soberón, Mario; Bravo, Alejandra; Tabashnik, Bruce E; Wu, Kongming

    2016-02-01

    Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Recent efforts to delay pest adaptation to Bt crops focus primarily on combinations of two or more Bt toxins that kill the same pest, but this approach is often compromised because resistance to one Bt toxin causes cross-resistance to others. Thus, integration of Bt toxins with alternative controls that do not exhibit such cross-resistance is urgently needed. The ideal scenario of negative cross-resistance, where selection for resistance to a Bt toxin increases susceptibility to alternative controls, has been elusive. Here we discovered that selection of the global crop pest, Helicoverpa armigera, for >1000-fold resistance to Bt toxin Cry1Ac increased susceptibility to abamectin and spineotram, insecticides derived from the soil bacteria Streptomyces avermitilis and Saccharopolyspora spinosa, respectively. Resistance to Cry1Ac did not affect susceptibility to the cyclodiene, organophospate, or pyrethroid insecticides tested. Whereas previous work demonstrated that the resistance to Cry1Ac in the strain analyzed here is conferred by a mutation disrupting an ATP-binding cassette protein named ABCC2, the new results show that increased susceptibility to abamectin is genetically linked with the same mutation. Moreover, RNAi silencing of HaABCC2 not only decreased susceptibility to Cry1Ac, it also increased susceptibility to abamectin. The mutation disrupting ABCC2 reduced removal of abamectin in live larvae and in transfected Hi5 cells. The results imply that negative cross-resistance occurs because the wild type ABCC2 protein plays a key role in conferring susceptibility to Cry1Ac and in decreasing susceptibility to abamectin. The negative cross-resistance between a Bt toxin and other bacterial insecticides reported here may facilitate more sustainable pest control.

  10. Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.

    PubMed

    Xu, G Wei; Toth, Julia I; da Silva, Sara R; Paiva, Stacey-Lynn; Lukkarila, Julie L; Hurren, Rose; Maclean, Neil; Sukhai, Mahadeo A; Bhattacharjee, Rabindra N; Goard, Carolyn A; Medeiros, Bruno; Gunning, Patrick T; Dhe-Paganon, Sirano; Petroski, Matthew D; Schimmer, Aaron D

    2014-01-01

    The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.

  11. Mutations in UBA3 Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924 in Human Leukemic Cells

    PubMed Central

    Xu, G. Wei; Toth, Julia I.; da Silva, Sara R.; Paiva, Stacey-Lynn; Lukkarila, Julie L.; Hurren, Rose; Maclean, Neil; Sukhai, Mahadeo A.; Bhattacharjee, Rabindra N.; Goard, Carolyn A.; Gunning, Patrick T.; Dhe-Paganon, Sirano; Petroski, Matthew D.; Schimmer, Aaron D.

    2014-01-01

    The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562MLN, R-U937MLN) were selected. R-K562MLN and R-U937MLN cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562MLN cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors. PMID:24691136

  12. Importance of sigma factor mutations in increased triclosan resistance in Salmonella Typhimurium.

    PubMed

    Gantzhorn, Mette Rørbæk; Olsen, John Elmerdahl; Thomsen, Line Elnif

    2015-05-19

    Salmonella enterica is the second most common foodborne pathogen. The use of biocides is crucial to prevent spread of foodborne pathogens, and it would be devastating for food safety if Salmonella would become resistant to the disinfectants used. Another concern is that exposure to disinfectants might lead to decreased susceptibility to antibiotics. The current study aimed to identify genetic changes causing high level triclosan resistance in S. enterica serovar Typhimurium and evaluate how these affected antibiotic resistance and efflux pump activity. Wild type strains S. Typhimurium 4/74 and DTU3 were adapted to increasing concentrations of the biocide triclosan by serial passage. High level triclosan resistant isolates (MIC > 1000 μg/ml) were obtained. Strains were genome sequenced, and SNPs in fabI, rpoS and rpoD were found to be associated with high level resistance. However, work with defined mutants revealed that a SNP in fabI was not sufficient to obtain high level resistance. This required additional mutations in the sigma factors rpoS or rpoD. The adapted strains showed triclosan-dependent increased efflux, increased fabI expression and reduced susceptibility towards the antibiotics enrofloxacin and sulphamethoxazole/trimethoprim. Medium level triclosan resistance could be obtained by fabI mutations in S. Typhimurium, however, high level resistance was found to require sigma factor mutations in addition to a fabI mutation. Reduced antibiotic sensitivity was observed for the adapted strains, which could be associated with increased efflux.

  13. Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer.

    PubMed

    Park, Hye-Jin; Park, Jeong-Bin; Lee, Sang-Jae; Song, Minjung

    2017-08-11

    Colon cancer is one of the most common types of cancer, and it has recently become a leading cause of death worldwide. Among colon cancers, the v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated form is notorious for its non-druggable features. Cetuximab, a monoclonal antibody that binds to the epidermal growth factor receptor, has been introduced as an antitumor therapy; however, secondary resistance and side effects significantly limit its effective use in these cancers. In this study, we prepared Phellinuslinteus on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. The combined treatment of PBR extract and cetuximab suppressed SW480 cell viability/proliferation, with the cells exhibiting altered cellular morphology and clonogenic potential. AnnexinV-fluorescein isothiocyanate/propidium iodide-stained flow cytometry and Western blotting were performed, and PBR extract combined with cetuximab treatment increased apoptosis of the SW480 cells and suppressed their KRAS protein expression. The potential of PBR as a synergistic anticancer agent was further investigated in a tumor-xenografted mouse model. Tumor growth was significantly suppressed with PBR extract and cetuximab co-treatment. In conclusion, PBR increased the sensitivity of KRAS-mutated colon cancer cells to cetuximab, which indicates the potential use of PBR as a medical food against colon cancer.

  14. Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer

    PubMed Central

    Park, Hye-Jin; Park, Jeong-Bin; Lee, Sang-Jae; Song, Minjung

    2017-01-01

    Colon cancer is one of the most common types of cancer, and it has recently become a leading cause of death worldwide. Among colon cancers, the v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated form is notorious for its non-druggable features. Cetuximab, a monoclonal antibody that binds to the epidermal growth factor receptor, has been introduced as an antitumor therapy; however, secondary resistance and side effects significantly limit its effective use in these cancers. In this study, we prepared Phellinuslinteus on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. The combined treatment of PBR extract and cetuximab suppressed SW480 cell viability/proliferation, with the cells exhibiting altered cellular morphology and clonogenic potential. AnnexinV–fluorescein isothiocyanate/propidium iodide–stained flow cytometry and Western blotting were performed, and PBR extract combined with cetuximab treatment increased apoptosis of the SW480 cells and suppressed their KRAS protein expression. The potential of PBR as a synergistic anticancer agent was further investigated in a tumor-xenografted mouse model. Tumor growth was significantly suppressed with PBR extract and cetuximab co-treatment. In conclusion, PBR increased the sensitivity of KRAS-mutated colon cancer cells to cetuximab, which indicates the potential use of PBR as a medical food against colon cancer. PMID:28800074

  15. Salmonella recD mutations increase recombination in a short sequence transduction assay.

    PubMed Central

    Miesel, L; Roth, J R

    1994-01-01

    We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease. Images PMID:8021190

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

    PubMed

    Czeizel, A

    1989-07-01

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

  17. Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products.

    PubMed

    Kristensen, Lasse S; Daugaard, Iben L; Christensen, Mariann; Hamilton-Dutoit, Stephen; Hager, Henrik; Hansen, Lise Lotte

    2010-12-01

    Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild-type homoduplexes. We have developed a fast COLD-PCR and high-resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen(®) KRAS mutation kit. The sensitivity was increased by 5- to 100-fold for melting temperature decreasing mutations when using COLD-PCR compared to standard PCR. Mutations, undetectable by the TheraScreen(®) kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. © 2010 Wiley-Liss, Inc.

  18. Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

    PubMed

    Butler, Jeff; Hooper, Kathryn A; Petrie, Stephen; Lee, Raphael; Maurer-Stroh, Sebastian; Reh, Lucia; Guarnaccia, Teagan; Baas, Chantal; Xue, Lumin; Vitesnik, Sophie; Leang, Sook-Kwan; McVernon, Jodie; Kelso, Anne; Barr, Ian G; McCaw, James M; Bloom, Jesse D; Hurt, Aeron C

    2014-04-01

    Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

  19. Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses

    PubMed Central

    Butler, Jeff; Hooper, Kathryn A.; Petrie, Stephen; Lee, Raphael; Maurer-Stroh, Sebastian; Reh, Lucia; Guarnaccia, Teagan; Baas, Chantal; Xue, Lumin; Vitesnik, Sophie; Leang, Sook-Kwan; McVernon, Jodie; Kelso, Anne; Barr, Ian G.; McCaw, James M.; Bloom, Jesse D.; Hurt, Aeron C.

    2014-01-01

    Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide. PMID:24699865

  20. A G protein alpha null mutation confers prolificacy potential in maize

    SciTech Connect

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-05-06

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.

  1. A G protein alpha null mutation confers prolificacy potential in maize

    DOE PAGES

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-05-06

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is importantmore » in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.« less

  2. A G protein alpha null mutation confers prolificacy potential in maize

    PubMed Central

    Urano, Daisuke; Jackson, David; Jones, Alan M.

    2015-01-01

    Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. The maize heterotrimeric G protein complex is important in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences. PMID:25948706

  3. Novel Timothy Syndrome Mutation Leading to Increase in CACNA1C Window Current

    PubMed Central

    Boczek, Nicole J.; Miller, Erin M.; Ye, Dan; Nesterenko, Vlad V.; Tester, David J.; Antzelevitch, Charles; Czosek, Richard J.; Ackerman, Michael J.; Ware, Stephanie M.

    2016-01-01

    Background Timothy syndrome (TS) is a rare multi-system genetic disorder characterized by a myriad of abnormalities including QT prolongation, syndactyly, and neurological symptoms. The predominant genetic causes are recurrent de novo missense mutations in exon 8/8A of the CACNA1C-encoded L-type calcium channel, however some cases remain genetically elusive. Objective To identify the genetic cause of TS in a case that did not harbor a CACNA1C mutation in exon 8/8A, and was negative for all other plausible genetic substrates. Methods Utilization of diagnostic exome sequencing to identify the genetic substrate responsible for our case of TS. The identified mutation was characterized using whole cell patch-clamp technique and the results of these analyses were modeled using a modified Luo-Rudy dynamic model to determine the effects on the cardiac action potential. Results Whole exome sequencing revealed a novel CACNA1C mutation, p.Ile1166Thr, in a young male with diagnosed TS. Functional electrophysiological analysis identified a novel mechanism of TS-mediated disease, with an overall loss of current density and a gain-of-function shift in activation, leading to an increased window current. Modeling studies of this variant predicted prolongation of the action potential, as well as the development of spontaneous early afterdepolarizations. Conclusion Through expanded whole exome sequencing, we have identified a novel genetic substrate for TS, p.Ile1166Thr-CACNA1C. Electrophysiological experiments combined with modeling studies have identified a novel TS mechanism through increased window current. Therefore, expanded genetic testing in cases of TS to the entire CACNA1C coding region, if initial targeted testing is negative, may be warranted. PMID:25260352

  4. Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

    PubMed

    Park, Sehee; Il Kim, Jin; Lee, Ilseob; Bae, Joon-Yong; Yoo, Kirim; Nam, Misun; Kim, Juwon; Sook Park, Mee; Song, Ki-Joon; Song, Jin-Won; Kee, Sun-Ho; Park, Man-Seong

    2017-09-07

    It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

  5. A red and far-red light receptor mutation confers resistance to the herbicide glyphosate

    PubMed Central

    Sharkhuu, Altanbadralt; Narasimhan, Meena L; Merzaban, Jasmeen S; Bressan, Ray A; Weller, Steve; Gehring, Chris

    2014-01-01

    Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant (gre1) was isolated and genetic analyses indicated that a dysfunctional red (R) and far-red (FR) light receptor, phytochrome B (phyB), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R:FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R:FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. PMID:24654847

  6. Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.

    PubMed

    Kennedy, Scott R; Salk, Jesse J; Schmitt, Michael W; Loeb, Lawrence A

    2013-01-01

    Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G → T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G → A and T → C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.

  7. Hyperferritinaemia-cataract syndrome: Worldwide mutations and phenotype of an increasingly diagnosed genetic disorder

    PubMed Central

    2010-01-01

    The hereditary hyperferritinaemia-cataract syndrome (HHCS) is characterised by an autosomal dominant cataract and high levels of serum ferritin without iron overload. The cataract develops due to L-ferritin deposits in the lens and its pulverulent aspect is pathognomonic. The syndrome is caused by mutations within the iron-responsive element of L-ferritin. These mutations prevent efficient binding of iron regulatory proteins 1 and 2 to the IRE in L-ferritin mRNA, resulting in an unleashed ferritin translation. This paper reviews all 31 mutations (27 single nucleotide transitions and four deletions) that have been described since 1995. Laboratory test showing hyperferritinaemia, normal serum iron and normal transferrin saturation are indicative for HHCS after exclusion of other causes of increased ferritin levels (inflammation, malignancy, alcoholic liver disease) and should prompt an ophthalmological consultation for diagnostic confirmation. Invasive diagnostics such as liver biopsy are not indicated. HHCS is an important differential diagnosis of hyperferritinaemia. Haematologists, gastroenterologists and ophthalmologists should be aware of this syndrome to spare patients from further invasive diagnosis (liver biopsy), and also from a false diagnosis of hereditary haemochromatosis followed by venesections. Patients diagnosed with HHCS should be counselled regarding the relative harmlessness of this genetic disease, with early cataract surgery as the only clinical consequence. PMID:20511138

  8. Essential Genes Embody Increased Mutational Robustness to Compensate for the Lack of Backup Genetic Redundancy

    PubMed Central

    Cohen, Osher; Oberhardt, Matthew; Yizhak, Keren; Ruppin, Eytan

    2016-01-01

    Genetic robustness is a hallmark of cells, occurring through many mechanisms and at many levels. Essential genes lack the common robustness mechanism of genetic redundancy (i.e., existing alongside other genes with the same function), and thus appear at first glance to leave cells highly vulnerable to genetic or environmental perturbations. Here we explore a hypothesis that cells might protect against essential gene loss through mechanisms that occur at various cellular levels aside from the level of the gene. Using Escherichia coli and Saccharomyces cerevisiae as models, we find that essential genes are enriched over non-essential genes for properties we call “coding efficiency” and “coding robustness”, denoting respectively a gene’s efficiency of translation and robustness to non-synonymous mutations. The coding efficiency levels of essential genes are highly positively correlated with their evolutionary conservation levels, suggesting that this feature plays a key role in protecting conserved, evolutionarily important genes. We then extend our hypothesis into the realm of metabolic networks, showing that essential metabolic reactions are encoded by more “robust” genes than non-essential reactions, and that essential metabolites are produced by more reactions than non-essential metabolites. Taken together, these results testify that robustness at the gene-loss level and at the mutation level (and more generally, at two cellular levels that are usually treated separately) are not decoupled, but rather, that cellular vulnerability exposed due to complete gene loss is compensated by increased mutational robustness. Why some genes are backed up primarily against loss and others against mutations still remains an open question. PMID:27997585

  9. Increased Use of Twitter at a Medical Conference: A Report and a Review of the Educational Opportunities

    PubMed Central

    Cumming, Grant P; Lee, Amanda J

    2012-01-01

    Background Most consider Twitter as a tool purely for social networking. However, it has been used extensively as a tool for online discussion at nonmedical and medical conferences, and the academic benefits of this tool have been reported. Most anesthetists still have yet to adopt this new educational tool. There is only one previously published report of the use of Twitter by anesthetists at an anesthetic conference. This paper extends that work. Objective We report the uptake and growth in the use of Twitter, a microblogging tool, at an anesthetic conference and review the potential use of Twitter as an educational tool for anesthetists. Methods A unique Twitter hashtag (#WSM12) was created and promoted by the organizers of the Winter Scientific Meeting held by The Association of Anaesthetists of Great Britain and Ireland (AAGBI) in London in January 2012. Twitter activity was compared with Twitter activity previously reported for the AAGBI Annual Conference (September 2011 in Edinburgh). All tweets posted were categorized according to the person making the tweet and the purpose for which they were being used. The categories were determined from a literature review. Results A total of 227 tweets were posted under the #WSM12 hashtag representing a 530% increase over the previously reported anesthetic conference. Sixteen people joined the Twitter stream by using this hashtag (300% increase). Excellent agreement (κ = 0.924) was seen in the classification of tweets across the 11 categories. Delegates primarily tweeted to create and disseminate notes and learning points (55%), describe which session was attended, undertake discussions, encourage speakers, and for social reasons. In addition, the conference organizers, trade exhibitors, speakers, and anesthetists who did not attend the conference all contributed to the Twitter stream. The combined total number of followers of those who actively tweeted represented a potential audience of 3603 people. Conclusions This

  10. Mutations in Nonessential eIF3k and eIF3l Genes Confer Lifespan Extension and Enhanced Resistance to ER Stress in Caenorhabditis elegans

    PubMed Central

    Reddy, Kirthi C.; Droste, Rita; Kim, Dennis H.

    2016-01-01

    The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. Here, we show that loss-of-function mutations in the genes encoding the conserved eIF3k and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16. Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1, ATF-6, and PEK-1, and independent of DAF-16. Our data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging. PMID:27690135

  11. Dependence On Glycolysis Sensitizes BRAF-mutated Melanomas For Increased Response To Targeted BRAF Inhibition

    PubMed Central

    Hardeman, Keisha N.; Peng, Chengwei; Paudel, Bishal B.; Meyer, Christian T.; Luong, Thong; Tyson, Darren R.; Young, Jamey D.; Quaranta, Vito; Fessel, Joshua P.

    2017-01-01

    Dysregulated metabolism can broadly affect therapy resistance by influencing compensatory signaling and expanding proliferation. Given many BRAF-mutated melanoma patients experience disease progression with targeted BRAF inhibitors, we hypothesized therapeutic response is related to tumor metabolic phenotype, and that altering tumor metabolism could change therapeutic outcome. We demonstrated the proliferative kinetics of BRAF-mutated melanoma cells treated with the BRAF inhibitor PLX4720 fall along a spectrum of sensitivity, providing a model system to study the interplay of metabolism and drug sensitivity. We discovered an inverse relationship between glucose availability and sensitivity to BRAF inhibition through characterization of metabolic phenotypes using nearly a dozen metabolic parameters in Principle Component Analysis. Subsequently, we generated rho0 variants that lacked functional mitochondrial respiration and increased glycolytic metabolism. The rho0 cell lines exhibited increased sensitivity to PLX4720 compared to the respiration-competent parental lines. Finally, we utilized the FDA-approved antiretroviral drug zalcitabine to suppress mitochondrial respiration and to force glycolysis in our cell line panel, resulting in increased PLX4720 sensitivity via shifts in EC50 and Hill slope metrics. Our data suggest that forcing tumor glycolysis in melanoma using zalcitabine or other similar approaches may be an adjunct to increase the efficacy of targeted BRAF therapy. PMID:28205616

  12. HLA-DR6 association confers increased resistance to T. rubrum onychomycosis in Mexican Mestizos.

    PubMed

    Asz-Sigall, Daniel; López-García, Lirio; Vega-Memije, María Elisa; Lacy-Niebla, Rosa María; García-Corona, Cristina; Ramírez-Rentería, Claudia; Granados, Julio; Villa, Antonio; Ameen, Mahreen; Arenas, Roberto

    2010-12-01

    Onychomycosis is multifactorial in origin. Studies have suggested an autosomal dominant pattern of inheritance and human leukocyte antigen DR4 (HLA-DR4) has been shown to protect against onychomycosis in an Ashkenazi Jewish population. This study investigates HLA class II association in a Mexican Mestizo population with Trichophyton rubrum onychomycosis. This was a prospective case-control study. Mexican Mestizos with a clinical diagnosis of onychomycosis and culture positive for T. rubrum were recruited, together with age- and sex-matched controls. First-degree relatives were also investigated for onychomycosis. Case-control samples were HLA typed by polymerase chain reaction sequence-specific primer based analysis. Twenty-one cases and 42 controls were recruited with a mean age of 40 years (range: 18-58 years). HLA-DR6 was found in seven (33%) cases and 19 (45%) controls [P < 0.023, odds ratio (OR) = 0.088, 95% confidence interval (CI): 0.01-0.71]. Six (29%) cases and three (7%) controls had an affected child (P < 0.043, OR = 9.15, 95% CI: 1.07-78.31), and 13 (62%) cases and 12 (29%) controls had an affected first-degree relative (P < 0.02, OR = 4.0, 95% CI: 1.1-14.3). These results suggest that HLA-DR6 confers protection against the development of onychomycosis in a Mexican Mestizo population. Having an affected first-degree relative significantly increases the risk of onychomycosis, suggesting genetic susceptibility. © 2010 The International Society of Dermatology.

  13. VEGF Promoter Polymorphism Confers an Increased Risk of Pulmonary Arterial Hypertension in a Chinese Population

    PubMed Central

    Zeng, Qingchun; Zhang, Peng; Li, Guoyang; Xie, Qiang; Cheng, Ying

    2017-01-01

    Purpose Evidence on the contribution of genes to the hereditary predisposition to pulmonary arterial hypertension (PAH) is limited. Materials and Methods In this study, we hypothesized that single nucleotide variants in vascular endothelial growth factor (VEGF) gene may alter gene function and expression and may be associated with PAH risk. Five putatively functional loci (rs699947C>A and rs833061T>C in the promoter, rs3025040C>T, rs10434G>A and rs3025053G>A in the 3'-UTR) in the VEGF gene were genotyped and analyzed in a retrospective study of 587 patients with PAH and 736 healthy subjects from southern China. Results We found that the rs833061T>C polymorphism was significantly associated with PAH risk, while the other single nucleotide polymorphisms were not. Compared to carriers with TT genotype, those with rs833061C variant genotype (CT/CC) had an increased risk of PAH (odds ratio=1.47, 95% confidence interval=1.18–1.83, p=0.001). Functional assays indicated that CT/CC variant genotype had significantly higher mRNA levels of VEGF in peripheral blood mononuclear cells than TT genotype (p=0.021). Luciferase reporter assay indicated that having a C allele conferred a significantly higher transcription activity than that with a T allele. Conclusion Our findings suggest that the functional polymorphism rs833061T>C in VEGF gene promoter modulates VEGF expression and may be a valuable biomarker for predicting PAH susceptibility. PMID:28120560

  14. Small-fiber neuropathy Nav1.8 mutation shifts activation to hyperpolarized potentials and increases excitability of dorsal root ganglion neurons.

    PubMed

    Huang, Jianying; Yang, Yang; Zhao, Peng; Gerrits, Monique M; Hoeijmakers, Janneke G J; Bekelaar, Kim; Merkies, Ingemar S J; Faber, Catharina G; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2013-08-28

    Idiopathic small-fiber neuropathy (I-SFN), clinically characterized by burning pain in distal extremities and autonomic dysfunction, is a disorder of small-caliber nerve fibers of unknown etiology with limited treatment options. Functional variants of voltage-gated sodium channel Nav1.7, encoded by SCN9A, have been identified in approximately one-third of I-SFN patients. These variants render dorsal root ganglion (DRG) neurons hyperexcitable. Sodium channel Nav1.8, encoded by SCN10A, is preferentially expressed in small-diameter DRG neurons, and produces most of the current underlying the upstroke of action potentials in these neurons. We previously demonstrated two functional variants of Nav1.8 that either enhance ramp current or shift activation in a hyperpolarizing direction, and render DRG neurons hyperexcitable, in I-SFN patients with no mutations of SCN9A. We have now evaluated additional I-SFN patients with no mutations in SCN9A, and report a novel I-SFN-related Nav1.8 mutation I1706V in a patient with painful I-SFN. Whole-cell voltage-clamp recordings in small DRG neurons demonstrate that the mutation hyperpolarizes activation and the response to slow ramp depolarizations. However, it decreases fractional channels resistant to fast inactivation and reduces persistent currents. Current-clamp studies reveal that mutant channels decrease current threshold and increase the firing frequency of evoked action potentials within small DRG neurons. These observations suggest that the effects of this mutation on activation and ramp current are dominant over the reduced persistent current, and show that these pro-excitatory gating changes confer hyperexcitability on peripheral sensory neurons, which may contribute to pain in this individual with I-SFN.

  15. Increased ventilatory response to exercise in symptomatic and asymptomatic LMNA mutation carriers: a follow-up study.

    PubMed

    Ollila, Laura; Heliö, Tiina; Sovijärvi, Anssi; Jalanko, Mikko; Kaartinen, Maija; Kuusisto, Johanna; Kärkkäinen, Satu; Jurkko, Raija; Reissell, Eeva; Palojoki, Eeva; Piirilä, Päivi

    2017-01-01

    LMNA mutations are an important cause of cardiomyopathy often leading to cardiac arrhythmias, heart failure and even heart transplantation. An increasing number of asymptomatic mutation carriers are identified, as family members of the index patients are screened. Our aim was to study the disease progression in asymptomatic LMNA mutation carriers and in patients with symptomatic cardiolaminopathy by repeated spiroergometric testing in a prospective clinical follow-up study. We studied 26 LMNA mutation carriers once a year during 5 years up to 6 times by spiroergometry, clinical assessment, laboratory tests and echocardiography. The 23 control subjects underwent clinical assessment and spiroergometry once. Twelve of the mutation carriers were asymptomatic, and 14 had some clinical manifestations of the mutation ranging from clinically relevant rhythm disturbances to DCM and heart failure. Compared to controls, the symptomatic carriers showed a higher slope of the ventilatory equivalent for CO2 (V˙E/V˙CO2 slope) and a lower fraction of end-tidal CO2 (FetCO2 ). The asymptomatic mutation carriers also showed an increased ventilatory response to exercise during the follow-up as indicated by increased V˙E/V˙CO2 slope and decreased FetCO2 . The study suggests that an increased ventilatory response during exercise might reveal a preclinical manifestation of DCM in LMNA mutation carriers. © 2015 The Authors. Clinical Physiology and Functional Imaging published by John Wiley & Sons Ltd on behalf of Scandinavian Society of Clinical Physiology and Nuclear Medicine.

  16. GBA mutations increase risk for Lewy body disease with and without Alzheimer disease pathology.

    PubMed

    Tsuang, Debby; Leverenz, James B; Lopez, Oscar L; Hamilton, Ronald L; Bennett, David A; Schneider, Julie A; Buchman, Aron S; Larson, Eric B; Crane, Paul K; Kaye, Jeffrey A; Kramer, Patricia; Woltjer, Randy; Kukull, Walter; Nelson, Peter T; Jicha, Gregory A; Neltner, Janna H; Galasko, Doug; Masliah, Eliezer; Trojanowski, John Q; Schellenberg, Gerard D; Yearout, Dora; Huston, Haley; Fritts-Penniman, Allison; Mata, Ignacio F; Wan, Jia Y; Edwards, Karen L; Montine, Thomas J; Zabetian, Cyrus P

    2012-11-06

    Mutations in the GBA gene occur in 7% of patients with Parkinson disease (PD) and are a well-established susceptibility factor for PD, which is characterized by Lewy body disease (LBD) neuropathologic changes (LBDNCs). We sought to determine whether GBA influences risk of dementia with LBDNCs, Alzheimer disease (AD) neuropathologic changes (ADNCs), or both. We screened the entire GBA coding region for mutations in controls and in subjects with dementia and LBDNCs and no or low levels of ADNCs (pure dementia with Lewy bodies [pDLB]), LBDNCs and high-level ADNCs (LBD-AD), and high-level ADNCs but without LBDNCs (AD). Among white subjects, pathogenic GBA mutations were identified in 6 of 79 pDLB cases (7.6%), 8 of 222 LBD-AD cases (3.6%), 2 of 243 AD cases (0.8%), and 3 of 381 controls (0.8%). Subjects with pDLB and LBD-AD were more likely to carry mutations than controls (pDLB: odds ratio [OR] = 7.6; 95% confidence interval [CI] = 1.8-31.9; p = 0.006; LBD-AD: OR = 4.6; CI = 1.2-17.6; p = 0.025), but there was no significant difference in frequencies between the AD and control groups (OR = 1.1; CI = 0.2-6.6; p = 0.92). There was a highly significant trend test across groups (χ(2)(1) = 19.3; p = 1.1 × 10(-5)), with the likelihood of carrying a GBA mutation increasing in the following direction: control/AD < LBD-AD < pDLB. GBA is a susceptibility gene across the LBD spectrum, but not in AD, and appears to convey a higher risk for PD and pDLB than for LBD-AD. PD and pDLB might be more similar to one another in genetic determinants and pathophysiology than either disease is to LBD-AD.

  17. Pore mutations in ammonium transporter AMT1 with increased electrogenic ammonium transport activity.

    PubMed

    Loqué, Dominique; Mora, Silvia I; Andrade, Susana L A; Pantoja, Omar; Frommer, Wolf B

    2009-09-11

    AMT/Mep ammonium transporters mediate high affinity ammonium/ammonia uptake in bacteria, fungi, and plants. The Arabidopsis AMT1 proteins mediate uptake of the ionic form of ammonium. AMT transport activity is controlled allosterically via a highly conserved cytosolic C terminus that interacts with neighboring subunits in a trimer. The C terminus is thus capable of modulating the conductivity of the pore. To gain insight into the underlying mechanism, pore mutants suppressing the inhibitory effect of mutations in the C-terminal trans-activation domain were characterized. AMT1;1 carrying the mutation Q57H in transmembrane helix I (TMH I) showed increased ammonium uptake but reduced capacity to take up methylammonium. To explore whether the transport mechanism was altered, the AMT1;1-Q57H mutant was expressed in Xenopus oocytes and analyzed electrophysiologically. AMT1;1-Q57H was characterized by increased ammonium-induced and reduced methylammonium-induced currents. AMT1;1-Q57H possesses a 100x lower affinity for ammonium (K(m)) and a 10-fold higher V(max) as compared with the wild type form. To test whether the trans-regulatory mechanism is conserved in archaeal homologs, AfAmt-2 from Archaeoglobus fulgidus was expressed in yeast. The transport function of AfAmt-2 also depends on trans-activation by the C terminus, and mutations in pore-residues corresponding to Q57H of AMT1;1 suppress nonfunctional AfAmt-2 mutants lacking the activating C terminus. Altogether, our data suggest that bacterial and plant AMTs use a conserved allosteric mechanism to control ammonium flux, potentially using a gating mechanism that limits flux to protect against ammonium toxicity.

  18. Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy.

    PubMed

    Berge, Knut Erik; Ose, Leiv; Leren, Trond P

    2006-05-01

    The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene encodes a proprotein convertase that causes degradation of cell surface low-density lipoprotein receptors (LDLRs). Mutations in the PCSK9 gene that disrupt the normal function of PCSK9 could therefore result in increased number of LDLRs and hypocholesterolemia. Also, the cholesterol-lowering effect of statins could be increased in subjects carrying mutations in the PCSK9 gene. We have screened 38 unrelated hypocholesterolemic subjects as well as 25 unrelated familial hypercholesterolemia (FH) heterozygotes who responded particularly well to statin therapy for mutations in the 12 exons of the PCSK9 gene by DNA sequencing. Six of the 38 (15.8%) hypocholesterolemic subjects were heterozygous for 1 of the 3 mutations R46L, G106R, or R237W in the PCSK9 gene. In the group of 25 FH heterozygotes who responded particularly well to statin therapy, 3 (8.8%) were heterozygous for mutations R46L or N157K in the PCSK9 gene. None of 441 hypercholesterolemic subjects without mutations in the LDLR gene or in the apolipoprotein B-100 gene possessed any of the 4 mutations. The 4 missense mutations R46L, G106R, N157K, and R237W are associated with hypocholesterolemia and possibly increased response to statin therapy.

  19. Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

    PubMed

    Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

    2006-07-01

    Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

  20. Mutations Conferring Resistance to SCH6, a Novel Hepatitis C Virus NS3/4A Protease Inhibitor: Reduced DNA Replication Fitness and Partial Rescue by Second-Site Mutations

    SciTech Connect

    Yi, MinKyung; Tong, Xiao; Skelton, Angela; Chase, Robert; Chen, Tong; Prongay, Andrew; Bogen, Stephane L.; Saksena, Anil K.; Njoroge, F. George; Veselenak, Ronald L.; Pyles, Richard B.; Bourne, Nigel; Malcolm, Bruce A.; Lemon, Stanley M.

    2008-06-30

    Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P{prime}-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

  1. Mutation G1629E Increases von Willebrand Factor Cleavage via a Cooperative Destabilization Mechanism.

    PubMed

    Aponte-Santamaría, Camilo; Lippok, Svenja; Mittag, Judith J; Obser, Tobias; Schneppenheim, Reinhard; Baldauf, Carsten; Gräter, Frauke; Budde, Ulrich; Rädler, Joachim O

    2017-01-10

    The large multimeric glycoprotein von Willebrand Factor (VWF) plays a pivotal adhesive role during primary hemostasis. VWF is cleaved by the protease ADAMTS13 as a down-regulatory mechanism to prevent excessive VWF-mediated platelet aggregation. For each VWF monomer, the ADAMTS13 cleavage site is located deeply buried inside the VWF A2 domain. External forces in vivo or denaturants in vitro trigger the unfolding of this domain, thereby leaving the cleavage site solvent-exposed and ready for cleavage. Mutations in the VWF A2 domain, facilitating the cleavage process, cause a distinct form of von Willebrand disease (VWD), VWD type 2A. In particular, the VWD type 2A Gly1629Glu mutation drastically accelerates the proteolytic cleavage activity, even in the absence of forces or denaturants. However, the effect of this mutation has not yet been quantified, in terms of kinetics or thermodynamics, nor has the underlying molecular mechanism been revealed. In this study, we addressed these questions by using fluorescence correlation spectroscopy, molecular dynamics simulations, and free energy calculations. The measured enzyme kinetics revealed a 20-fold increase in the cleavage rate for the Gly1629Glu mutant compared with the wild-type VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites of the VWF multimer at the same time. According to our simulations and free energy calculations, the Gly1629Glu mutation causes structural perturbation in the A2 domain and thereby destabilizes the domain by ∼10 kJ/mol, promoting its unfolding. Taken together, the enhanced proteolytic activity of Gly1629Glu can be readily explained by an increased availability of the ADAMTS13 cleavage site through A2-domain-fold thermodynamic destabilization. Our study puts forward the Gly1629Glu mutant as a very efficient enzyme substrate for ADAMTS13 activity assays. Copyright © 2017 Biophysical Society

  2. Mutations in blaKPC-3 That Confer Ceftazidime-Avibactam Resistance Encode Novel KPC-3 Variants That Function as Extended-Spectrum β-Lactamases.

    PubMed

    Haidar, Ghady; Clancy, Cornelius J; Shields, Ryan K; Hao, Binghua; Cheng, Shaoji; Nguyen, M Hong

    2017-05-01

    We identified four blaKPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant blaKPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop. Copyright © 2017 American Society for Microbiology.

  3. Mutations in Succinate Dehydrogenase Subunit C Increase Radiosensitivity and Bystander Responses

    NASA Astrophysics Data System (ADS)

    Zhou, Hongning; Hei, Tom K.

    Although radiation-induced bystander effect is well studied in the past decade, the precise mech-anisms are still unclear. It is likely that a combination of pathways involving both primary and secondary signaling processes is involved in producing a bystander effect. There is recent evidence that mitochondria play a critical role in bystander responses. Recently studies found that a mutation in succinate dehydrogenese subunit C (SDHC), an integral membrane protein in complex II of the electron transport chain, resulted in increased superoxide, oxidative stress, apoptosis, tumorigenesis, and genomic instability, indicating that SDHC play a critical role in maintaining mitochondrial function. In the present study, using Chinese hamster fibroblasts (B1 cells) and the mutants (B9 cells) containing a single base substitution that produced a premature stop codon resulting in a 33-amino acid COOH-terminal truncation of the SDHC protein, we found that B9 cells had an increase in intracellular superoxide content, nitric oxide species, and mitochondrial membrane potential when compared with wild type cells. After irradiated with a grade of doses of gamma rays, B9 cells show an increased radiosensitivity, especially at high doses. The HPRT- mutant yield after gamma-ray irradiation in B9 cells was significantly higher than that of B1 cells. A single, 3Gy dose of gamma-rays increased the background mutant level by more than 4 fold. In contrast, the mutant induction was less than 2 fold in B1 cells. In addition, B9 cells produced a higher bystander mutagenesis after alpha particle irradiation than the B1 cells. Furthermore, pretreated with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide scavenger, significantly decreased the bystander effect. Our findings demonstrate that a mutation in SDHC increases radiosensitivity in both directly irradiated cells and in neighboring bystander cells, and mito-chondrial function play an essential role in

  4. Microsatellite instability induced mutations in DNA repair genes CtIP and MRE11 confer hypersensitivity to poly (ADP-ribose) polymerase inhibitors in myeloid malignancies

    PubMed Central

    Gaymes, Terry J.; Mohamedali, Azim M.; Patterson, Miranda; Matto, Nazia; Smith, Alexander; Kulasekararaj, Austin; Chelliah, Rajani; Curtin, Nicola; Farzaneh, Farzin; Shall, Sydney; Mufti, Ghulam J.

    2013-01-01

    Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukemia and myelodysplastic syndrome correlated with chromosomal instability and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity through disruption of DNA repair function. Acute myeloid leukemia cell lines (n=12) and primary cell samples (n=18), and bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients (n=63) were profiled for microsatellite instability using fluorescent fragment polymerase chain reaction. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, Western blotting and gene expression analysis was used to study the functional consequences of mutations. Acute myeloid leukemia cell lines (4 of 12, 33%) and primary samples (2 of 18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukemia samples and cell lines demonstrated a downregulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11 of 56 (20%) patients with de novo high-risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4 of them (36%) possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP

  5. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

    PubMed Central

    2010-01-01

    Background Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. Results A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. Conclusions This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations. PMID:20846421

  6. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites.

    PubMed

    Hunt, Paul; Martinelli, Axel; Modrzynska, Katarzyna; Borges, Sofia; Creasey, Alison; Rodrigues, Louise; Beraldi, Dario; Loewe, Laurence; Fawcett, Richard; Kumar, Sujai; Thomson, Marian; Trivedi, Urmi; Otto, Thomas D; Pain, Arnab; Blaxter, Mark; Cravo, Pedro

    2010-09-16

    Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum. A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme. This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.

  7. Inhibition of MEK Confers Hypersensitivity to X-radiation in the context of BRAF mutation in a Model of Childhood Astrocytoma

    PubMed Central

    Studebaker, Adam; Bondra, Kathryn; Seum, Star; Shen, Changxian; Phelps, Doris A.; Chronowski, Christopher; Leasure, Justin; Smith, Paul D.; Kurmasheva, Raushan T.; Mo, Xiaokui; Fouladi, Maryam; Houghton, Peter J.

    2015-01-01

    Purpose Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment, however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma. Experimental Design The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts. Results Inhibition of MEK signaling by selumetinib, suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity. Conclusions Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae. PMID:25981859

  8. cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B.

    PubMed Central

    Ishiguro, J; Saitou, A; Durán, A; Ribas, J C

    1997-01-01

    The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguro and Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C. Under any of these restrictive treatments, cells swell up and finally lyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched. The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes. All of these data suggest a relationship between the cps1+ gene and cell wall synthesis. A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis. PMID:9401022

  9. Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

    PubMed Central

    Berrazeg, M.; Jeannot, K.; Ntsogo Enguéné, Véronique Yvette; Broutin, I.; Loeffert, S.; Fournier, D.

    2015-01-01

    Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting. PMID:26248364

  10. eis Promoter C14G and C15G Mutations Do Not Confer Kanamycin Resistance in Mycobacterium tuberculosis.

    PubMed

    Pholwat, Suporn; Stroup, Suzanne; Heysell, Scott; Ogarkov, Oleg; Zhdanova, Svetlana; Ramakrishnan, Girija; Houpt, Eric

    2016-12-01

    We studied the significance of particular eis mutations on Mycobacterium tuberculosis drug resistance using a specialized transduction strategy. Recombinant strains harboring eis promoter mutations C14T, C12T, and G10A exhibited kanamycin resistance with MICs of 40, 10, and 20 μg/ml, respectively, while recombinant strains harboring C14G and C15G mutations were kanamycin susceptible (MIC, 2.5 to 5 μg/ml). Each of the eis mutants tested remained amikacin susceptible (MIC, 0.5 to 4 μg/ml). The identification of specific eis mutations is needed for accurate genotypic susceptibility testing for kanamycin.

  11. Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

    PubMed Central

    Bommakanti, Ananth S.; Lindahl, Lasse; Zengel, Janice M.

    2008-01-01

    The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both Gram-positive and Gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes. PMID:18218702

  12. Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae.

    PubMed

    Bommakanti, Ananth S; Lindahl, Lasse; Zengel, Janice M

    2008-03-01

    The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.

  13. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

    PubMed Central

    Liu, Ta-Ming; Woyach, Jennifer A.; Zhong, Yiming; Lozanski, Arletta; Lozanski, Gerard; Dong, Shuai; Strattan, Ethan; Lehman, Amy; Zhang, Xiaoli; Jones, Jeffrey A.; Flynn, Joseph; Andritsos, Leslie A.; Maddocks, Kami; Jaglowski, Samantha M.; Blum, Kristie A.; Byrd, John C.; Dubovsky, Jason A.

    2015-01-01

    Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib’s capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance. PMID:25972157

  14. A novel nonsense mutation in androgen receptor confers resistance to CYP17 inhibitor treatment in prostate cancer

    PubMed Central

    Han, Dong; Gao, Shuai; Valencia, Kevin; Owiredu, Jude; Han, Wanting; de Waal, Eric; Macoska, Jill A; Cai, Changmeng

    2017-01-01

    The standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that blocks transcriptional activity of androgen receptor (AR). However, ADT invariably leads to the development of castration-resistant PCa (CRPC) with restored activity of AR. CRPC can be further treated with CYP17 inhibitors to block androgen synthesis pathways, but most patients still relapse after a year of such treatment. The mechanisms that drive this progression are not fully understood, but AR activity, at least in a subset of cancers, appears to be restored again. Importantly, AR mutations are more frequently detected in this type of cancer. By analyzing tumor biopsy mRNA from CRPC patients who had developed resistance to CYP17 inhibitor treatment, we have identified a novel nonsense mutation (Q784*) at the ligand binding domain (LBD) of AR, which produces a C-terminal truncated AR protein that lacks intact LBD. This AR-Q784* mutant is transcriptionally inactive, but it is constitutively expressed in the nucleus and can bind to DNA in the absence of androgen. Significantly, our results show that AR-Q784* can heterodimerize with, and enhance the transcriptional activity of, full-length AR. Moreover, expressing AR-Q784* in an AR positive PCa cell line enhances the chromatin binding of endogenous AR and the recruitment of p300 coactivator under the low androgen condition, leading to increased cell growth. This activity of AR-Q784* mimics the function of some AR splice variants, indicating that CYP17 inhibitor treatment in CRPC may select for LBD-truncated forms of AR to restore AR signaling. PMID:28036278

  15. Rhbdf2 mutations increase its protein stability and drive EGFR hyperactivation through enhanced secretion of amphiregulin

    PubMed Central

    Hosur, Vishnu; Johnson, Kenneth R.; Burzenski, Lisa M.; Stearns, Timothy M.; Maser, Richard S.; Shultz, Leonard D.

    2014-01-01

    The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains. PMID:24825892

  16. Rhbdf2 mutations increase its protein stability and drive EGFR hyperactivation through enhanced secretion of amphiregulin.

    PubMed

    Hosur, Vishnu; Johnson, Kenneth R; Burzenski, Lisa M; Stearns, Timothy M; Maser, Richard S; Shultz, Leonard D

    2014-05-27

    The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains.

  17. Low pesticide rates may hasten the evolution of resistance by increasing mutation frequencies.

    PubMed

    Gressel, Jonathan

    2011-03-01

    At very low pesticide rates, a certain low proportion of pests may receive a sublethal dose, are highly stressed by the pesticide and yet survive. Stress is a general enhancer of mutation rates. Thus, the survivors are likely to have more than normal mutations, which might include mutations leading to pesticide resistance, both for multifactorial (polygenic, gene amplification, sequential allelic mutations) and for major gene resistance. Management strategies should consider how to eliminate the subpopulation of pests with the high mutation rates, but the best strategy is probably to avoid too low application rates of pesticides from the outset.

  18. Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays.

    PubMed

    André, E; Goeminne, L; Colmant, A; Beckert, P; Niemann, S; Delmee, M

    2017-04-01

    Neither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. We designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. The technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. We propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  19. Increased activation of blood coagulation in pregnant women with the Factor V Leiden mutation.

    PubMed

    Kjellberg, Ulla; van Rooijen, Marianne; Bremme, Katarina; Hellgren, Margareta

    2014-10-01

    The risk of venous thromboembolism is enhanced in pregnant carriers of the Factor V Leiden mutation. The primary aim of the study was to compare prothrombin fragments 1+2, soluble fibrin and D-dimer levels in pregnant Factor V Leiden mutation carriers with those in non-carriers. Secondary aims were to evaluate whether these biomarkers could predict placenta-mediated complications or venous thromboembolism, and to study blood coagulation after caesarean section with thromboprophylaxis and after vaginal delivery without thromboprophylaxis. Prothrombin fragments 1+2, soluble fibrin and D-dimer levels were studied longitudinally in 476 carriers with singleton pregnancies from gestational weeks 23-25 until 8-10 weeks postpartum. Prothrombin fragments 1+2 and D-dimer levels gradually increased during pregnancy. D-dimer levels were higher in carriers, both during pregnancy and puerperium, compared to non-carriers. D-dimer levels above 0.5mg/l were found in about 30% and 20% of the heterozygous carriers at 4-5 and 8-10 weeks postpartum, respectively. Soluble fibrin levels were mainly unchanged during pregnancy, with no difference between carriers and non-carriers. Biomarker levels were similar in carriers with uncomplicated and complicated pregnancies. Higher D-dimer levels indicate increased blood coagulation and fibrinolysis activity in carriers. The high proportion of carriers with D-dimer levels exceeding 0.5mg/l postpartum must be considered when assessing the probability of venous thromboembolism. Large overlaps in biomarker levels in normal and complicated pregnancies suggest that these biomarkers cannot be used as predictors. Thromboprophylaxis following caesarean section may prevent increased activation of blood coagulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. A calcium-dependent potassium current is increased by a single-gene mutation in Paramecium.

    PubMed

    Hennessey, T M; Kung, C

    1987-01-01

    The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential.

  1. Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo

    PubMed Central

    Kuo, Cheng-Ju; Chen, Jenn-Wei; Chiu, Hao-Chieh; Teng, Ching-Hao; Hsu, Tai-I; Lu, Pei-Jung; Syu, Wan-Jr; Wang, Sin-Tian; Chou, Ting-Chen; Chen, Chang-Shi

    2016-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen causing severe diseases in humans worldwide. Currently, there is no specific treatment available for EHEC infection and the use of conventional antibiotics is contraindicated. Therefore, identification of potential therapeutic targets and development of effective measures to control and treat EHEC infection are needed. Lipopolysaccharides (LPS) are surface glycolipids found on the outer membrane of gram-negative bacteria, including EHEC, and LPS biosynthesis has long been considered as potential anti-bacterial target. Here, we demonstrated that the EHEC rfaD gene that functions in the biosynthesis of the LPS inner core is required for the intestinal colonization and pathogenesis of EHEC in vivo. Disruption of the EHEC rfaD confers attenuated toxicity in Caenorhabditis elegans and less bacterial colonization in the intestine of C. elegans and mouse. Moreover, rfaD is also involved in the control of susceptibility of EHEC to antimicrobial peptides and host intestinal immunity. It is worth noting that rfaD mutation did not interfere with the growth kinetics when compared to the wild-type EHEC cells. Taken together, we demonstrated that mutations of the EHEC rfaD confer hypersusceptibility to host intestinal innate immunity in vivo, and suggested that targeting the RfaD or the core LPS synthesis pathway may provide alternative therapeutic regimens for EHEC infection. PMID:27570746

  2. Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation

    PubMed Central

    Fanning, Sean W; Mayne, Christopher G; Dharmarajan, Venkatasubramanian; Carlson, Kathryn E; Martin, Teresa A; Novick, Scott J; Toy, Weiyi; Green, Bradley; Panchamukhi, Srinivas; Katzenellenbogen, Benita S; Tajkhorshid, Emad; Griffin, Patrick R; Shen, Yang; Chandarlapaty, Sarat; Katzenellenbogen, John A; Greene, Geoffrey L

    2016-01-01

    Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition. DOI: http://dx.doi.org/10.7554/eLife.12792.001 PMID:26836308

  3. Mouse somatic mutation orthologous to MELAS A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene confers respiration defects.

    PubMed

    Shimizu, Akinori; Enoki, Shunkei; Ishikawa, Kaori; Mito, Takayuki; Obata, Kanae; Nagashima, Ruriko; Yonekawa, Hiromichi; Nakada, Kazuto; Hayashi, Jun-Ichi

    2015-11-27

    We searched for mtDNA harboring somatic mutations in mouse B82 cells, and found an A2748G mutation orthologous to the A3302G mutation in tRNA(Leu(UUR)) gene reported in a patient with MELAS, the most prevalent mitochondrial disease. We isolated subclones of B82 cells until we obtained one subclone harboring >95% A2748G mtDNA. Cytoplasmic transfer of A2748G mtDNA resulted in cotransfer of A2748G mtDNA and respiration defects into mouse ES cells. Thus, A2748G mtDNA is responsible for respiration defects, and the ES cells harboring A2748G mtDNA may be useful for generation of transmitochondrial mice harboring A2748G mtDNA as potential disease models of MELAS.

  4. Mutations in the feline immunodeficiency virus envelope glycoprotein confer resistance to a dominant-negative fragment of Tsg101 by enhancing infectivity and cell-to-cell virus transmission.

    PubMed

    Luttge, Benjamin G; Panchal, Prashant; Puri, Vinita; Checkley, Mary Ann; Freed, Eric O

    2014-04-01

    The Pro-Ser-Ala-Pro (PSAP) motif in the p2 domain of feline immunodeficiency virus (FIV) Gag is required for efficient virus release, virus replication, and Gag binding to the ubiquitin-E2-variant (UEV) domain of Tsg101. As a result of this direct interaction, expression of an N-terminal fragment of Tsg101 containing the UEV domain (referred to as TSG-5') inhibits FIV release. In these respects, the FIV p2(Gag) PSAP motif is analogous to the PTAP motif of HIV-1 p6(Gag). To evaluate the feasibility of a late domain-targeted inhibition of virus replication, we created an enriched Crandell-Rees feline kidney (CRFK) cell line (T5'(hi)) that stably expresses high levels of TSG-5'. Here we show that mutations in either the V3 loop or the second heptad repeat (HR2) domain of the FIV envelope glycoprotein (Env) rescue FIV replication in T5'(hi) cells without increasing FIV release efficiency. TSG-5'-resistance mutations in Env enhance virion infectivity and the cell-cell spread of FIV when diffusion is limited using a semi-solid growth medium. These findings show that mutations in functional domains of Env confer TSG-5'-resistance, which we propose enhances specific infectivity and the cell-cell transmission of virus to counteract inefficient virus release. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking. © 2013.

  5. Long-range DHPS mutations unexpectedly increase Mycobacterium chimaera susceptibility to sulfonamides.

    PubMed

    Gotthard, Guillaume; Muhammed Ameen, Sirwan; Drancourt, Michel; Chabriere, Eric

    2013-12-01

    The two closely related mycobacteria, Mycobacterium intracellulare and Mycobacterium chimaera, exhibit a more than two-fold difference in their in vitro susceptibility to sulfonamides. Sulfonamides are antibiotics targeting the 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS) enzyme involved in the folate synthesis pathway. Comparing the DHPS gene sequence in six M. intracellulare and M. chimaera types trains and clinical isolates yielded only four amino acid changes. In silico structural modelling surprisingly indicated that these amino acids are not located in the active site of DHPS and do not interact directly with sulfonamides. Unexpectedly, these amino acids in distal positions may play a key role in the increased sulfonamide susceptibility observed in M. chimaera compared with M. intracellulare. This example illustrates how three-dimensional models could help to identify distal mutations capable of modulating enzymatic activity. Copyright © 2013 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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

    SciTech Connect

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

    2010-03-15

    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.

  7. Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort.

    PubMed

    Singh, Jaya; Mishra, Avshesh; Pandian, Arunachalam Jayamuruga; Mallipatna, Ashwin C; Khetan, Vikas; Sripriya, S; Kapoor, Suman; Agarwal, Smita; Sankaran, Satish; Katragadda, Shanmukh; Veeramachaneni, Vamsi; Hariharan, Ramesh; Subramanian, Kalyanasundaram; Mannan, Ashraf U

    2016-01-01

    Retinoblastoma (Rb) is the most common primary intraocular cancer of childhood and one of the major causes of blindness in children. India has the highest number of patients with Rb in the world. Mutations in the RB1 gene are the primary cause of Rb, and heterogeneous mutations are distributed throughout the entire length of the gene. Therefore, genetic testing requires screening of the entire gene, which by conventional sequencing is time consuming and expensive. In this study, we screened the RB1 gene in the DNA isolated from blood or saliva samples of 50 unrelated patients with Rb using the TruSight Cancer panel. Next-generation sequencing (NGS) was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect germline pathogenic mutations in 66% (33/50) of the cases, 12 of which were novel. We were able to detect all types of mutations, including missense, nonsense, splice site, indel, and structural variants. When we considered bilateral Rb cases only, the mutation detection rate increased to 100% (22/22). In unilateral Rb cases, the mutation detection rate was 30% (6/20). Our study suggests that NGS-based approaches increase the sensitivity of mutation detection in the RB1 gene, making it fast and cost-effective compared to the conventional tests performed in a reflex-testing mode.

  8. Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort

    PubMed Central

    Singh, Jaya; Mishra, Avshesh; Pandian, Arunachalam Jayamuruga; Mallipatna, Ashwin C.; Khetan, Vikas; Sripriya, S.; Kapoor, Suman; Agarwal, Smita; Sankaran, Satish; Katragadda, Shanmukh; Veeramachaneni, Vamsi; Hariharan, Ramesh; Subramanian, Kalyanasundaram

    2016-01-01

    Purpose Retinoblastoma (Rb) is the most common primary intraocular cancer of childhood and one of the major causes of blindness in children. India has the highest number of patients with Rb in the world. Mutations in the RB1 gene are the primary cause of Rb, and heterogeneous mutations are distributed throughout the entire length of the gene. Therefore, genetic testing requires screening of the entire gene, which by conventional sequencing is time consuming and expensive. Methods In this study, we screened the RB1 gene in the DNA isolated from blood or saliva samples of 50 unrelated patients with Rb using the TruSight Cancer panel. Next-generation sequencing (NGS) was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. Results We were able to detect germline pathogenic mutations in 66% (33/50) of the cases, 12 of which were novel. We were able to detect all types of mutations, including missense, nonsense, splice site, indel, and structural variants. When we considered bilateral Rb cases only, the mutation detection rate increased to 100% (22/22). In unilateral Rb cases, the mutation detection rate was 30% (6/20). Conclusions Our study suggests that NGS-based approaches increase the sensitivity of mutation detection in the RB1 gene, making it fast and cost-effective compared to the conventional tests performed in a reflex-testing mode. PMID:27582626

  9. The BRAF{sup T1799A} mutation confers sensitivity of thyroid cancer cells to the BRAF{sup V600E} inhibitor PLX4032 (RG7204)

    SciTech Connect

    Xing, Joanna; Liu, Ruixin; Xing, Mingzhao; Trink, Barry

    2011-01-28

    Research highlights: {yields} Exciting therapeutic potential has been recently reported for the BRAF{sup V600E} inhibitor PLX4032 in melanoma. {yields} We tested the effects of PLX4032 on the growth of thyroid cancer cells which often harbor the BRAF{sup V600E} mutation. {yields} We observed a potent BRAF{sup V600E}-dependent inhibition of thyroid cancer cells by PLX4032. {yields} We thus demonstrated an important therapeutic potential of PLX4032 for thyroid cancer. -- Abstract: Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF{sup V600E}, as a result of the BRAF{sup T1799A} mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF{sup V600E}-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF{sup T1799A} mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF{sup T1799A} mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC{sub 50} values (0.115-1.156 {mu}M) in BRAF{sup V600E} mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC{sub 50} values (56.674-1349.788 {mu}M). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF{sup T1799A} mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF{sup T1799A} mutation-selective therapeutic agent for thyroid cancer.

  10. A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.

    PubMed Central

    Moses, A C; Rosen, H N; Moller, D E; Tsuzaki, S; Haddow, J E; Lawlor, J; Liepnieks, J J; Nichols, W C; Benson, M D

    1990-01-01

    In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4. Images PMID:1979335

  11. Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

    PubMed

    Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

    2009-03-01

    Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

  12. Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae.

    PubMed

    Tapia, Hugo; Young, Lindsey; Fox, Douglas; Bertozzi, Carolyn R; Koshland, Douglas

    2015-05-12

    Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.

  13. The RET p.G533C mutation confers predisposition to multiple endocrine neoplasia type 2A in a Brazilian kindred and is able to induce a malignant phenotype in vitro and in vivo.

    PubMed

    Oliveira, Mariana N L; Hemerly, Jefferson P; Bastos, André U; Tamanaha, Rosana; Latini, Flavia R M; Camacho, Cléber P; Impellizzeri, Anelise; Maciel, Rui M B; Cerutti, Janete M

    2011-09-01

    We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation. Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma. Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood. Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

  14. E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient

    PubMed Central

    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

    2015-01-01

    Background. 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. Methods. 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. Results. 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. Conclusions. 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. PMID:25985905

  15. Differences in Reversion of Resistance Mutations to Wild-Type under Structured Treatment Interruption and Related Increase in Replication Capacity

    PubMed Central

    Paquet, Agnes C.; Baxter, John; Weidler, Jodi; Lie, Yolanda; Lawrence, Jody; Kim, Rose; Bates, Michael; Coakley, Eoin; Chappey, Colombe

    2011-01-01

    Background The CPCRA 064 study examined the effect of structured treatment interruption (STI) of up to 4 months followed by salvage treatment in patients failing therapy with multi-drug resistant HIV. We examined the relationship between the reversion rate of major reverse transcriptase (RT) resistance-associated mutations and change in viral replication capacity (RC). The dataset included 90 patients with RC and genotypic data from virus samples collected at 0 (baseline), 2 and 4 months of STI. Principal Findings Rapid shift towards wild-type RC was observed during the first 2 months of STI. Median RC increased from 47.5% at baseline to 86.0% at 2 months and to 97.5% at 4 months. Between baseline and 2 months of STI, T215F had the fastest rate of reversion (41%) and the reversion of E44D and T69D was associated with the largest changes in RC. Among the most prevalent RT mutations, M184V had the fastest rate of reversion from baseline to 2 months (40%), and its reversion was associated with the largest increase in RC. Most rates of reversion increased between 2 months and 4 months, but the change in RC was more limited as it was already close to 100%. The highest frequency of concurrent reversion was found for L100I and K103N. Mutagenesis tree models showed that M184V, when present, was overall the first mutation to revert among all the RT mutations reported in the study. Conclusion Longitudinal analysis of combined phenotypic and genotypic data during STI showed a large amount of variability in prevalence and reversion rates to wild-type codons among the RT resistance-associated mutations. The rate of reversion of these mutations may depend on the extent of RC increase as well as the co-occurring reversion of other mutations belonging to the same mutational pathway. PMID:21297946

  16. Role of a Novel I1781T Mutation and Other Mechanisms in Conferring Resistance to Acetyl-CoA Carboxylase Inhibiting Herbicides in a Black-Grass Population

    PubMed Central

    Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Dale, Richard P.; McIndoe, Eddie

    2013-01-01

    Background Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2). Methodology/Principal Findings Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele. Conclusion/Significance This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances

  17. The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in Saccharomyces cerevisiae under high-pressure and low-temperature conditions.

    PubMed

    Nagayama, Ai; Kato, Chiaki; Abe, Fumiyoshi

    2004-04-01

    Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2(E27F)) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2(D563N)) and HPG2-3 (Tat2(E570K)) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.

  18. Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

    PubMed Central

    Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua

    2016-01-01

    ABSTRACT Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human

  19. Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae

    PubMed Central

    Tapia, Hugo; Young, Lindsey; Fox, Douglas; Bertozzi, Carolyn R.; Koshland, Douglas

    2015-01-01

    Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms. PMID:25918381

  20. Detection of JAK2 V617F mutation increases the diagnosis of myeloproliferative neoplasms

    PubMed Central

    ZHANG, SHU-PENG; LI, HUI; LAI, REN-SHENG

    2015-01-01

    The Janus kinase (JAK)2 gene, which is located on chromosome 9p24, is involved in the signaling transduction pathways of the hematopoietic and immune system. Mutations in the JAK2 gene have served as disease markers for myeloproliferative neoplasms (MPNs). The aim of the present study was to investigate the occurrence of the JAK2 gene mutation in 140 clinical samples, and to evaluate its clinical significance in MPNs and other hematological diseases. Genomic DNA was extracted from the peripheral blood leukocytes or bone marrow karyocytes of 140 clinical samples, which included 130 patients with various types of hematological disease and 10 control patients. In addition, exons 12 and 14 of the JAK2 gene were analyzed by direct sequencing and the mutation rates of various MPN subtypes were evaluated. Of the 140 samples, exons 12 and 14 were tested in 74 samples, however, exon 14 only was tested in 66 samples. No mutations were identified in exon 12. The V617F mutation rate in polycythemia vera was 82.1% (23/28), and the mutation rates in essential thrombocythemia histiocytosis, primary myelofibrosis and other MPNs were 53.1% (17/32), 40.0% (4/10) and 60.0% (6/10), respectively. Therefore, the total mutation rate of the JAK2 gene in MPN was 62.5% (50/80). For non-MPN hematological diseases, four V617F mutations were detected in samples of leukocytosis of unknown origin (4/12), however, no JAK2 V617F mutations were identified in the 10 controls. Therefore, JAK2 V617F mutations may present a novel marker for diagnosis of MPNs. Furthermore, the direct sequencing method appeared to be satisfactory for the clinical gene testing of hematological samples. PMID:25624900

  1. Mutation at residue 376 of ALS confers tribenuron-methyl resistance in flixweed (Descurainia sophia) populations from Hebei Province, China.

    PubMed

    Xu, Xian; Liu, Guiqiao; Chen, Silong; Li, Binghua; Liu, Xiaomin; Wang, Xiaoyun; Fan, Cuiqin; Wang, Guiqi; Ni, Hanwen

    2015-11-01

    The acetolactate synthase (ALS) inhibitor tribenuron has been used continuously for approximately twenty years as an herbicide in winter wheat fields in China. Flixweed (Descurainia sophia) has evolved resistance to tribenuron, due to multiple amino acid mutations at the 197th residue of ALS. In this study, the molecular basis of tribenuron resistance was investigated using two resistant populations, Xingtai (XT) and Shijiazhuang (SJ), and two susceptible populations, Cangzhou (CZ) and Handan (HD). Whole-plant tests and ALS activity assays showed that the two resistant populations were highly resistant to tribenuron. Targeted amplification of ALS genes from the four populations showed that there were two ALS genes in each population, and both of them were expressed in flixweed; the full coding lengths of the two ALS genes were 1998bp and 2004bp. Mutations related to tribenuron resistance in flixweed were located in only the 1998bp paralog. An ALS activity assay showed that the resistant population SJ displayed slight cross-resistance to florasulam, with a resistance factor of 4.81, but the resistant population XT did not have cross-resistance to florasulam. The resistant population XT was found to carry the previously reported mutation Pro197Ser, but the resistant population SJ carried a different mutation, Asp376Glu, known from other weeds but novel in flixweed. Our results demonstrated that multiple versions of ALS genes exist in flixweed and that mutations at multiple sites may result in ALS-inhibitor resistance in this weed.

  2. TP53 mutation hits energy metabolism and increases glycolysis in breast cancer

    PubMed Central

    Munkácsy, Gyöngyi; Horváth, Gergő; Nagy, Ádám M.; Ambrus, Attila; Hauser, Péter; Szabó, András; Tretter, László; Győrffy, Balázs

    2016-01-01

    Promising new hallmarks of cancer is alteration of energy metabolism that involves molecular mechanisms shifting cancer cells to aerobe glycolysis. Our goal was to evaluate the correlation between mutation in the commonly mutated tumor suppressor gene TP53 and metabolism. We established a database comprising mutation and RNA-seq expression data of the TCGA repository and performed receiver operating characteristics (ROC) analysis to compare expression of each gene between TP53 mutated and wild type samples. All together 762 breast cancer samples were evaluated of which 215 had TP53 mutation. Top up-regulated metabolic genes include glycolytic enzymes (e.g. HK3, GPI, GAPDH, PGK1, ENO1), glycolysis regulator (PDK1) and pentose phosphate pathway enzymes (PGD, TKT, RPIA). Gluconeogenesis enzymes (G6PC3, FBP1) were down-regulated. Oxygen consumption and extracellular acidification rates were measured in TP53 wild type and mutant breast cell lines with a microfluorimetric analyzer. Applying metabolic inhibitors in the presence and absence of D-glucose and L-glutamine in cell culture experiments resulted in higher glycolytic and mitochondrial activity in TP53 mutant breast cancer cell lines. In summary, TP53 mutation influences energy metabolism at multiple levels. Our results provide evidence for the synergistic activation of multiple hallmarks linking to these the mutation status of a key driver gene. PMID:27582538

  3. BRCA Mutations Increase Fertility in Families at Hereditary Breast/Ovarian Cancer Risk

    PubMed Central

    Kwiatkowski, Fabrice; Arbre, Marie; Bidet, Yannick; Laquet, Claire; Uhrhammer, Nancy; Bignon, Yves-Jean

    2015-01-01

    Background Deleterious mutations in the BRCA genes are responsible for a small, but significant, proportion of breast and ovarian cancers (5 - 10 %). Proof of de novo mutations in hereditary breast/ovarian cancer (HBOC) families is rare, in contrast to founder mutations, thousands of years old, that may be carried by as much as 1 % of a population. Thus, if mutations favoring cancer survive selection pressure through time, they must provide advantages that compensate for the loss of life expectancy. Method This hypothesis was tested within 2,150 HBOC families encompassing 96,325 individuals. Parameters included counts of breast/ovarian cancer, age at diagnosis, male breast cancer and other cancer locations. As expected, well-known clinical parameters discriminated between BRCA-mutated families and others: young age at breast cancer, ovarian cancer, pancreatic cancer and male breast cancer. The major fertility differences concerned men in BRCA-mutated families: they had lower first and mean age at paternity, and fewer remained childless. For women in BRCA families, the miscarriage rate was lower. In a logistic regression including clinical factors, the different miscarriage rate and men's mean age at paternity remained significant. Results Fertility advantages were confirmed in a subgroup of 746 BRCA mutation carriers and 483 non-carriers from BRCA mutated families. In particular, female carriers were less often nulliparous (9.1 % of carriers versus 16.0 %, p = 0.003) and had more children (1.8 ± 1.4 SD versus 1.5 ± 1.3, p = 0.002) as well as male carriers (1.7 ± 1.3 versus 1.4 ± 1.3, p = 0.024). Conclusion Although BRCA mutations shorten the reproductive period due to cancer mortality, they compensate by improving fertility both in male and female carriers. PMID:26047126

  4. SFTPC mutations cause SP-C degradation and aggregate formation without increasing ER stress.

    PubMed

    Thurm, Tobias; Kaltenborn, Eva; Kern, Sunčana; Griese, Matthias; Zarbock, Ralf

    2013-08-01

    Mutations in the gene encoding surfactant protein C (SP-C) cause familial and sporadic interstitial lung disease (ILD), which is associated with considerable morbidity and mortality. Unfortunately, effective therapeutic options are still lacking due to a very limited understanding of pathomechanisms. Knowledge of mutant SP-C proprotein (proSP-C) trafficking, processing, intracellular degradation and aggregation is a crucial prerequisite for the development of specific therapies to correct aberrant trafficking and processing of proSP-C and to hinder accumulation of cytotoxic aggregates. To identify possible starting points for therapeutic intervention, we stably transfected A549 alveolar epithelial cells with several proSP-C mutations previously found in patients suffering from ILD. Effects of mutant proSP-C were assessed by Western blotting, immunofluorescence and Congo red staining. A group of mutations (p.I73T, p.L110R, p.A116D and p.L188Q) resulted in aberrant proSP-C products, which were at least partially trafficked to lamellar bodies. Another group of mutations (p.P30L and p.P115L) was arrested in the endoplasmic reticulum (ER). Except for p.I73T, all mutations led to accumulation of intracellular Congo red-positive aggregates. Enhanced ER stress was detectable in none of these stably transfected cells. Different SP-C mutations have unique consequences for alveolar epithelial cell biology. As these cannot be predicted based upon the localization of the mutation, our data emphasize the importance of studying individual mutations in detail in order to develop mutation-specific therapies. © 2013 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.

  5. Single ABCA3 Mutations Increase Risk for Neonatal Respiratory Distress Syndrome

    PubMed Central

    Wegner, Daniel J.; DePass, Kelcey; Heins, Hillary; Druley, Todd E.; Mitra, Robi D.; An, Ping; Zhang, Qunyuan; Nogee, Lawrence M.; Cole, F. Sessions; Hamvas, Aaron

    2012-01-01

    BACKGROUND AND OBJECTIVE: Neonatal respiratory distress syndrome (RDS) due to pulmonary surfactant deficiency is heritable, but common variants do not fully explain disease heritability. METHODS: Using next-generation, pooled sequencing of race-stratified DNA samples from infants ≥34 weeks’ gestation with and without RDS (n = 513) and from a Missouri population-based cohort (n = 1066), we scanned all exons of 5 surfactant-associated genes and used in silico algorithms to identify functional mutations. We validated each mutation with an independent genotyping platform and compared race-stratified, collapsed frequencies of rare mutations by gene to investigate disease associations and estimate attributable risk. RESULTS: Single ABCA3 mutations were overrepresented among European-descent RDS infants (14.3% of RDS vs 3.7% of non-RDS; P = .002) but were not statistically overrepresented among African-descent RDS infants (4.5% of RDS vs 1.5% of non-RDS; P = .23). In the Missouri population-based cohort, 3.6% of European-descent and 1.5% of African-descent infants carried a single ABCA3 mutation. We found no mutations among the RDS infants and no evidence of contribution to population-based disease burden for SFTPC, CHPT1, LPCAT1, or PCYT1B. CONCLUSIONS: In contrast to lethal neonatal RDS resulting from homozygous or compound heterozygous ABCA3 mutations, single ABCA3 mutations are overrepresented among European-descent infants ≥34 weeks’ gestation with RDS and account for ∼10.9% of the attributable risk among term and late preterm infants. Although ABCA3 mutations are individually rare, they are collectively common among European- and African-descent individuals in the general population. PMID:23166334

  6. Hypomorphic, homozygous mutations in Phosphoglucomutase 3 impair immunity and increase serum IgE levels

    PubMed Central

    Sassi, Atfa; Lazaroski, Sandra; Wu, Gang; Haslam, Stuart M.; Fliegauf, Manfred; Mellouli, Fethi; Patiroglu, Turkan; Unal, Ekrem; Ozdemir, Mehmet Akif; Jouhadi, Zineb; Khadir, Khadija; Ben-Khemis, Leila; Ben-Ali, Meriem; Ben-Mustapha, Imen; Borchani, Lamia; Pfeifer, Dietmar; Jakob, Thilo; Khemiri, Monia; Asplund, A. Charlotta; Gustafsson, Manuela O.; Lundin, Karin E.; Falk-Sörqvist, Elin; Moens, Lotte N.; Gungor, Hatice Eke; Engelhardt, Karin R.; Dziadzio, Magdalena; Stauss, Hans; Fleckenstein, Bernhard; Meier, Rebecca; Prayitno, Khairunnadiya; Maul-Pavicic, Andrea; Schaffer, Sandra; Rakhmanov, Mirzokhid; Henneke, Philipp; Kraus, Helene; Eibel, Hermann; Kölsch, Uwe; Nadifi, Sellama; Nilsson, Mats; Bejaoui, Mohamed; Schäffer, Alejandro A.; Edvard Smith, C. I.; Dell, Anne; Barbouche, Mohamed-Ridha; Grimbacher, Bodo

    2016-01-01

    Background Recurrent bacterial and fungal infections, eczema and elevated serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in STAT3 and DOCK8, involved in signal transduction pathways. However, glycosylation defects have not been described in HIES. One crucial enzyme in the glycosylation pathway is Phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of UDP-GlcNAc which is required for the biosynthesis of N-glycans. Objective To elucidate the genetic cause in HIES patients who do not carry mutations in STAT3 or DOCK8. Methods After establishing a linkage interval by SNP-chip genotyping and homozygosity mapping in two HIES families from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by Western blotting and glycosylation was profiled by mass spectrometry. Results Mutational analysis of candidate genes in a 11.9 Mb linkage region on chromosome 6 shared by two multiplex families identified two homozygous mutations in PGM3 which segregated with the disease status and followed a recessive inheritance trait. The mutations predict amino acid changes in Phosphoglucomutase-3; PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in two other affected families, respectively. These hypomorphic mutations have impact on the biosynthetic reactions involving UDP-GlcNAc. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-/tetra-antennary N-glycans. T cell proliferation and differentiation was impaired in patients. Most patients showed developmental delay and many had psychomotor retardation. Conclusion Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity, as biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper

  7. Assessing the Risk That Phytophthora melonis Can Develop a Point Mutation (V1109L) in CesA3 Conferring Resistance to Carboxylic Acid Amide Fungicides

    PubMed Central

    Chen, Lei; Zhu, Shusheng; Lu, Xiaohong; Pang, Zhili; Cai, Meng; Liu, Xili

    2012-01-01

    The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC50 values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10−7 and 1×10−6, respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC50 values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations. PMID:22848705

  8. Assessing the risk that Phytophthora melonis can develop a point mutation (V1109L) in CesA3 conferring resistance to carboxylic acid amide fungicides.

    PubMed

    Chen, Lei; Zhu, Shusheng; Lu, Xiaohong; Pang, Zhili; Cai, Meng; Liu, Xili

    2012-01-01

    The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) µg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI  =  mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.

  9. Cyclic ichthyosis with epidermolytic hyperkeratosis: A phenotype conferred by mutations in the 2B domain of keratin K1.

    PubMed Central

    Sybert, V P; Francis, J S; Corden, L D; Smith, L T; Weaver, M; Stephens, K; McLean, W H

    1999-01-01

    Bullous congenital ichthyosiform erythroderma (BCIE) is characterized by blistering and erythroderma in infancy and by erythroderma and ichthyosis thereafter. Epidermolytic hyperkeratosis is a hallmark feature of light and electron microscopy. Here we report on four individuals from two families with a unique clinical disorder with histological findings of epidermolytic hyperkeratosis. Manifesting erythema and superficial erosions at birth, which improved during the first few months of life, affected individuals later developed palmoplantar hyperkeratosis with patchy erythema and scale elsewhere on the body. Three affected individuals exhibit dramatic episodic flares of annular, polycyclic erythematous plaques with scale, which coalesce to involve most of the body surface. The flares last weeks to months. In the interim periods the skin may be normal, except for palmoplantar hyperkeratosis. Abnormal keratin-filament aggregates were observed in suprabasal keratinocytes from both probands, suggesting that the causative mutation might reside in keratin K1 or keratin K10. In one proband, sequencing of K1 revealed a heterozygous mutation, 1436T-->C, predicting a change of isoleucine to threonine in the highly conserved helix-termination motif. In the second family, a heterozygous mutation, 1435A-->T, was found in K1, predicting an isoleucine-to-phenylalanine substitution in the same codon. Both mutations were excluded in both a control population and all unaffected family members tested. These findings reveal that a clinical phenotype distinct from classic BCIE but with similar histology can result from K1 mutations and that mutations at this codon give rise to a clinically unique condition. PMID:10053007

  10. Increased Aggregation Is More Frequently Associated to Human Disease-Associated Mutations Than to Neutral Polymorphisms

    PubMed Central

    De Baets, Greet; Van Doorn, Loic; Rousseau, Frederic; Schymkowitz, Joost

    2015-01-01

    Protein aggregation is a hallmark of over 30 human pathologies. In these diseases, the aggregation of one or a few specific proteins is often toxic, leading to cellular degeneration and/or organ disruption in addition to the loss-of-function resulting from protein misfolding. Although the pathophysiological consequences of these diseases are overt, the molecular dysregulations leading to aggregate toxicity are still unclear and appear to be diverse and multifactorial. The molecular mechanisms of protein aggregation and therefore the biophysical parameters favoring protein aggregation are better understood. Here we perform an in silico survey of the impact of human sequence variation on the aggregation propensity of human proteins. We find that disease-associated variations are statistically significantly enriched in mutations that increase the aggregation potential of human proteins when compared to neutral sequence variations. These findings suggest that protein aggregation might have a broader impact on human disease than generally assumed and that beyond loss-of-function, the aggregation of mutant proteins involved in cancer, immune disorders or inflammation could potentially further contribute to disease by additional burden on cellular protein homeostasis. PMID:26340370

  11. fur (-) mutation increases the survival time of Escherichia coli under photooxidative stress in aquatic environments.

    PubMed

    Darcan, C; Aydin, Ebru

    2012-09-01

    We investigated the survival of the wild type Escherichia coli (QC771) and fur- mutant strain (QC1732) under photooxidative stress in different water sources. The survival of fur- mutant and wild type E. coli was seen as a significant decrease in the visible light samples in the presence of methylene blue (MB). The fur-E. coli strain lived longer than the wild type E. coli strain on exposure to MB and visible light, which generates singlet oxygen, in both lake water (48-h) and pure water (16-h). It is interesting to note that the survival of both wild type and the fur- mutant strain was more protected at 24 °C than at other temperatures. The Fur protein does not have any relation to the entry of E. coli into the viable but nonculturable state (VBNC) under photooxidative stress. This is the first study which shows that fur- mutation increases the resistance of E. coli to photooxidative stress in aquatic environments, and the Fur protein does not have any relation to the entry of E. coli into the VBNC state.

  12. Mutation rate is reduced by increased dosage of mutL gene in Escherichia coli K-12.

    PubMed

    Galán, Juan-Carlos; Turrientes, María-Carmen; Baquero, María-Rosario; Rodríguez-Alcayna, Manuel; Martínez-Amado, Jorge; Martínez, José-Luis; Baquero, Fernando

    2007-10-01

    A variable but substantial proportion of wild Escherichia coli isolates present consistently lower mutation frequencies than that found in the ensemble of strains. The genetic mechanisms responsible for the hypo-mutation phenotype are much less known than those involved in hyper-mutation. Changes in E. coli mutation frequencies derived from the gene-copy effect of mutS, mutL, mutH, uvrD, mutT, mutY, mutM, mutA, dnaE, dnaQ, and rpoS are explored. When present in a very high copy number ( approximately 300 copies cell(-1)), mutL, mutH, and mutA gene copies yielded >/=twofold decrease in mutation rates determined by Luria-Delbrück fluctuation tests. Nevertheless, when the copy number was not such high ( approximately 15 copies cell(-1)), only mutL results in a consistent twofold decrease in the mutation rate. This reduction seems to be independent from the RecA background, phase of growth, or from the presence of proficient MutS. An increase in mutL gene copies was also able to partially compensate the hypermutator phenotype of a mutS-defective E. coli derivative.

  13. PSCA gene variants (rs2294008 and rs2978974) confer increased susceptibility of gallbladder carcinoma in females.

    PubMed

    Rai, Rajani; Sharma, Kiran L; Misra, Sanjeev; Kumar, Ashok; Mittal, Balraj

    2013-11-10

    PSCA is a tissue specific tumor suppressor or oncogene which has been found to be associated with several human tumors including gallbladder cancer. It is considered to be involved in the cell-proliferation inhibition and/or cell-death induction activity. Therefore, we aimed to investigate the role of PSCA gene polymorphisms in gallbladder cancer risk in North Indian population. A total of 405 gallbladder cancer patients and 247 healthy controls were included in the case-control study for risk prediction. We examined the association of two functional SNPs, rs2294008 and rs2978974 in PSCA gene by genotyping using Taqman allelic discrimination assays. Statistical analysis was done using SPSS software, version 17. Linkage disequilibrium and haplotype analysis was done with the help of SNPstats software. FDR test was used to correct for multiple comparisons. No significant associations of rs2294008 and rs2978974 genetic variants of the PSCA gene were found with GBC risk at allele, genotype or haplotype levels. Stratifying the subjects on the basis of gallstone also did not show any significant result. However, on gender stratification, we found a significant association of Trs2294008-Grs2978974 haplotype with higher risk of GBC in females (FDR Pcorr=0.021, OR=1.6). In contrary, Trs2294008-A rs2978974 haplotype conferred significant lower risk in males (FDR Pcorr=0.013; OR=0.25). These findings suggest that PSCA genetic variants may have a significant effect on GBC susceptibility in a gender specific manner. © 2013.

  14. Increased Artemis levels confer radioresistance to both high and low LET radiation exposures.

    PubMed

    Sridharan, Deepa M; Whalen, Mary K; Almendrala, Donna; Cucinotta, Francis A; Kawahara, Misako; Yannone, Steven M; Pluth, Janice M

    2012-06-19

    Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

  15. Increased Artemis levels confer radioresistance to both high and low LET radiation exposures

    PubMed Central

    2012-01-01

    Background Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation. Results Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage. Conclusions These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies. PMID:22713703

  16. G2385R and I2020T Mutations Increase LRRK2 GTPase Activity

    PubMed Central

    Jang, Jihoon; Joe, Eun-hye; Son, Ilhong; Seol, Wongi

    2016-01-01

    The LRRK2 mutation is a major causal mutation in familial Parkinson's disease. Although LRRK2 contains functional GTPase and kinase domains and their activities are altered by pathogenic mutations, most studies focused on LRRK2 kinase activity because the most prevalent mutant, G2019S, enhances kinase activity. However, the G2019S mutation is extremely rare in the Asian population. Instead, the G2385R mutation was reported as a major risk factor in the Asian population. Similar to other LRRK2 studies, G2385R studies have also focused on kinase activity. Here, we investigated GTPase activities of G2385R with other LRRK2 mutants, such as G2019S, R1441C, and I2020T, as well as wild type (WT). Our results suggest that both I2020T and G2385R contain GTPase activities stronger than that of WT. A kinase assay using the commercial recombinant proteins showed that I2020T harbored stronger activity, whereas G2385R had weaker activity than that of WT, as reported previously. This is the first report of LRRK2 I2020T and G2385R GTPase activities and shows that most of the LRRK2 mutations that are pathogenic or a risk factor altered either kinase or GTPase activity, suggesting that their physiological consequences are caused by altered enzyme activities. PMID:27314038

  17. No evidence of increased mutation rates at microsatellite loci in offspring of A-bomb survivors.

    PubMed

    Kodaira, M; Ryo, H; Kamada, N; Furukawa, K; Takahashi, N; Nakajima, H; Nomura, T; Nakamura, N

    2010-02-01

    To evaluate the genetic effects of A-bomb radiation, we examined mutations at 40 microsatellite loci in exposed families (father-mother-offspring, mostly uni-parental exposures), which consisted of 66 offspring having a mean paternal dose of 1.87 Gy and a mean maternal dose of 1.27 Gy. The control families consisted of 63 offspring whose parents either were exposed to low doses of radiation (< 0.01 Gy) or were not in the cities of Hiroshima or Nagasaki at the time of the bombs. We found seven mutations in the exposed alleles (7/2,789; mutation rate 0.25 x 10(-2)/locus/generation) and 26 in the unexposed alleles (26/7,465; 0.35 x 10(-2)/locus/generation), which does not indicate an effect from parental exposure to radiation. Although we could not assign the parental origins of four mutations, the conclusion may hold since even if we assume that these four mutations had occurred in the exposed alleles, the estimated mean mutation rate would be 0.39 x 10(-2) in the exposed group [(7 + 4)/2,789)], which is slightly higher than 0.35 x 10(-2) in the control group, but the difference is not statistically significant.

  18. Impact of individual mutations on increased fitness in adaptively evolved strains of Escherichia coli.

    PubMed

    Applebee, M Kenyon; Herrgård, Markus J; Palsson, Bernhard Ø

    2008-07-01

    We measured the relative fitness among a set of experimentally evolved Escherichia coli strains differing by a small number of adaptive mutations by directly measuring allelic frequencies in head-to-head competitions using a mass spectrometry-based method. We compared the relative effects of mutations to the same or similar genes acquired in multiple strains when expressed in allele replacement strains. We found that the strongest determinant of fitness among the evolved strains was the impact of beneficial mutations to the RNA polymerase beta and beta' subunit genes. We also identified several examples of epistatic interactions between rpoB/C and glpK mutations and identified two other mutations that are beneficial only in the presence of previously acquired mutations but that have little or no adaptive benefit to the wild-type strain. Allele frequency estimation is shown to be a highly sensitive method for measuring selection rates during competitions between strains differing by as little as a single-nucleotide polymorphism and may be of great use for investigating epistatic interactions.

  19. The Y137H mutation of VvCYP51 gene confers the reduced sensitivity to tebuconazole in Villosiclava virens

    PubMed Central

    Wang, Fei; Lin, Yang; Yin, Wei-Xiao; Peng, You-Liang; Schnabel, Guido; Huang, Jun-Bin; Luo, Chao-Xi

    2015-01-01

    Management of rice false smut disease caused by Villosiclava virens is dependent on demethylation inhibitor (DMI) fungicides. Investigation of molecular mechanisms of resistance is therefore of upmost importance. In this study the gene encoding the target protein for DMI fungicides (VvCYP51) was cloned and investigated. The VvCYP51 gene in the resistant mutant revealed both a change from tyrosine to histidine at position 137 (Y137H) and elevated gene expression compared to the parental isolate. In order to determine which of these mechanisms was responsible for the reduced sensitivity to DMI fungicide tebuconazole, transformants expressing the mutated or the wild type VvCYP51 gene were generated. Transformants carrying the mutated gene were more resistant to tebuconazole compared to control transformants lacking the mutation, but the expression of the VvCYP51 gene was not significantly correlated with EC50 values. The wild type VvCYP51 protein exhibited stronger affinity for tebuconazole compared to the VvCYP51/Y137H in both molecular docking analysis and experimental binding assays. The UV-generated mutant as well as transformants expressing the VvCYP51/Y137H did not exhibit significant fitness penalties based on mycelial growth and spore germination, suggesting that isolates resistant to DMI fungicides based on the Y137H mutation may develop and be competitive in the field. PMID:26631591

  20. Two single mutations in the fusion protein of Newcastle disease virus confer hemagglutinin-neuraminidase independent fusion promotion and attenuate the pathogenicity in chickens.

    PubMed

    Ji, Yanhong; Liu, Tao; Jia, Yane; Liu, Bin; Yu, Qingzhong; Cui, Xiaole; Guo, Fengfeng; Chang, Huiyun; Zhu, Qiyun

    2017-09-01

    The fusion (F) protein of Newcastle disease virus (NDV) affects viral infection and pathogenicity through mediating membrane fusion. Previously, we found NDV with increased fusogenic activity in which contained T458D or G459D mutation in the F protein. Here, we investigated the effects of these two mutations on viral infection, fusogenicity and pathogenicity. Syncytium formation assays indicated that T458D or G459D increased the F protein cleavage activity and enhanced cell fusion with or without the presence of HN protein. The T458D- or G459D-mutated NDV resulted in a decrease in virus replication or release from cells. The animal study showed that the pathogenicity of the mutated NDVs was attenuated in chickens. These results indicate that these two single mutations in F altered or diminished the requirement of HN for promoting membrane fusion. The increased fusogenic activity may disrupt the cellular machinery and consequently decrease the virus replication and pathogenicity in chickens. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa.

    PubMed

    Wondji, Charles S; Dabire, Roch K; Tukur, Zainab; Irving, Helen; Djouaka, Rousseau; Morgan, John C

    2011-07-01

    Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the γ-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique. The distribution of the Rdl(R) mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa

    PubMed Central

    Wondji, Charles S.; Dabire, Roch K.; Tukur, Zainab; Irving, Helen; Djouaka, Rousseau; Morgan, John C.

    2011-01-01

    Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the γ-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique. The distribution of the RdlR mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions. PMID:21501685

  3. In Utero Exposure to Benzo[a]Pyrene Increases Mutation Burden in the Soma and Sperm of Adult Mice

    PubMed Central

    Meier, Matthew J.; O’Brien, Jason M.; Beal, Marc A.; Allan, Beverly; Yauk, Carole L.; Marchetti, Francesco

    2016-01-01

    Background: Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear. Objectives: We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) has an impact on the burden and distribution of mutations in adult mice. Methods: We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development. Results: Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of first filial generation (F1) males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on the reproductive function of F1 males. Conclusion: In utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently affecting both the genetic health of the F1 and the population gene pool. Citation: Meier MJ, O’Brien JM, Beal MA, Allan B, Yauk CL, Marchetti F. 2017. In utero exposure to benzo[a]pyrene increases mutation burden in the soma and sperm of adult mice. Environ Health Perspect 125:82–88; http://dx.doi.org/10.1289/EHP211 PMID:27448386

  4. OPTN 691_692insAG is a founder mutation causing recessive ALS and increased risk in heterozygotes

    PubMed Central

    Goldstein, Orly; Nayshool, Omri; Nefussy, Beatrice; Traynor, Bryan J.; Renton, Alan E.; Gana-Weisz, Mali; Drory, Vivian E.

    2016-01-01

    Objective: To detect genetic variants underlying familial and sporadic amyotrophic lateral sclerosis (ALS). Methods: We analyzed 2 founder Jewish populations of Moroccan and Ashkenazi origins and ethnic matched controls. Exome sequencing of 2 sisters with ALS from Morocco was followed by genotyping the identified causative null mutation in 379 unrelated patients with ALS and 1,000 controls. The shared risk haplotype was characterized using whole-genome single nucleotide polymorphism array. Results: We identified 5 unrelated patients with ALS homozygous for the null 691_692insAG mutation in the optineurin gene (OPTN), accounting for 5.8% of ALS of Moroccan origin and 0.3% of Ashkenazi. We also identified a high frequency of heterozygous carriers among patients with ALS, 8.7% and 2.9%, respectively, compared to 0.75% and 1.0% in controls. The risk of carriers for ALS was significantly increased, with odds ratio of 13.46 and 2.97 in Moroccan and Ashkenazi Jews, respectively. We determined that 691_692insAG is a founder mutation in the tested populations with a minimal risk haplotype of 58.5 Kb, encompassing the entire OPTN gene. Conclusions: Our data show that OPTN 691_692insAG mutation is a founder mutation in Moroccan and Ashkenazi Jews. This mutation causes autosomal recessive ALS and significantly increases the risk to develop the disease in heterozygous carriers, suggesting both a recessive mode of inheritance and a dominant with incomplete penetrance. These data emphasize the important role of OPTN in ALS pathogenesis, and demonstrate the complex genetics of ALS, as the same mutation leads to different phenotypes and appears in 2 patterns of inheritance. PMID:26740678

  5. Digenic inheritance novel mutations in SCN5a and SNTA1 increase late I(Na) contributing to LQT syndrome.

    PubMed

    Hu, Rou-Mu; Tan, Bi-Hua; Orland, Kate M; Valdivia, Carmen R; Peterson, Amber; Pu, Jielin; Makielski, Jonathan C

    2013-04-01

    SCN5A and SNTA1 are reported susceptible genes for long QT syndrome (LQTS). This study was designed to elucidate a plausible pathogenic arrhythmia mechanism for the combined novel mutations R800L-SCN5A and A261V-SNTA1 on cardiac sodium channels. A Caucasian family with syncope and marginally prolonged QT interval was screened for LQTS-susceptibility genes and found to harbor the R800L mutation in SCN5A and A261V mutation in SNTA1, and those with both mutations had the strongest clinical phenotype. The mutations were engineered into the most common splice variant of human SCN5A and SNTA1 cDNA, respectively, and sodium current (INa) was characterized in human embryonic kidney 293 cells cotransfected with neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b). Peak INa densities were unchanged for wild-type (WT) and for mutant channels containing R800L-SCN5A, A261V-SNTA1, or R800L-SCN5A plus A261V-SNTA1. However, late INa for either single mutant was moderately increased two- to threefold compared with WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1 significantly enhanced the INa late/peak ratio by 5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The gain-of-function effect could be blocked by the N(G)-monomethyl-l-arginine, a nNOS inhibitor. We conclude that novel mutations in SCN5A and SNTA1 jointly exert a nNOS-dependent gain-of-function on SCN5A channels, which may consequently prolong the action potential duration and lead to LQTS phenotype.

  6. Strategic priorities for increasing physical activity among adults age 50 and older: the national blueprint consensus conference summary report.

    PubMed

    Sheppard, Lisa; Senior, Jane; Park, Chae Hee; Mockenhaupt, Robin; Bazzarre, Terry; Chodzko-Zajko, Wojtek

    2003-12-01

    On May 1, 2001, a coalition of national organizations released a major planning document designed to develop a national strategy for the promotion of physically active lifestyles among the mid-life and older adult population. The National Blueprint: Increasing Physical Activity Among Adults Age 50 and Older was developed with input from 46 organizations with expertise in health, medicine, social and behavioral sciences, epidemiology, gerontology/geriatrics, clinical science, public policy, marketing, medical systems, community organization, and environmental issues. The Blueprint notes that, despite a wealth of evidence about the benefits of physical activity for mid-life and older persons, there has been little success in convincing age 50+ Americans to adopt physically active lifestyles. The Blueprint identifies barriers in the areas of research, home and community programs, medical systems, public policy and advocacy, and marketing and communications. In addition to identifying barriers, the Blueprint proposes a number of concrete strategies that could be employed in order to overcome the barriers to physical activity in society at large. This report summarizes the outcome of the National Blueprint Consensus Conference that was held in October 2002. In this conference, representatives of more than 50 national organizations convened in Washington, D.C. with the goal of identifying high priority and high feasibility strategies which would advance the National Blueprint and which could be initiated within the next 12 to 24 months. Participants in the consensus conference were assigned to one of five breakout groups: home and community, marketing, medical systems, public policy, and research. Each breakout group was charged with identifying the three highest priority strategies within their area for effectively increasing physical activity levels in the mid-life and older adult population. In addition to the 15 strategies identified by the breakout groups, three

  7. Increasing Collaborative Efforts in Career Education, K-12. A Series of Mini-Conferences. Phase I. Final Report.

    ERIC Educational Resources Information Center

    Coley, Walton, Ed.

    During 1977-78, a series of Office of Career Education "mini-conferences" was held, each conference devoted to attacking the concept of collaboration in career education from a different perspective. The notes from this set of mini-conferences form the body of this document. (A companion document, CE 020 111, describes the follow-up…

  8. Increased progerin expression associated with unusual LMNA mutations causes severe progeroid syndromes.

    PubMed

    Moulson, Casey L; Fong, Loren G; Gardner, Jennifer M; Farber, Emily A; Go, Gloriosa; Passariello, Annalisa; Grange, Dorothy K; Young, Stephen G; Miner, Jeffrey H

    2007-09-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a rare precocious aging syndrome caused by mutations in LMNA that lead to synthesis of a mutant form of prelamin A, generally called progerin, that cannot be processed to mature lamin A. Most HGPS patients have a recurrent heterozygous de novo mutation in exon 11 of LMNA, c.1824C>T/p.G608G; this synonymous mutation activates a nearby cryptic splice donor site, resulting in synthesis of the mutant prelamin A, progerin, which lacks 50 amino acids within the carboxyl-terminal domain. Abnormal splicing is incomplete, so the mutant allele produces some normally-spliced transcripts. Nevertheless, the synthesis of progerin is sufficient to cause misshapen nuclei in cultured cells and severe disease phenotypes in affected patients. Here we present two patients with extraordinarily severe forms of progeria caused by unusual mutations in LMNA. One had a splice site mutation (c.1968+1G>A; or IVS11+1G>A), and the other had a novel synonymous coding region mutation (c.1821G>A/p.V607V). Both mutations caused very frequent use of the same exon 11 splice donor site that is activated in typical HGPS patients. As a consequence, the ratios of progerin mRNA and protein to wild-type were higher than in typical HGPS patients. Fibroblasts from both patients exhibited nuclear shape abnormalities typical of HGPS, and cells treated with a protein farnesyltransferase inhibitor exhibited fewer misshapen nuclei. Thus, farnesyltransferase inhibitors may prove to be useful even when progerin expression levels are higher than those in typical HGPS patients.

  9. Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.

    PubMed

    Lomri, A; Lemonnier, J; Hott, M; de Parseval, N; Lajeunie, E; Munnich, A; Renier, D; Marie, P J

    1998-03-15

    Apert syndrome, associated with fibroblast growth factor receptor (FGFR) 2 mutations, is characterized by premature fusion of cranial sutures. We analyzed proliferation and differentiation of calvaria cells derived from Apert infants and fetuses with FGFR-2 mutations. Histological analysis revealed premature ossification, increased extent of subperiosteal bone formation, and alkaline phosphatase- positive preosteoblastic cells in Apert fetal calvaria compared with age-matched controls. Preosteoblastic calvaria cells isolated from Apert infants and fetuses showed normal cell growth in basal conditions or in response to exogenous FGF-2. In contrast, the number of alkaline phosphatase- positive calvaria cells was fourfold higher than normal in mutant fetal calvaria cells with the most frequent Apert FGFR-2 mutation (Ser252Trp), suggesting increased maturation rate of cells in the osteoblastic lineage. Biochemical and Northern blot analyses also showed that the expression of alkaline phosphatase and type 1 collagen were 2-10-fold greater than normal in mutant fetal calvaria cells. The in vitro production of mineralized matrix formed by immortalized mutant fetal calvaria cells cultured in aggregates was also increased markedly compared with control immortalized fetal calvaria cells. The results show that Apert FGFR-2 mutations lead to an increase in the number of precursor cells that enter the osteogenic pathway, leading ultimately to increased subperiosteal bone matrix formation and premature calvaria ossification during fetal development, which establishes a connection between the altered genotype and cellular phenotype in Apert syndromic craniosynostosis.

  10. A missense mutation in the fabB (beta-ketoacyl-acyl carrier protein synthase I) gene confers tiolactomycin resistance to Escherichia coli.

    PubMed

    Jackowski, Suzanne; Zhang, Yong-Mei; Price, Allen C; White, Stephen W; Rock, Charles O

    2002-05-01

    Thiolactomycin (TLM) is an antibiotic that inhibits bacterial type II fatty acid synthesis at the condensing enzyme step, and beta-ketoacyl-acyl carrier protein synthase I (FabB) is the relevant target in Escherichia coli. TLM resistance is associated with the upregulation of efflux pumps. Therefore, a tolC knockout mutant (strain ANS1) was constructed to eliminate the contribution of type I secretion systems to TLM resistance. Six independent TLM-resistant clones of strain ANS1 were isolated, and all possessed the same missense mutation in the fabB gene (T1168G) that directed the expression of a mutant protein, FabB(F390V). FabB(F390V) was resistant to TLM in vitro. Leucine is the only other amino acid found at position 390 in nature, and the Staphylococcus aureus FabF protein, which contains this substitution, was sensitive to TLM. Structural modeling predicted that the CG2 methyl group of the valine side chain interfered with the positioning of the C11 methyl on the isoprenoid side chain of TLM in the binary complex, whereas the absence of a bulky methyl group on the leucine side chain permitted TLM binding. These data illustrate that missense mutations that introduce valine at position 390 confer TLM resistance while maintaining the vital catalytic properties of FabB.

  11. Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare).

    PubMed

    Kreis, M; Shewry, P R; Forde, B G; Rahman, S; Miflin, B J

    1983-08-01

    We have analyzed the molecular nature of the Riso 56 mutation that occurs in barley. This mutation results in a depression of hordein accumulation in the grain and consequently in a higher overall lysine content. In particular, the amount of B hordein, which is encoded by the complex locus Hor-2, is decreased by about 75% because of the absence of the major components. The synthesis of certain minor polypeptides, with properties similar to the major B hordeins, remains unaffected. Analysis of endosperm RNA, by in vitro translation and hybridization to various cloned cDNAs derived from hordein mRNA, shows that mRNA for the major B hordeins is not present in the endosperm. Hybridization of a B hordein cDNA clone to gel-fractionated restriction digests of mutant and wild-type DNA indicates that at least 85 kb of DNA has been deleted from the Hor-2 locus in the high-lysine mutant.

  12. Treatment-emergent mutations in NAEβ confer resistance to the NEDD8-activating enzyme inhibitor MLN4924.

    PubMed

    Milhollen, Michael A; Thomas, Michael P; Narayanan, Usha; Traore, Tary; Riceberg, Jessica; Amidon, Benjamin S; Bence, Neil F; Bolen, Joseph B; Brownell, James; Dick, Lawrence R; Loke, Huay-Keng; McDonald, Alice A; Ma, Jingya; Manfredi, Mark G; Sells, Todd B; Sintchak, Mike D; Yang, Xiaofeng; Xu, Qing; Koenig, Erik M; Gavin, James M; Smith, Peter G

    2012-03-20

    MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

  13. Evolutionary Action Score of TP53 Identifies High-Risk Mutations Associated with Decreased Survival and Increased Distant Metastases in Head and Neck Cancer.

    PubMed

    Neskey, David M; Osman, Abdullah A; Ow, Thomas J; Katsonis, Panagiotis; McDonald, Thomas; Hicks, Stephanie C; Hsu, Teng-Kuei; Pickering, Curtis R; Ward, Alexandra; Patel, Ameeta; Yordy, John S; Skinner, Heath D; Giri, Uma; Sano, Daisuke; Story, Michael D; Beadle, Beth M; El-Naggar, Adel K; Kies, Merrill S; William, William N; Caulin, Carlos; Frederick, Mitchell; Kimmel, Marek; Myers, Jeffrey N; Lichtarge, Olivier

    2015-04-01

    TP53 is the most frequently altered gene in head and neck squamous cell carcinoma, with mutations occurring in over two-thirds of cases, but the prognostic significance of these mutations remains elusive. In the current study, we evaluated a novel computational approach termed evolutionary action (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk, and validated this system in both in vivo and in vitro models. Patients with high-risk TP53 mutations had the poorest survival outcomes and the shortest time to the development of distant metastases. Tumor cells expressing high-risk TP53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastases. We also documented an association between the presence of high-risk mutations and decreased expression of TP53 target genes, highlighting key cellular pathways that are likely to be dysregulated by this subset of p53 mutations that confer particularly aggressive tumor behavior. Overall, our work validated EAp53 as a novel computational tool that may be useful in clinical prognosis of tumors harboring p53 mutations. ©2015 American Association for Cancer Research.

  14. Evolutionary Action score of TP53 (EAp53) identifies high risk mutations associated with decreased survival and increased distant metastases in head and neck cancer

    PubMed Central

    Neskey, David M.; Osman, Abdullah A.; Ow, Thomas J.; Katsonis, Panagiotis; McDonald, Thomas; Hicks, Stephanie C.; Hsu, Teng-Kuei; Pickering, Curtis R.; Ward, Alexandra; Patel, Ameeta; Yordy, John S.; Skinner, Heath D.; Giri, Uma; Sano, Daisuke; Story, Michael D.; Beadle, Beth M.; El-Naggar, Adel K.; Kies, Merrill S.; William, William N.; Caulin, Carlos; Frederick, Mitchell; Kimmel, Marek; Myers, Jeffrey N.; Lichtarge, Olivier

    2015-01-01

    TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC) with mutations occurring in over two third of cases, but the prognostic significance of these mutations remains elusive. In the current study, we evaluated a novel computational approach termed Evolutionary Action (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk, and validated this system in both in vivo and in vitro models. Patients with high risk TP53 mutations had the poorest survival outcomes and the shortest time to the development of distant metastases. Tumor cells expressing high risk TP53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastases. We also documented an association between the presence of high risk mutations and decreased expression of TP53 target genes, highlighting key cellular pathways that are likely to be dysregulated by this subset of p53 mutations which confer particularly aggressive tumor behavior. Overall, our work validated EAp53 as a novel computational tool that may be useful in clinical prognosis of tumors harboring p53 mutations. PMID:25634208

  15. A targeted dominant negative mutation of the thyroid hormone α1 receptor causes increased mortality, infertility, and dwarfism in mice

    PubMed Central

    Kaneshige, Masahiro; Suzuki, Hideyo; Kaneshige, Kumiko; Cheng, Jun; Wimbrow, Heather; Barlow, Carrolee; Willingham, Mark C.; Cheng, Sheue-yann

    2001-01-01

    Mutations in the thyroid hormone receptor β (TRβ) gene result in resistance to thyroid hormone. However, it is unknown whether mutations in the TRα gene could lead to a similar disease. To address this question, we prepared mutant mice by targeting mutant thyroid hormone receptor kindred PV (PV) mutation to the TRα gene locus by means of homologous recombination (TRα1PV mice). The PV mutation was derived from a patient with severe resistance to thyroid hormone that has a frameshift of the C-terminal 14 aa of TRβ1. We knocked in the same PV mutation to the corresponding TRα gene locus to compare the phenotypes of TRα1PV/+ mice with those of TRβPV/+ mice. TRα1PV/+ mice were viable, indicating that the mutation of the TRα gene is not embryonic lethal. In drastic contrast to the TRβPV/+ mice, which do not exhibit a growth abnormality, TRα1PV/+ mice were dwarfs. These dwarfs exhibited increased mortality and reduced fertility. In contrast to TRβPV/+ mice, which have a hyperactive thyroid, TRα1PV/+ mice exhibited mild thyroid failure. The in vivo pattern of abnormal regulation of T3 target genes in TRα1PV/+ mice was unique from those of TRβPV/+ mice. The distinct phenotypes exhibited by TRα1PV/+ and TRβPV/+ mice indicate that the in vivo functions of TR mutants are isoform-dependent. The TRα1PV/+ mice may be used as a tool to uncover human diseases associated with mutations in the TRα gene and, furthermore, to understand the molecular mechanisms by which TR isoforms exert their biological activities. PMID:11734632

  16. Fitness Conferred by BCR-ABL Kinase Domain Mutations Determines the Risk of Pre-Existing Resistance in Chronic Myeloid Leukemia

    PubMed Central

    Skaggs, Brian; Gorre, Mercedes; Sawyers, Charles L.; Michor, Franziska

    2011-01-01

    Chronic myeloid leukemia (CML) is the first human malignancy to be successfully treated with a small molecule inhibitor, imatinib, targeting a mutant oncoprotein (BCR-ABL). Despite its successes, acquired resistance to imatinib leads to reduced drug efficacy and frequent progression of disease. Understanding the characteristics of pre-existing resistant cells is important for evaluating the benefits of first-line combination therapy with second generation inhibitors. However, due to limitations of assay sensitivity, determining the existence and characteristics of resistant cell clones at the start of therapy is difficult. Here we combined a mathematical modeling approach using branching processes with experimental data on the fitness changes (i.e., changes in net reproductive rate) conferred by BCR-ABL kinase domain mutations to investigate the likelihood, composition, and diversity of pre-existing resistance. Furthermore, we studied the impact of these factors on the response to tyrosine kinase inhibitors. Our approach predicts that in most patients, there is at most one resistant clone present at the time of diagnosis of their disease. Interestingly, patients are no more likely to harbor the most aggressive, pan-resistant T315I mutation than any other resistance mutation; however, T315I cells on average establish larger-sized clones at the time of diagnosis. We established that for patients diagnosed late, the relative benefit of combination therapy over monotherapy with imatinib is significant, while this benefit is modest for patients with a typically early diagnosis time. These findings, after pre-clinical validation, will have implications for the clinical management of CML: we recommend that patients with advanced-phase disease be treated with combination therapy with at least two tyrosine kinase inhibitors. PMID:22140458

  17. BRCA1 founder mutations do not contribute to increased risk of gastric cancer in the Polish population.

    PubMed

    Ławniczak, Małgorzata; Jakubowska, Anna; Białek, Andrzej; Lubiński, Jan; Jaworska-Bieniek, Katarzyna; Kaczmarek, Katarzyna; Starzyńska, Teresa

    2016-01-01

    Gastric cancer (GC) is part of the spectrum of diseases linked to BRCA1 and BRCA2 mutations that increase the risk of breast and ovarian cancer. Data suggesting an increased risk of developing GC among BRCA1 and BRCA2 mutation carriers are based almost exclusively on indirect studies. The objective was to assess in a direct study whether there is a relationship between GC and selected recurrent BRCA1 and BRCA2 mutations in the Polish population. Three hundred seventeen GC patients (193 males and 124 females; mean age 59.5 ± 12.8 y) diagnosed at the Department of Gastroenterology at the Pomeranian Medical University were included in this retrospective study. All patients were genotyped for 3 BRCA1 Polish founder mutations (5382insC, C61G and 4153delA) as well as for 9 known recurrent mutations in BRCA1 and BRCA2 genes. Genotyping was performed using allele-specific oligonucleotide polymerase chain reaction (ASA-PCR) for 4153delA and 5382insC, restriction fragment length polymorphism (PCR-RFLP) for C61G and TaqMan real-time PCR for 185delAG, 3819del5, 3875del4, 5370C > T, 886delGT, 4075delGT, 5467insT, 6174delT and 8138del5. Among tested mutations one founder BRCA1 mutation 5382insC was detected in two of 317 (0.63 %) GC cases. A comparison of frequency of detected BRCA1 founder mutations in GC patients to previously described 4570 Polish controls (0.63 % vs. 0.48 %) failed to indicate an increased risk of GC in the mutation carriers (OR = 1.3; 95 % CI 0.3-5.6, p = 0.71). A comparison of frequency of GC male cases and male controls (1.0 % vs. 0.43 %,OR = 1.5; 95 % CI 0.3-6.4, p = 0.61) allowed to formulate the same conclusion that there is no increased risk for GC for males. None of the 9 recurrent BRCA1 and BRCA2 mutations has been detected in tested GC patients. The current study indicates that founder BRCA1 mutations reported in Polish breast/ovarian cancer patients do not contribute to increased GC risk. The nine tested recurrent

  18. I223R Mutation in Influenza A(H1N1)pdm09 Neuraminidase Confers Reduced Susceptibility to Oseltamivir and Zanamivir and Enhanced Resistance with H275Y

    PubMed Central

    Abou-Jaoudé, Georges; Scemla, Anne; Ribaud, Patricia; Mercier-Delarue, Séverine; Caro, Valérie; Enouf, Vincent; Simon, François; Molina, Jean-Michel; van der Werf, Sylvie

    2012-01-01

    Background Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility. Methods The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0–15) and zanamivir (days 15–25 and 70–80). Results Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6–69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin. Conclusions Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines

  19. You Are What You Eat: Within-Subject Increases in Fruit and Vegetable Consumption Confer Beneficial Skin-Color Changes

    PubMed Central

    Whitehead, Ross D.; Re, Daniel; Xiao, Dengke; Ozakinci, Gozde; Perrett, David I.

    2012-01-01

    Background Fruit and vegetable consumption and ingestion of carotenoids have been found to be associated with human skin-color (yellowness) in a recent cross-sectional study. This carotenoid-based coloration contributes beneficially to the appearance of health in humans and is held to be a sexually selected cue of condition in other species. Methodology and Principal Findings Here we investigate the effects of fruit and vegetable consumption on skin-color longitudinally to determine the magnitude and duration of diet change required to change skin-color perceptibly. Diet and skin-color were recorded at baseline and after three and six weeks, in a group of 35 individuals who were without makeup, self-tanning agents and/or recent intensive UV exposure. Six-week changes in fruit and vegetable consumption were significantly correlated with changes in skin redness and yellowness over this period, and diet-linked skin reflectance changes were significantly associated with the spectral absorption of carotenoids and not melanin. We also used psychophysical methods to investigate the minimum color change required to confer perceptibly healthier and more attractive skin-coloration. Modest dietary changes are required to enhance apparent health (2.91 portions per day) and attractiveness (3.30 portions). Conclusions Increased fruit and vegetable consumption confers measurable and perceptibly beneficial effects on Caucasian skin appearance within six weeks. This effect could potentially be used as a motivational tool in dietary intervention. PMID:22412966

  20. You are what you eat: within-subject increases in fruit and vegetable consumption confer beneficial skin-color changes.

    PubMed

    Whitehead, Ross D; Re, Daniel; Xiao, Dengke; Ozakinci, Gozde; Perrett, David I

    2012-01-01

    Fruit and vegetable consumption and ingestion of carotenoids have been found to be associated with human skin-color (yellowness) in a recent cross-sectional study. This carotenoid-based coloration contributes beneficially to the appearance of health in humans and is held to be a sexually selected cue of condition in other species. Here we investigate the effects of fruit and vegetable consumption on skin-color longitudinally to determine the magnitude and duration of diet change required to change skin-color perceptibly. Diet and skin-color were recorded at baseline and after three and six weeks, in a group of 35 individuals who were without makeup, self-tanning agents and/or recent intensive UV exposure. Six-week changes in fruit and vegetable consumption were significantly correlated with changes in skin redness and yellowness over this period, and diet-linked skin reflectance changes were significantly associated with the spectral absorption of carotenoids and not melanin. We also used psychophysical methods to investigate the minimum color change required to confer perceptibly healthier and more attractive skin-coloration. Modest dietary changes are required to enhance apparent health (2.91 portions per day) and attractiveness (3.30 portions). Increased fruit and vegetable consumption confers measurable and perceptibly beneficial effects on Caucasian skin appearance within six weeks. This effect could potentially be used as a motivational tool in dietary intervention.

  1. A short insertion mutation disrupts genesis of miR-16 and causes increased body weight in domesticated chicken

    PubMed Central

    Jia, Xinzheng; Lin, Huiran; Nie, Qinghua; Zhang, Xiquan; Lamont, Susan J.

    2016-01-01

    Body weight is one of the most important quantitative traits with high heritability in chicken. We previously mapped a quantitative trait locus (QTL) for body weight by genome-wide association study (GWAS) in an F2 chicken resource population. To identify the causal mutations linked to this QTL, expression profiles were determined on livers of high-weight and low-weight chicken lines by microarray. Combining the expression pattern with SNP effects by GWAS, miR-16 was identified as the most likely potential candidate with a 3.8-fold decrease in high-weight lines. Re-sequencing revealed that a 54-bp insertion mutation in the upstream region of miR-15a-16 displayed high allele frequencies in high-weight commercial broiler line. This mutation resulted in lower miR-16 expression by introducing three novel splicing sites instead of the missing 5′ terminal splicing of mature miR-16. Elevating miR-16 significantly inhibited DF-1 chicken embryo cell proliferation, consistent with a role in suppression of cellular growth. The 54-bp insertion was significantly associated with increased body weight, bone size and muscle mass. Also, the insertion mutation tended towards fixation in commercial broilers (Fst > 0.4). Our findings revealed a novel causative mutation for body weight regulation that aids our basic understanding of growth regulation in birds. PMID:27808177

  2. Possible introgression of the VRTN mutation increasing vertebral number, carcass length and teat number from Chinese pigs into European pigs

    PubMed Central

    Yang, Jie; Huang, Lusheng; Yang, Ming; Fan, Yin; Li, Lin; Fang, Shaoming; Deng, Wenjiang; Cui, Leilei; Zhang, Zhen; Ai, Huashui; Wu, Zhenfang; Gao, Jun; Ren, Jun

    2016-01-01

    Vertnin (VRTN) variants have been associated with the number of thoracic vertebrae in European pigs, but the association has not been evidenced in Chinese indigenous pigs. In this study, we first performed a genome-wide association study in Chinese Erhualian pigs using one VRTN candidate causative mutation and the Illumina Porcine 60K SNP Beadchips. The VRTN mutation is significantly associated with thoracic vertebral number in this population. We further show that the VRTN mutation has pleiotropic and desirable effects on teat number and carcass (body) length across four diverse populations, including Erhualian, White Duroc × Erhualian F2 population, Duroc and Landrace pigs. No association was observed between VRTN genotype and growth and fatness traits in these populations. Therefore, testing for the VRTN mutation in pig breeding schemes would not only increase the number of vertebrae and nipples, but also enlarge body size without undesirable effects on growth and fatness traits, consequently improving pork production. Further, by using whole-genome sequence data, we show that the VRTN mutation was possibly introgressed from Chinese pigs into European pigs. Our results provide another example showing that introgressed Chinese genes greatly contributed to the development and production of modern European pig breeds. PMID:26781738

  3. IDH1R132H Mutation Increases U87 Glioma Cell Sensitivity to Radiation Therapy in Hypoxia

    PubMed Central

    Wang, Xiao-Wei; Labussière, Marianne; Valable, Samuel; Pérès, Elodie A.; Guillamo, Jean-Sébastien; Sanson, Marc

    2014-01-01

    Objective. IDH1 codon 132 mutation (mostly Arg132His) is frequently found in gliomas and is associated with longer survival. However, it is still unclear whether IDH1 mutation renders the cell more vulnerable to current treatment, radio- and chemotherapy. Materials and Methods. We transduced U87 with wild type IDH1 or IDH1R132H expressing lentivirus and analyzed the radiosensitivity (dose ranging 0 to 10 Gy) under normoxia (20% O2) and moderate hypoxia (1% O2). Results. We observed that IDH1R132H U87 cells grow faster in hypoxia and were more sensitive to radiotherapy (in terms of cell mortality and colony formation assay) compared to nontransduced U87 and IDH1wt cells. This effect was not observed in normoxia. Conclusion. These data suggest that IDH1R132H mutation increases radiosensitivity in mild hypoxic conditions. PMID:24895549

  4. Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

    PubMed Central

    Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Jaiswal, Alok; Zhou, Yong; Liang, Hong; Lectez, Benoit; Kopra, Kari; Guzmán, Camilo; Härmä, Harri; Hancock, John F; Aittokallio, Tero; Abankwa, Daniel

    2015-01-01

    Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer. DOI: http://dx.doi.org/10.7554/eLife.08905.001 PMID:26274561

  5. Novel MTND1 mutations cause isolated exercise intolerance, complex I deficiency and increased assembly factor expression

    PubMed Central

    Gorman, Grainne S.; Blakely, Emma L.; Hornig-Do, Hue-Tran; Tuppen, Helen A.L.; Greaves, Laura C.; He, Langping; Baker, Angela; Falkous, Gavin; Newman, Jane; Trenell, Michael I.; Lecky, Bryan; Petty, Richard K.; Turnbull, Doug M.; McFarland, Robert

    2015-01-01

    We describe the clinical, biochemical and molecular characterization of two adults with progressive exercise intolerance and severe isolated mitochondrial complex I (CI) deficiency due to novel MTND1 mutations. We demonstrate compensatory CI assembly factor up-regulation probably partially rescuing the clinical phenotype. PMID:25626417

  6. Evidence for increased somatic cell mutations at the glycophorin A locus in atomic bomb survivors.

    PubMed

    Langlois, R G; Bigbee, W L; Kyoizumi, S; Nakamura, N; Bean, M A; Akiyama, M; Jensen, R H

    1987-04-24

    A recently developed assay for somatic cell mutations was used to study survivors of the atomic bomb at Hiroshima. This assay measures the frequency of variant erythrocytes produced by erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Significant linear relations between variant frequency (VF) and radiation exposure were observed for three different variant cell phenotypes. The spontaneous and induced VFs agree with previous measurements of radiation-induced mutagenesis in other systems; this evidence supports a mutational origin for variant cells characterized by a loss of GPA expression and suggests that the GPA assay system may provide a cumulative dosimeter of past radiation exposures. VFs for some survivors differ dramatically from the calculated dose response, and these deviations appear to result primarily from statistical fluctuations in the number of mutations in the stem-cell pool. These fluctuations allow one to estimate the number of long-lived hemopoietic stem cells in humans.

  7. Mutations Affecting Starch Synthase III in Arabidopsis Alter Leaf Starch Structure and Increase the Rate of Starch Synthesis1

    PubMed Central

    Zhang, Xiaoli; Myers, Alan M.; James, Martha G.

    2005-01-01

    The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis. PMID:15908598

  8. Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation.

    PubMed

    Zhang, Shirong; Zheng, Xiaoliang; Huang, Haixiu; Wu, Kan; Wang, Bing; Chen, Xufeng; Ma, Shenglin

    2015-03-20

    Afatinib is a second-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and has shown a significant clinical benefit in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the potential therapeutic effects of afatinib combining with other modalities, including ionizing radiation (IR), are not well understood. In this study, we developed a gefitinib-resistant cell subline (PC-9-GR) with a secondary EGFR mutation (T790M) from NSCLC PC-9 cells after chronic exposures to increasing doses of gefitinib. The presence of afatinib significantly increases the cell killing effect of radiation in PC-9-GR cells harboring acquired T790M, but not in H1975 cells with de novo T790M or in H460 cells that express wild-type EGFR. In PC-9-GR cells, afatinib remarkable blocks baseline of EGFR and ERK phosphorylations, and causes delay of IR-induced AKT phosphorylation. Afatinib treatment also leads to increased apoptosis and suppressed DNA damage repair in irradiated PC-9-GR cells, and enhanced tumor growth inhibition when combined with IR in PC-9-GR xenografts. Our findings suggest a potential therapeutic impact of afatinib as a radiation sensitizer in lung cancer cells harboring acquired T790M mutation, providing a rationale for a clinical trial with combination of afatinib and radiation in NSCLCs with EGFR T790M mutation.

  9. Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1.

    PubMed

    Jang, Su Jin; Wi, Soo Jin; Choi, Yoo Jin; An, Gynheung; Park, Ky Young

    2012-09-01

    A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H(2)O(2). The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenously-applied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H(2)O(2). Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.

  10. Role of key genetic mutations on increasing migration of brain cancer cells through confinement.

    PubMed

    Bui, Loan; Bhuiyan, Sayem H; Hendrick, Alissa; Chuong, Cheng-Jen; Kim, Young-Tae

    2017-09-01

    Uncontrolled invasive cancer cell migration is among the major challenges for the treatment and management of brain cancer. Although the genetic profiles of brain cancer cells have been well characterized, the relationship between the genetic mutations and the cells' mobility has not been clearly understood. In this study, using microfluidic devices that provide a wide range of physical confinements from 20 × 5 μm(2) to 3 × 5 μm(2) in cross sections, we studied the effect of physical confinement on the migratory capacity of cell lines with different types of mutations. Human glioblastoma and genetically modified mouse astrocytes were used. Human glioblastoma cells with EGFRvIII mutation were found to exhibit high degree of migratory capacity in narrow confinement. From mouse astrocytes, cells with triple mutations (p53-/- PTEN-/- BRAF) were found to exhibit the highest level of migratory capacity in narrow confinement compared to both double (p53-/- PTEN-/-) and single (p53-/-) mutant cells. Furthermore, when treating the triple mutant astrocytes with AZD-6244, an inhibitor of the RAF/MEK/ERK pathway, we found significant reduction in migration through the confined channels when compared to that of controls (83% decrease in 5 × 5 μm(2) and 86% in 3 × 5 μm(2) channels). Our data correlate genetic mutations from different cell lines to their motility in different degrees of confinement. Our results also suggest a potential therapeutic target such as BRAF oncogene for inhibition of brain cancer invasion.

  11. Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin.

    PubMed

    Huang, Jiwei; Zhang, Xinhua; Liu, Dun; Wei, Xiaofeng; Shang, Xuan; Xiong, Fu; Yu, Lihua; Yin, Xiaolin; Xu, Xiangmin

    2015-10-01

    Krüppel-like factor 1 (KLF1) regulates erythroid lineage commitment, globin switching, and the terminal maturation of red blood cells. Variants in human KLF1 have been identified as an important causative factor in a wide spectrum of phenotypes. This study investigated two unrelated male children in China who had refractory anemia associated with poikilocythemia. These were accompanied by an upregulation of biochemical markers of hemolysis, along with abnormal hemoglobin (Hb) level and elevated reticulocyte counts. Next-generation sequencing revealed that the patients were compound heterozygotes for a KLF1 frameshift mutation c.525_526insCGGCGCC (p.(Gly176ArgfsTer179)) and one of two missense variants, c.892 G>C (p.(Ala298Pro)) and c.1012C>T (p.(Pro338Ser)). The subjects had microcytic hypochromic anemia, and their healthy parents had single mutation. The two missense mutations affected a highly conserved codon in the zinc finger DNA-binding domain of KLF1, but the protein stability was unaffected in K-562 cells. A KLF1-targeted promoter-reporter assay showed that the two mutations reduce the expression of the HBB, BCL11A, and CD44 genes involved in erythropoiesis, with consequent dyserythropoiesis and an α/non-α chain imbalance. A systematic analysis was performed of the phenotypes associated with the KLF1 mutations in the two families, and the clinical characteristics and differential diagnoses of the disease are presented. This is the first report of an autosomal recessive anemia presenting with microcytic hypochromia, abnormal Hb profile, and other distinctive erythrocyte phenotypes, and provides insight into the multiple roles of KLF1 during erythropoiesis.

  12. Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

    PubMed Central

    Huang, Jiwei; Zhang, Xinhua; Liu, Dun; Wei, Xiaofeng; Shang, Xuan; Xiong, Fu; Yu, Lihua; Yin, Xiaolin; Xu, Xiangmin

    2015-01-01

    Krüppel-like factor 1 (KLF1) regulates erythroid lineage commitment, globin switching, and the terminal maturation of red blood cells. Variants in human KLF1 have been identified as an important causative factor in a wide spectrum of phenotypes. This study investigated two unrelated male children in China who had refractory anemia associated with poikilocythemia. These were accompanied by an upregulation of biochemical markers of hemolysis, along with abnormal hemoglobin (Hb) level and elevated reticulocyte counts. Next-generation sequencing revealed that the patients were compound heterozygotes for a KLF1 frameshift mutation c.525_526insCGGCGCC (p.(Gly176ArgfsTer179)) and one of two missense variants, c.892 G>C (p.(Ala298Pro)) and c.1012C>T (p.(Pro338Ser)). The subjects had microcytic hypochromic anemia, and their healthy parents had single mutation. The two missense mutations affected a highly conserved codon in the zinc finger DNA-binding domain of KLF1, but the protein stability was unaffected in K-562 cells. A KLF1-targeted promoter–reporter assay showed that the two mutations reduce the expression of the HBB, BCL11A, and CD44 genes involved in erythropoiesis, with consequent dyserythropoiesis and an α/non-α chain imbalance. A systematic analysis was performed of the phenotypes associated with the KLF1 mutations in the two families, and the clinical characteristics and differential diagnoses of the disease are presented. This is the first report of an autosomal recessive anemia presenting with microcytic hypochromia, abnormal Hb profile, and other distinctive erythrocyte phenotypes, and provides insight into the multiple roles of KLF1 during erythropoiesis. PMID:25585695

  13. JAK3 deregulation by activating mutations confers invasive growth advantage in extranodal nasal-type natural killer cell lymphoma.

    PubMed

    Bouchekioua, A; Scourzic, L; de Wever, O; Zhang, Y; Cervera, P; Aline-Fardin, A; Mercher, T; Gaulard, P; Nyga, R; Jeziorowska, D; Douay, L; Vainchenker, W; Louache, F; Gespach, C; Solary, E; Coppo, P

    2014-02-01

    Extranodal, nasal-type natural killer (NK)/T-cell lymphoma (NKCL) is an aggressive malignancy with poor prognosis in which, usually, signal transducer and activator of transcription 3 (STAT3) is constitutively activated and oncogenic. Here, we demonstrate that STAT3 activation mostly results from constitutive Janus kinase (JAK)3 phosphorylation on tyrosine 980, as observed in three of the four tested NKCL cell lines and in 20 of the 23 NKCL tumor samples under study. In one of the cell lines and in 4 of 19 (21%) NKCL primary tumor samples, constitutive JAK3 activation was related to an acquired mutation (A573V or V722I) in the JAK3 pseudokinase domain. We then show that constitutive activation of the JAK3/STAT3 pathway has a major role in NKCL cell growth and survival and in the invasive phenotype. Indeed, NKCL cell growth was slowed down in vitro by targeting JAK3 with chemical inhibitors or small-interfering RNAs. In a human NKCL xenograft mouse model, tumor growth was significantly delayed by the JAK3 inhibitor CP-690550. Altogether, the constitutive activation of JAK3, which can result from JAK3-activating mutations, is a frequent feature of NKCL that deserves to be tested as a therapeutic target.

  14. Somatic Mutation of the SNP rs11614913 and Its Association with Increased MIR 196A2 Expression in Breast Cancer.

    PubMed

    Zhao, Huanhuan; Xu, Jingman; Zhao, Dan; Geng, Meijuan; Ge, Haize; Fu, Li; Zhu, Zhengmao

    2016-02-01

    Common genetic variants (single-nucleotide polymorphisms [SNPs]) in microRNA genes may alter their maturation or expression, resulting in varied functional consequences. Several studies have evaluated the association between the SNP rs11614913 and cancer risk in diverse populations and in a range of cancers, with contradictory outcomes. In this study, we examined 114 paired samples (tumor and normal tissues) from breast cancer patients to study the genotype distribution and somatic mutation of the SNP in MIR 196A2 (rs11614913 C-T). In addition, we evaluated their influence on the mature MIR 196A2 expression. We found that 14% (16/114) of tumors underwent somatic mutation of the SNP rs11614913. Moreover, the CT heterozygous and the CC homozygous states of SNP rs11614913 were more prone to mutation, while the TT homozygous state appeared to be resistant. We further detected a significant increase (p = 0.002) in mature MIR 196A2 expression in breast cancer. In particular, we found a significant association between the occurrence of SNP rs11614913 mutation and high expression (p = 0.0002). In addition, the mature MIR 196A2 expression level was significantly associated with the higher tumor grade (p = 0.004). Taken together, our results seem to demonstrate that somatic mutation of SNP rs11614913 in MIR 196A2 can have an influence on its expression. In addition, it indicated that an unknown mechanism is responsible for both the mutation of SNP rs11614913 and the dysregulation of mature MIR 196A2 expression.

  15. Familial mutations in fibrinogen Aα (FGA) chain identified in renal amyloidosis increase in vitro amyloidogenicity of FGA fragment.

    PubMed

    Sivalingam, Vishwanath; Patel, Basant K

    2016-08-01

    Amyloidoses are clinical disorders where deposition of β-sheet rich, misfolded protein aggregates called amyloid occurs in vital organs like brain, kidney, liver or heart etc. Aggregation of several proteins such as immunoglobulin light chain, fibrinogen Aα chain (FGA) and lysozyme have been found to be associated with renal amyloidosis. Fibrinogen amyloidosis (AFib) is predominantly familial and is associated with the deposition of mutant FGA amyloid, primarily in kidneys. Over ten substitution and frame-shift mutations in FGA have been identified from AFib patients. Whether wild-type FGA is also involved in AFib is yet unknown. The affected tissues from AFib patients usually show ∼10 kDA peptide from C-terminal 80 amino acid residues of mutant FGA. Notably, this region also encompasses all known disease-related mutations. Whether these point mutations increase the amyloidogenicity of FGA leading to disease progression, have not been studied yet. Here, we have investigated the role of two disease-related mutations in affecting amyloidogenic propensity of an FGA(496-581) fragment. We found that at physiological pH, the wild-type FGA(496-581) fragment remains monomeric, whereas its E540V mutant forms amyloid-like fibrils as observed by AFM. Also, FGA(496-581) harbouring another familial mutation, R554L, converts in vitro into globular, β-sheet rich aggregates, showing amyloid-like properties. These findings suggest that familial mutations in FGA may have role in renal amyloidosis via enhanced amyloid formation.

  16. Nickel accumulation in lung tissues is associated with increased risk of p53 mutation in lung cancer patients.

    PubMed

    Chiou, Yu-Hu; Wong, Ruey-Hong; Chao, Mu-Rong; Chen, Chih-Yi; Liou, Saou-Hsing; Lee, Huei

    2014-10-01

    Occupational exposure to nickel compounds has been associated with lung cancer. The correlation between high nickel levels and increased risk of lung cancer has been previously reported in a case-control study. This study assessed whether nickel exposure increased the occurrence of p53 mutations due to DNA repair inhibition by nickel. A total of 189 lung cancer patients were enrolled to determine nickel levels in tumor-adjacent normal lung tissues and p53 mutation status in lung tumors through atomic absorption spectrometry and direct sequencing, respectively. Nickel levels in p53 mutant patients were significantly higher than those in p53 wild-type patients. When patients were divided into high- and low-nickel subgroups by median nickel level, the high-nickel subgroup of patients had an odds ratio (OR) of 3.25 for p53 mutation risk relative to the low-nickel subgroup patients. The OR for p53 mutation risk of lifetime non-smokers, particularly females, in the high-nickel subgroup was greater than that in the low-nickel subgroup. To determine whether nickel affected DNA repair capacity, we conducted the host cell reactivation assay in A549 and H1975 lung cancer cells and showed that the DNA repair activity was reduced by nickel chloride in a dose-dependent manner. This was associated with elevated production of hydrogen peroxide-induced 8-oxo-deoxyguanosine. Therefore, increased risk of p53 mutation due to defective DNA repair caused by high nickel levels in lung tissues may be one mechanism by which nickel exposure contributes to lung cancer development, especially in lifetime female non-smokers.

  17. Recurrent HOXB13 mutations in the Dutch population do not associate with increased breast cancer risk

    PubMed Central

    Liu, Jingjing; Prager–van der Smissen, Wendy J. C.; Schmidt, Marjanka K.; Collée, J. Margriet; Cornelissen, Sten; Lamping, Roy; Nieuwlaat, Anja; Foekens, John A.; Hooning, Maartje J.; Verhoef, Senno; van den Ouweland, Ans M. W.; Hogervorst, Frans B. L.; Martens, John W. M.; Hollestelle, Antoinette

    2016-01-01

    The HOXB13 p.G84E mutation has been firmly established as a prostate cancer susceptibility allele. Although HOXB13 also plays a role in breast tumor progression, the association of HOXB13 p.G84E with breast cancer risk is less evident. Therefore, we comprehensively interrogated the entire HOXB13 coding sequence for mutations in 1,250 non-BRCA1/2 familial breast cancer cases and 800 controls. We identified two predicted deleterious missense mutations, p.G84E and p.R217C, that were recurrent among breast cancer cases and further evaluated their association with breast cancer risk in a larger study. Taken together, 4,520 familial non-BRCA1/2 breast cancer cases and 3,127 controls were genotyped including the cases and controls of the whole gene screen. The concordance rate for the genotyping assays compared with Sanger sequencing was 100%. The prostate cancer risk allele p.G84E was identified in 18 (0.56%) of 3,187 cases and 16 (0.70%) of 2,300 controls (OR = 0.81, 95% CI = 0.41–1.59, P = 0.54). Additionally, p.R217C was identified in 10 (0.31%) of 3,208 cases and 2 (0.087%) of 2,288 controls (OR = 3.57, 95% CI = 0.76–33.57, P = 0.14). These results imply that none of the recurrent HOXB13 mutations in the Dutch population are associated with breast cancer risk, although it may be worthwhile to evaluate p.R217C in a larger study. PMID:27424772

  18. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  19. ARID5B polymorphism confers an increased risk to acquire specific MLL rearrangements in early childhood leukemia

    PubMed Central

    2014-01-01

    Background Acute leukemia in early age (EAL) is characterized by acquired genetic alterations such as MLL rearrangements (MLL-r). The aim of this case-controlled study was to investigate whether single nucleotide polymorphisms (SNPs) of IKZF1, ARID5B, and CEBPE could be related to the onset of EAL cases (<24 months-old at diagnosis). Methods The SNPs (IKZF1 rs11978267, ARID5B rs10821936 and rs10994982, CEBPE rs2239633) were genotyped in 265 cases [169 acute lymphoblastic leukemia (ALL) and 96 acute myeloid leukaemia (AML)] and 505 controls by Taqman allelic discrimination assay. Logistic regression was used to evaluate the association between SNPs of cases and controls, adjusted on skin color and/or age. The risk was determined by calculating odds ratios (ORs) with 95% confidence interval (CI). Results Children with the IKZF1 SNP had an increased risk of developing MLL-germline ALL in white children. The heterozygous/mutant genotype in ARID5B rs10994982 significantly increased the risk for MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively). The heterozygous genotype in ARID5B rs10821936 increased the risk for MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). Furthermore, ARID5B rs10821936 conferred increased risk for MLL-MLLT3 positive cases (OR 7.10, 95% CI:1.54-32.68). Our data do not show evidence that CEBPE rs2239633 confers increased genetic susceptibility to EAL. Conclusions IKZF1 and CEBPE variants seem to play a minor role in genetic susceptibility to EAL, while ARID5B rs10821936 increased the risk of MLL-MLLT3. This result shows that genetic susceptibility could be associated with the differences regarding MLL breakpoints and partner genes. PMID:24564228

  20. Hydrogen Peroxide Removes TRPM4 Current Desensitization Conferring Increased Vulnerability to Necrotic Cell Death*

    PubMed Central

    Simon, Felipe; Leiva-Salcedo, Elías; Armisén, Ricardo; Riveros, Ana; Cerda, Oscar; Varela, Diego; Eguiguren, Ana Luisa; Olivero, Pablo; Stutzin, Andrés

    2010-01-01

    Necrosis is associated with an increase in plasma membrane permeability, cell swelling, and loss of membrane integrity with subsequent release of cytoplasmic constituents. Severe redox imbalance by overproduction of reactive oxygen species is one of the main causes of necrosis. Here we demonstrate that H2O2 induces a sustained activity of TRPM4, a Ca2+-activated, Ca2+-impermeant nonselective cation channel resulting in an increased vulnerability to cell death. In HEK 293 cells overexpressing TRPM4, H2O2 was found to eliminate in a dose-dependent manner TRPM4 desensitization. Site-directed mutagenesis experiments revealed that the Cys1093 residue is crucial for the H2O2-mediated loss of desensitization. In HeLa cells, which endogenously express TRPM4, H2O2 elicited necrosis as well as apoptosis. H2O2-mediated necrosis but not apoptosis was abolished by replacement of external Na+ ions with sucrose or the non-permeant cation N-methyl-d-glucamine and by knocking down TRPM4 with a shRNA directed against TRPM4. Conversely, transient overexpression of TRPM4 in HeLa cells in which TRPM4 was previously silenced re-established vulnerability to H2O2-induced necrotic cell death. In addition, HeLa cells exposed to H2O2 displayed an irreversible loss of membrane potential, which was prevented by TRPM4 knockdown. PMID:20884614

  1. Suppression of Phospholipase Dγs Confers Increased Aluminum Resistance in Arabidopsis thaliana

    PubMed Central

    Zhao, Jian; Wang, Cunxi; Bedair, Mohamed; Welti, Ruth; W. Sumner, Lloyd; Baxter, Ivan; Wang, Xuemin

    2011-01-01

    Aluminum (Al) toxicity is the major stress in acidic soil that comprises about 50% of the world's arable land. The complex molecular mechanisms of Al toxicity have yet to be fully determined. As a barrier to Al entrance, plant cell membranes play essential roles in plant interaction with Al, and lipid composition and membrane integrity change significantly under Al stress. Here, we show that phospholipase Dγs (PLDγs) are induced by Al stress and contribute to Al-induced membrane lipid alterations. RNAi suppression of PLDγ resulted in a decrease in both PLDγ1 and PLDγ2 expression and an increase in Al resistance. Genetic disruption of PLDγ1 also led to an increased tolerance to Al while knockout of PLDγ2 did not. Both RNAi-suppressed and pldγ1-1 mutants displayed better root growth than wild-type under Al stress conditions, and PLDγ1-deficient plants had less accumulation of callose, less oxidative damage, and less lipid peroxidation compared to wild-type plants. Most phospholipids and glycolipids were altered in response to Al treatment of wild-type plants, whereas fewer changes in lipids occurred in response to Al stress in PLDγ mutant lines. Our results suggest that PLDγs play a role in membrane lipid modulation under Al stress and that high activities of PLDγs negatively modulate plant tolerance to Al. PMID:22163277

  2. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

    SciTech Connect

    Biedermann, K.A.; Sun, J.R.; Giaccia, A.J.; Tosto, L.M.; Brown, J.M. )

    1991-02-15

    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.

  3. CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model

    PubMed Central

    Wert, Katherine J.; Bassuk, Alexander G.; Wu, Wen-Hsuan; Gakhar, Lokesh; Coglan, Diana; Mahajan, MaryAnn; Wu, Shu; Yang, Jing; Lin, Chyuan-Sheng; Tsang, Stephen H.; Mahajan, Vinit B.

    2015-01-01

    A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5R243L only in the retina. The resulting hCAPN5R243L transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5R243L mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5R243L mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients. PMID:25994508

  4. Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins

    PubMed Central

    Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

    2014-01-01

    In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. PMID:24942546

  5. Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

    PubMed

    Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

    2014-08-01

    In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  6. Morphine Increases Lamivudine- and Nevirapine-Induced Human Immunodeficiency Virus-1 Drug-Resistant Mutations In Vitro.

    PubMed

    Liang, Bingyu; Jiang, Junjun; Pan, Peijiang; Chen, Rongfeng; Zhuang, Daomin; Zhao, Fangning; Chen, Hui; Huang, Jiegang; Su, Qijian; Cao, Cunwei; Li, Jingyun; Liang, Hao; Ye, Li

    2017-04-01

    Epidemiological studies have demonstrated that the human immunodeficiency virus (HIV)-1 drug-resistant rate among injecting drug users is higher than that in other HIV-1-positive populations, which is generally believed to be largely due to clinical nonadherence. Little is known, however, about whether heroin abuse has a direct impact on the generation of HIV-1 drug-resistant mutations. In this study, we investigated the impacts of morphine, the active metabolite of heroin, on HIV-1 infection/replication and HIV-1 drug-resistant mutations through an in vitro HIV-1-CD4(+) T cell system under selective pressure from two typical antiviral drugs, Lamivudine and Nevirapine. We found that morphine treatment of MT4 cells (a CD4(+) T cell line) significantly increased HIV-1 III B (a T-tropic viral strain) infection and replication in MT4 cells, and the effect of morphine on HIV-1 was mediated through an opioid receptor. More importantly, our results showed that morphine treatment not only induced more drug-resistant mutations under selective pressure from antiretroviral drugs but also shortened the mutations' generation time, compared with the control groups that were treated with antiretroviral drugs alone. Although the in vivo relevance remains to be determined, these findings provide direct in vitro evidence to support the possibility that heroin abuse itself can act as an independent factor contributing to the generation of HIV-1 drug resistance during clinical antiretroviral therapy. Therapeutic guidelines should consider this issue for heroin users with HIV infection.

  7. Subclinical Nonautoimmune Hyperthyroidism in a Family Segregates with a Thyrotropin Receptor Mutation with Weakly Increased Constitutive Activity

    PubMed Central

    Chen, Chun-Rong; Higashiyama, Takuya; Mizutori-Sasai, Yumiko; Ito, Mitsuru; Kubota, Sumihisa; Amino, Nobuyuki; Miyauchi, Akira; Rapoport, Basil

    2010-01-01

    Background Subclinical hyperthyroidism is usually associated with Graves' disease or toxic nodular goiter. Here we report a family with hereditary subclinical hyperthyroidism caused by a constitutively activating germline mutation of the thyrotropin receptor (TSHR) gene. Methods The proband was a 64-year-old Japanese woman who presented with a thyroid nodule and was found to be euthyroid with a suppressed serum TSH. The nodule was not hot. Although antibodies to thyroid peroxidase and thyroglobulin antibodies were present, TSHR antibodies were not detected by TSH-binding inhibition or by bioassay. Two of her middle-aged sons, but not her daughter, also had subclinical hyperthyroidism without TSHR antibodies. Without therapy, the clinical condition of the affected individuals remained unchanged over 3 years without development of overt hyperthyroidism. Results A novel heterozygous TSHR point mutation causing a glutamic acid to lysine substitution at codon 575 (E575K) in the second extracellular loop was detected in the three family members with subclinical hyperthyroidism, but was absent in her one daughter with normal thyroid function. In vitro functional studies of the E575K TSHR mutation demonstrated a weak, but significant, increase in constitutive activation of the cAMP pathway. Conclusion Although hereditary nonautoimmune overt hyperthyroidism is very rare, TSHR activating mutations as a cause of subclinical hyperthyroidism may be more common and should be considered in the differential diagnosis, especially if familial. PMID:20929407

  8. Oxidative Stress Increases the Blood Brain Barrier Permeability Resulting in Increased Incidence of Brain Metastasis in BRCA Mutation Carriers

    DTIC Science & Technology

    2012-02-01

    to HBMEC showed increase in ROS levels as compared to control, and this increased in ROS formation was abrogated by the antioxidant uric acid , UA...in HBMEC permeability was observed by ROS and these changes were inhibited in the presence of UA antioxidant, uric acid , indicating the involvement

  9. Marked increase in biofilm-derived rough pneumococcal variants and rifampin-resistant strains not due to hex gene mutations.

    PubMed

    McEllistrem, M Catherine; Scott, Jennifer R; Zuniga-Castillo, Jacobo; Khan, Saleem A

    2009-06-01

    Otitis, pneumonia, and meningitis are tissue-based pneumococcal infections that can be associated with biofilms. The emergence of phenotypic rough variants, also known as acapsular small-colony variants, is essential for pneumococcal biofilm formation. These rough variants can increase nearly 100-fold in biofilms over time and can arise through single nucleotide polymorphisms (SNPs), deletions, or tandem duplications in the first gene of the capsular operon, cps3D. We detected a 100-fold increase in rifampin-resistant (Rif(r)) mutants in biofilms compared to planktonic cultures using a nonvaccine serotype 3 strain, which is causing an increasing number of cases of otitis in the 7-valent pneumococcal conjugate vaccine era. Since both rough variants and Rif(r) strains can arise through SNPs, they could emerge due to alteration of the mismatch repair (MMR) system. The Hex system, a pneumococcal MMR system, repairs mismatches during replication and transformation. In this study, no mutations were detected in the hexAB gene sequences among several rough variants with unique mutations in the cps3D gene. Within a hexA null mutant grown in broth, we detected only a 17.5-fold increase in rough variants compared to the wild-type parental strain. Taken together, these data suggest that mutations in the hex genes and modulation of hexA activity are unlikely to account for the generation of biofilm-derived rough variants.

  10. Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis▿

    PubMed Central

    Venkatesan, Ranga N.; Treuting, Piper M.; Fuller, Evan D.; Goldsby, Robert E.; Norwood, Thomas H.; Gooley, Ted A.; Ladiges, Warren C.; Preston, Bradley D.; Loeb, Lawrence A.

    2007-01-01

    Mammalian DNA polymerase δ (Pol δ) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol δ. Homozygous Pold1L604G/L604G and Pold1L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1+/L604G mice did not differ, while that of Pold1+/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1+/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase δ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer. PMID:17785453

  11. Ankylosing spondylitis confers substantially increased risk of clinical spine fractures: a nationwide case-control study.

    PubMed

    Prieto-Alhambra, D; Muñoz-Ortego, J; De Vries, F; Vosse, D; Arden, N K; Bowness, P; Cooper, C; Diez-Perez, A; Vestergaard, P

    2015-01-01

    Ankylosing spondylitis (AS) leads to osteopenia/osteoporosis and spine rigidity. We conducted a case-control study and found that AS-affected patients have a 5-fold and 50% increased risk of clinical spine and all clinical fractures, respectively. Excess risk of both is highest in the first years and warrants an early bone health assessment after diagnosis. Ankylosing spondylitis (AS) is related to spine rigidity and reduced bone mass, but data on its impact on fracture risk are scarce. We aimed to study the association between AS and clinical fractures using a case-control design. From the Danish Health Registries, we identified all subjects who sustained a fracture in the year 2000 (cases) and matched up to three controls by year of birth, gender and region. Clinically diagnosed AS was identified using International Classification of Diseases, 8th revision (ICD-8; 71249), and International Classification of Diseases, 10th revision (ICD-10; M45) codes. We also studied the impact of AS duration. Conditional logistic regression was used to estimate crude and adjusted odds ratios (ORs) for non-traumatic fractures (any site, clinical spine and non-vertebral) according to AS status and time since AS diagnosis. Multivariate models were adjusted for fracture history, socio-economic status, previous medical consultations, alcoholism and use of oral glucocorticoids. We identified 139/124,655 (0.11%) AS fracture cases, compared to 271/373,962 (0.07%) AS controls. Unadjusted (age- and gender-matched) odds ratio (OR) were 1.54 [95% confidence interval (95%CI) 1.26-1.89] for any fracture, 5.42 [2.50-11.70] for spine and 1.39 [1.12-1.73] for non-vertebral fracture. The risk peaked in the first 2.5 years following AS diagnosis: OR 2.69 [1.84-3.92] for any fracture. Patients with AS have a 5-fold higher risk of clinical spine fracture and a 35% increased risk of non-vertebral fracture. This excess risk peaks early, in the first 2.5 years of AS disease. Patients should be assessed

  12. Resistance to the Novel Fungicide Pyrimorph in Phytophthora capsici: Risk Assessment and Detection of Point Mutations in CesA3 That Confer Resistance

    PubMed Central

    Pang, Zhili; Shao, Jingpeng; Chen, Lei; Lu, Xiaohong; Hu, Jian; Qin, Zhaohai; Liu, Xili

    2013-01-01

    Pyrimorph is a novel fungicide with high activity against the plant pathogen Phytophthora capsici. We investigated the risk that P. capsici can develop resistance to pyrimorph. The baseline sensitivities of 226 P. capsici isolates, tested by mycelial growth inhibition, showed a unimodal distribution with a mean EC50 value of 1.4261 (±0.4002) µg/ml. Twelve pyrimorph-resistant mutants were obtained by repeated exposure to pyrimorph in vitro with a frequency of approximately 1×10−4. The resistance factors of the mutants ranged from 10.67 to 56.02. Pyrimorph resistance of the mutants was stable after 10 transfers on pyrimorph-free medium. Fitness in sporulation, cystospore germination, and pathogenicity in the pyrimorph-resistant mutants was similar to or less than that in the parental wild-type isolates. On detached pepper leaves and pepper plants treated with the recommended maximum dose of pyrimorph, however, virulence was greater for mutants with a high level of pyrimorph resistance than for the wild type. The results suggest that the risk of P. capsici developing resistance to pyrimorph is low to moderate. Among mutants with a high level of pyrimorph resistance, EC50 values for pyrimorph and CAA fungicides flumorph, dimethomorph, and mandipropamid were positively correlated. This indicated that point mutations in cellulose synthase 3 (CesA3) may confer resistance to pyrimorph. Comparison of CesA3 in isolates with a high level of pyrimorph resistance and parental isolates showed that an amino acid change from glutamine to lysine at position 1077 resulted in stable, high resistance in the mutants. Based on the point mutations, an allele-specific PCR method was developed to detect pyrimorph resistance in P. capsici populations. PMID:23431382

  13. Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels*

    PubMed Central

    Tang, Qiong-Yao; Larry, Trevor; Hendra, Kalen; Yamamoto, Erica; Bell, Jessica; Cui, Meng; Logothetis, Diomedes E.; Boland, Linda M.

    2015-01-01

    All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81–104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404–404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification. PMID:25957411

  14. Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification.

    PubMed

    Steain, Megan C; Dwyer, Dominic E; Hurt, Aeron C; Kol, Chenda; Saksena, Nitin K; Cunningham, Anthony L; Wang, Bin

    2009-12-01

    In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.

  15. Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

    PubMed

    Lall, Rahul K; Syed, Deeba N; Khan, Mohammad Imran; Adhami, Vaqar M; Gong, Yuansheng; Lucey, John A; Mukhtar, Hasan

    2016-09-01

    We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Powdery Mildew Resistance Conferred by Loss of the ENHANCED DISEASE RESISTANCE1 Protein Kinase Is Suppressed by a Missense Mutation in KEEP ON GOING, a Regulator of Abscisic Acid Signaling1[W][OA

    PubMed Central

    Wawrzynska, Anna; Christiansen, Katy M.; Lan, Yinan; Rodibaugh, Natalie L.; Innes, Roger W.

    2008-01-01

    Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling. PMID:18815384

  17. Powdery mildew resistance conferred by loss of the ENHANCED DISEASE RESISTANCE1 protein kinase is suppressed by a missense mutation in KEEP ON GOING, a regulator of abscisic acid signaling.

    PubMed

    Wawrzynska, Anna; Christiansen, Katy M; Lan, Yinan; Rodibaugh, Natalie L; Innes, Roger W

    2008-11-01

    Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling.

  18. Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons.

    PubMed

    Gopal, Pallavi P; Nirschl, Jeffrey J; Klinman, Eva; Holzbaur, Erika L F

    2017-03-21

    Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.

  19. Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons

    PubMed Central

    Gopal, Pallavi P.; Nirschl, Jeffrey J.; Holzbaur, Erika L. F.

    2017-01-01

    Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid–liquid phase separation and liquid-like non–membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation. PMID:28265061

  20. Patient Participation at Health Care Conferences: Engaged Patients Increase Information Flow, Expand Propagation, and Deepen Engagement in the Conversation of Tweets Compared to Physicians or Researchers.

    PubMed

    Utengen, Audun; Rouholiman, Dara; Gamble, Jamison G; Grajales Iii, Francisco Jose; Pradhan, Nisha; Staley, Alicia C; Bernstein, Liza; Young, Sean D; Clauson, Kevin A; Chu, Larry F

    2017-08-17

    Health care conferences present a unique opportunity to network, spark innovation, and disseminate novel information to a large audience, but the dissemination of information typically stays within very specific networks. Social network analysis can be adopted to understand the flow of information between virtual social communities and the role of patients within the network. The purpose of this study is to examine the impact engaged patients bring to health care conference social media information flow and how they expand dissemination and distribution of tweets compared to other health care conference stakeholders such as physicians and researchers. From January 2014 through December 2016, 7,644,549 tweets were analyzed from 1672 health care conferences with at least 1000 tweets who had registered in Symplur's Health Care Hashtag Project from 2014 to 2016. The tweet content was analyzed to create a list of the top 100 influencers by mention from each conference, who were then subsequently categorized by stakeholder group. Multivariate linear regression models were created using stepwise function building to identify factors explaining variability as predictor variables for the model in which conference tweets were taken as the dependent variable. Inclusion of engaged patients in health care conference social media was low compared to that of physicians and has not significantly changed over the last 3 years. When engaged patient voices are included in health care conferences, they greatly increase information flow as measured by total tweet volume (beta=301.6) compared to physicians (beta=137.3, P<.001), expand propagation of information tweeted during a conference as measured by social media impressions created (beta=1,700,000) compared to physicians (beta=270,000, P<.001), and deepen engagement in the tweet conversation as measured by replies to their tweets (beta=24.4) compared to physicians (beta=5.5, P<.001). Social network analysis of hubs and authorities

  1. Increased mutant frequency and altered mutation spectrum of the lacI transgene in Wilson disease rats with hepatitis.

    PubMed

    Sone, H; Li, Y J; Ishizuka, M; Aoki, Y; Nagao, M

    2000-09-15

    The mutant strain Long-Evans Cinnamon (LEC) rat, which accumulates copper in the liver because of a mutation in the Atp7b gene, encoding a copper-ATPase, is a model of Wilson disease. It spontaneously develops hepatitis, and subsequently hepatocellular carcinoma and cholangiofibrosis. Excess intracellular copper has been thought to induce DNA damage through reactive oxygen species produced by Cu (II)/Cu (I) redox cycling, and also by direct interaction with DNA. We have developed lacI transgenic Wilson disease (WND-B) rats by mating LEC with Big Blue F344 rats carrying a lambda shuttle vector harboring the lacI gene. lacI mutations of the livers of C-B heterozygous (Atp7b w/m, lacI) and WND-B homozygous (Atp7b m/m, lacI) rats at 6, 24, and 40 weeks of ages were analyzed. Mutant frequencies in the WND-B rats were 2.0 +/- 0.7 x 10(-5), 5.3 +/- 0.9 x 10(-5), and 5.3 +/- 1.0 x 10(-5), respectively, significantly higher than those of C-B rats. Nucleotide sequence analysis revealed that the frequency of deletion mutations of more than two nucleotides were much higher, 15% in WND-B rats, but only 2% in C-B rats. In addition, the average size of deletion was larger in the former. Loss of oligonucleotide-repeat units was specific and relatively frequent in WND-B rats. This type of mutation might be implicated in the induction of DNA strand scissions by reactive oxygen species. These findings suggest that the increase in mutant frequencies and/or the specific type of mutation according to copper accumulation play a crucial role in hepatocarcinogenesis in LEC rats.

  2. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia.

    PubMed

    Minson, Katherine A; Smith, Catherine C; DeRyckere, Deborah; Libbrecht, Clara; Lee-Sherick, Alisa B; Huey, Madeline G; Lasater, Elisabeth A; Kirkpatrick, Gregory D; Stashko, Michael A; Zhang, Weihe; Jordan, Craig T; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V; Earp, H Shelton; Shah, Neil P; Graham, Douglas K

    2016-03-01

    FMS-like tyrosine kinase 3-targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD-mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

  3. The MERTK/FLT3 inhibitor MRX-2843 overcomes resistance-conferring FLT3 mutations in acute myeloid leukemia

    PubMed Central

    Minson, Katherine A.; Smith, Catherine C.; Libbrecht, Clara; Lee-Sherick, Alisa B.; Huey, Madeline G.; Lasater, Elisabeth A.; Kirkpatrick, Gregory D.; Stashko, Michael A.; Zhang, Weihe; Jordan, Craig T.; Kireev, Dmitri; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton; Shah, Neil P.; Graham, Douglas K.

    2016-01-01

    FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML. PMID:27158668

  4. Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

    PubMed Central

    Eshaghi, Alireza; Shalhoub, Sarah; Rosenfeld, Paul; Li, Aimin; Higgins, Rachel R.; Stogios, Peter J.; Savchenko, Alexei; Bastien, Nathalie; Li, Yan; Rotstein, Coleman

    2014-01-01

    Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission. PMID:25246391

  5. Mineral particle size in children with osteogenesis imperfecta type I is not increased independently of specific collagen mutations.

    PubMed

    Fratzl-Zelman, Nadja; Schmidt, Ingo; Roschger, Paul; Glorieux, Francis H; Klaushofer, Klaus; Fratzl, Peter; Rauch, Frank; Wagermaier, Wolfgang

    2014-03-01

    Osteogenesis imperfecta (OI) type I represents the mildest form of OI and is usually caused by two classes of autosomal dominant mutations in collagen type I: haploinsufficiency leading to a reduced quantity of structurally normal collagen (quantitative mutation), or sequence abnormalities generating structurally aberrant collagen chains (qualitative mutation). An abnormally high bone matrix mineralization has been observed in all OI cases investigated so far, independently of mutation type. This raises the question whether the increased amount of mineral is due to mineral particles growing to larger sizes or to a higher number of more densely packed particles. For this reason, we revisit the problem by investigating the mineral particle size in cancellous bone from two subsets of the previously analyzed biopsies (patient's age: 2-4.2 and 7.6-11years) comparing OI quantitative mutations (n=5), OI qualitative mutations (n=5) and controls (n=6). We used a combined small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) setup with a beam diameter of 10μm of synchrotron radiation, which allows the determination of mineral particle characteristics in 10μm thick sections at the same positions where the matrix mineralization density was previously determined. The thickness parameter of mineral particles (T) was obtained from SAXS data and the mineral volume fraction was calculated from the mean calcium content of the bone matrix determined by quantitative back-scattered electron imaging (qBEI). The combination of these two quantities allowed calculating the true particle width (W) of the plate-like mineral crystals. T was larger in the older than in the younger age-group independently of genotype (p<0.004) and was larger in the controls than in each OI group. The qBEI results showed that the mineral volume fraction increased from 32.45wt.% in controls to 36.44wt.% in both OI groups (corresponding to a 12% increase in relative terms). Combining these

  6. Caveolin-1 (P132L), a common breast cancer mutation, confers mammary cell invasiveness and defines a novel stem cell/metastasis-associated gene signature.

    PubMed

    Bonuccelli, Gloria; Casimiro, Mathew C; Sotgia, Federica; Wang, Chenguang; Liu, Manran; Katiyar, Sanjay; Zhou, Jie; Dew, Elliott; Capozza, Franco; Daumer, Kristin M; Minetti, Carlo; Milliman, Janet N; Alpy, Fabien; Rio, Marie-Christine; Tomasetto, Catherine; Mercier, Isabelle; Flomenberg, Neal; Frank, Philippe G; Pestell, Richard G; Lisanti, Michael P

    2009-05-01

    Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-alpha as predicted, because only estrogen receptor-alpha-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis.

  7. Caveolin-1 (P132L), a Common Breast Cancer Mutation, Confers Mammary Cell Invasiveness and Defines a Novel Stem Cell/Metastasis-Associated Gene Signature

    PubMed Central

    Bonuccelli, Gloria; Casimiro, Mathew C.; Sotgia, Federica; Wang, Chenguang; Liu, Manran; Katiyar, Sanjay; Zhou, Jie; Dew, Elliott; Capozza, Franco; Daumer, Kristin M.; Minetti, Carlo; Milliman, Janet N.; Alpy, Fabien; Rio, Marie-Christine; Tomasetto, Catherine; Mercier, Isabelle; Flomenberg, Neal; Frank, Philippe G.; Pestell, Richard G.; Lisanti, Michael P.

    2009-01-01

    Here we used the Met-1 cell line in an orthotopic transplantation model in FVB/N mice to dissect the role of the Cav-1(P132L) mutation in human breast cancer. Identical experiments were performed in parallel with wild-type Cav-1. Cav-1(P132L) up-regulated the expression of estrogen receptor-α as predicted, because only estrogen receptor-α-positive patients have been shown to harbor Cav-1(P132L) mutations. In the context of primary tumor formation, Cav-1(P132L) behaved as a loss-of-function mutation, lacking any tumor suppressor activity. In contrast, Cav-1(P132L) caused significant increases in cell migration, invasion, and experimental metastasis, consistent with a gain-of-function mutation. To identify possible molecular mechanism(s) underlying this invasive gain-of-function activity, we performed unbiased gene expression profiling. From this analysis, we show that the Cav-1(P132L) expression signature contains numerous genes that have been previously associated with cell migration, invasion, and metastasis. These include i) secreted growth factors and extracellular matrix proteins (Cyr61, Plf, Pthlh, Serpinb5, Tnc, and Wnt10a), ii) proteases that generate EGF and HGF (Adamts1 and St14), and iii) tyrosine kinase substrates and integrin signaling/adapter proteins (Akap13, Cdcp1, Ddef1, Eps15, Foxf1a, Gab2, Hs2st1, and Itgb4). Several of the P132L-specific genes are also highly expressed in stem/progenitor cells or are associated with myoepithelial cells, suggestive of an epithelial-mesenchymal transition. These results directly support clinical data showing that patients harboring Cav-1 mutations are more likely to undergo recurrence and metastasis. PMID:19395651

  8. The G1138A mutation rate in the fibroblast growth factor receptor 3 (FGFR3) gene is increased in cells carrying the t (4; 14) translocation.

    PubMed

    Reddy, P L; Grewal, R P

    2009-04-22

    Spontaneous mutations are a common phenomenon, occurring in both germ-line and somatic genomes. They may have deleterious consequences including the development of genetic disorders or, when occurring in somatic tissues, may participate in the process of carcinogenesis. Similar to many mutational hotspots, the G1138A mutation in the fibroblast growth factor receptor 3 (FGFR3) gene occurs at a CpG site. In germ-line tissues, the G1138A mutation results in achondroplasia and has one of the highest spontaneous mutation rates in the human genome. Although not at the G1138A site, there are increased rates of other somatic mutations in the FGFR3 gene that have been reported in multiple myeloma cases associated with a translocation, t (4; 14). The chromosome-4 break points in this translocation are clustered in a 70-kb region centromeric to the FGFR3 gene. We hypothesized that this translocation may impact the mutation rate at the G1138A site. We employed a semi-quantitative polymerase chain reaction-based assay to measure the frequency of this mutation in multiple myeloma cell lines carrying t (4; 14) translocation. Analysis of these cell lines varied from no change to a 10-fold increase in the mutation frequency compared with normal controls. In general, there was an increase in the G1138A mutational frequency suggesting that chromosomal rearrangement can affect the stability of the CpG hotspots.

  9. LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

    PubMed

    Tomoyasu, Toshifumi; Imaki, Hidenori; Masuda, Sachiko; Okamoto, Ayumi; Kim, Hyejin; Waite, Richard D; Whiley, Robert A; Kikuchi, Ken; Hiramatsu, Keiichi; Tabata, Atsushi; Nagamune, Hideaki

    2013-09-01

    Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

  10. Dominantly inherited myotonia congenita resulting from a mutation that increases open probability of the muscle chloride channel CLC-1.

    PubMed

    Richman, David P; Yu, Yawei; Lee, Ting-Ting; Tseng, Pang-Yen; Yu, Wei-Ping; Maselli, Ricardo A; Tang, Chih-Yung; Chen, Tsung-Yu

    2012-12-01

    Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (P (o)) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common-gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased P (o) of the channel's fast-gate, from ~0.4 in the wild type to >0.9 in the mutant at -90 mV. In contrast, the mutant exhibits a minimal effect on the P (o) of the common-gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast-gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a fivefold increase in chloride conductance in the wild type to a twofold increase in the mutant-this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.

  11. The TMEM43 Newfoundland mutation p.S358L causing ARVC-5 was imported from Europe and increases the stiffness of the cell nucleus.

    PubMed

    Milting, Hendrik; Klauke, Bärbel; Christensen, Alex Hoerby; Müsebeck, Jörg; Walhorn, Volker; Grannemann, Sören; Münnich, Tamara; Šarić, Tomo; Rasmussen, Torsten Bloch; Jensen, Henrik Kjærulf; Mogensen, Jens; Baecker, Carolin; Romaker, Elena; Laser, Kai Thorsten; zu Knyphausen, Edzard; Kassner, Astrid; Gummert, Jan; Judge, Daniel P; Connors, Sean; Hodgkinson, Kathy; Young, Terry-L; van der Zwaag, Paul A; van Tintelen, J Peter; Anselmetti, Dario

    2015-04-07

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic condition caused predominantly by mutations within desmosomal genes. The mutation leading to ARVC-5 was recently identified on the island of Newfoundland and caused by the fully penetrant missense mutation p.S358L in TMEM43. Although TMEM43-p.S358L mutation carriers were also found in the USA, Germany, and Denmark, the genetic relationship between North American and European patients and the disease mechanism of this mutation remained to be clarified. We screened 22 unrelated ARVC patients without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43-p.S358L founder mutation. Due to its deleterious clinical phenotype, this mutation should be checked in any case of ARVC-related genotyping. It appears that the increased stiffness of the cell nucleus might be related to the massive loss of cardiomyocytes, which is typically found in ventricles of ARVC hearts. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  12. A Novel Mutation p.A59P in N-Terminal Domain of Methyl-CpG-Binding Protein 2 Confers Phenotypic Variability in 3 Cases of Tunisian Rett Patients: Clinical Evaluations and In Silico Investigations.

    PubMed

    Kharrat, Marwa; Hsairi, Ines; Fendri-Kriaa, Nourhene; Kenoun, Houda; Othmen, Houda Ben; Ben Mahmoud, Afif; Ghorbel, Rania; Abid, Imen; Triki, Chahnez; Fakhfakh, Faiza

    2015-11-01

    Rett syndrome is a monogenic X-linked dominant neurodevelopmental disorder related to mutation in MECP2, which encodes the methyl-CpG-binding protein MeCP2. The aim of this study was to search for mutations of MECP2 gene in Tunisian Rett patients and to evaluate the impact of the found variants on structural and functional features of MeCP2. The result of mutation analysis revealed that 3 Rett patients shared the same novel heterozygous point mutation c.175G>C (p.A59P). The p.A59P mutation was located in a conserved amino acid in the N-terminal segment of MeCP2. This novel mutation confers a phenotypic variability with different clinical severity scores (3, 8, and 9) and predicted by Sift and PolyPhen to be damaging. Modeling results showed that p.A59P adds 2 hydrogen bonds and changes the structural conformation of MeCP2 with a significant root mean square deviation value (9.66 Å), suggesting that this mutation could probably affect the conformation, function and stability of MeCP2.

  13. Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6-N-Glycosylation and Increased Susceptibility to Galectin-3 Binding

    PubMed Central

    Priglinger, Claudia S.; Obermann, Jara; Szober, Christoph M.; Merl-Pham, Juliane; Ohmayer, Uli; Behler, Jennifer; Gruhn, Fabian; Kreutzer, Thomas C.; Wertheimer, Christian; Geerlof, Arie; Priglinger, Siegfried G.; Hauck, Stefanie M.

    2016-01-01

    Epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells is a crucial event in the onset of proliferative vitreoretinopathy (PVR), the most common reason for treatment failure in retinal detachment surgery. We studied alterations in the cell surface glycan expression profile upon EMT of RPE cells and focused on its relevance for the interaction with galectin-3 (Gal-3), a carbohydrate binding protein, which can inhibit attachment and spreading of human RPE cells in a dose- and carbohydrate-dependent manner, and thus bares the potential to counteract PVR-associated cellular events. Lectin blot analysis revealed that EMT of RPE cells in vitro confers a glycomic shift towards an abundance of Thomsen-Friedenreich antigen, poly-N-acetyllactosamine chains, and complex-type branched N-glycans. Using inhibitors of glycosylation we found that both, binding of Gal-3 to the RPE cell surface and Gal-3-mediated inhibition of RPE attachment and spreading, strongly depend on the interaction of Gal-3 with tri- or tetra-antennary complex type N-glycans and sialylation of glycans but not on complex-type O-glycans. Importantly, we found that β1,6 N-acetylglucosaminyltransferase V (Mgat5), the key enzyme catalyzing the synthesis of tetra- or tri-antennary complex type N-glycans, is increased upon EMT of RPE cells. Silencing of Mgat5 by siRNA and CRISPR-Cas9 genome editing resulted in reduced Gal-3 binding. We conclude from these data that binding of recombinant Gal-3 to the RPE cell surface and inhibitory effects on RPE attachment and spreading largely dependent on interaction with Mgat5 modified N-glycans, which are more abundant on dedifferentiated than on the healthy, native RPE cells. Based on these findings we hypothesize that EMT of RPE cells in vitro confers glycomic changes, which account for high affinity binding of recombinant Gal-3, particularly to the cell surface of myofibroblastic RPE. From a future perspective recombinant Gal-3 may disclose a

  14. Targeted Mutation of the Gene for Cellular Glutathione Peroxidase (Gpx1) Increases Noise-Induced Hearing Loss in Mice

    PubMed Central

    McFadden, Sandra L.; Ding, Da-Lian; Lear, Patricia M.; Ho, Ye-Shih

    2000-01-01

    Reactive oxygen species (ROS) and oxidative stress have been implicated in cochlear injury following loud noise and ototoxins. Genetic mutations that impair antioxidant defenses would be expected to increase cochlear injury following acute insults and to contribute to cumulative injury that presents as age-related hearing loss. We examined whether genetically based deficiency of cellular glutathione peroxidase, a major antioxidant enzyme, increases noise-induced hearing loss in mice. Two-month-old "knockout" mice with a targeted inactivating mutation of the gene coding for glutathione peroxidase (Gpx1) and wild type controls were exposed to broadband noise for one hour at 110 dB SPL. Auditory brainstem response (ABR) thresholds at test frequencies ranging from 5 to 40 kHz were obtained two and four weeks after exposure to determine the stable permanent component of the hearing loss. Depending on test frequency, Gpx1 knockout mice showed up to 16 dB higher ABR thresholds prior to noise exposure, and up to 15 dB greater noise-induced hearing loss, compared with controls. Within the cochlear base, there was also a significant contribution of the knockout to inner and outer hair cell loss, as well as nerve fiber loss. Our results support a link between genetic impairment of antioxidant defenses, vulnerability of the cochlea injury, and cochlear degeneration. Such impairment produces characteristics expected of some mutations associated with age-related hearing loss and offers one possible mechanism for their action. PMID:11545230

  15. Kras mutations increase telomerase activity and targeting telomerase is a promising therapeutic strategy for Kras-mutant NSCLC

    PubMed Central

    Shi, Bowen; Zhang, Lianmin; Qian, Dong; Li, Chenguang; Zhang, Hua; Wang, Shengguang; Zhu, Jinfang; Gao, Liuwei; Zhang, Qiang; Jia, Bin; Hao, Ligang; Wang, Changli; Zhang, Bin

    2017-01-01

    As shortened telomeres inhibit tumor formation and prolong life span in a KrasG12D mouse lung cancer model, we investigated the implications of telomerase in Kras-mutant NSCLC. We found that Kras mutations increased TERT (telomerase reverse transcriptase) mRNA expression and telomerase activity and telomere length in both immortalized bronchial epithelial cells (BEAS-2B) and lung adenocarcinoma cells (Calu-3). MEK inhibition led to reduced TERT expression and telomerase activity. Furthermore, telomerase inhibitor BIBR1532 shortened telomere length and inhibited mutant Kras-induced long-term proliferation, colony formation and migration capabilities of BEAS-2B and Calu-3 cells. Importantly, BIBR1532 sensitized oncogenic Kras expressing Calu-3 cells to chemotherapeutic agents. The Calu-3-KrasG12D xenograft mouse model confirmed that BIBR1532 enhanced the antitumor efficacy of paclitaxel in vivo. In addition, higher TERT expression was seen in Kras-mutant NSCLC than that with wild-type Kras. Our data suggest that Kras mutations increase telomerase activity and telomere length by activating the RAS/MEK pathway, which contributes to an aggressive phenotype of NSCLC. Kras mutations-induced lung tumorigenesis and chemoresistance are attenuated by telomerase inhibition. Targeting telomerase/telomere may be a promising therapeutic strategy for patients with Kras-mutant NSCLC. PMID:27329725

  16. Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population.

    PubMed

    Yen, H-L; McKimm-Breschkin, J L; Choy, K-T; Wong, D D Y; Cheung, P P H; Zhou, J; Ng, I H; Zhu, H; Webby, R J; Guan, Y; Webster, R G; Peiris, J S M

    2013-07-16

    We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2'-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which

  17. Microenvironment mediated alterations to metabolic pathways confer increased chemo-resistance in CD133+ tumor initiating cells

    PubMed Central

    Nomura, Alice; Dauer, Patricia; Gupta, Vineet; McGinn, Olivia; Arora, Nivedita; Majumdar, Kaustav; III, Charles Uhlrich; Dalluge, Joseph; Dudeja, Vikas; Saluja, Ashok; Banerjee, Sulagna

    2016-01-01

    Chemoresistance in pancreatic cancer has been attributed to tumor-initiating cells (TICs), a minor sub-population of tumor cells. However, the mechanism of chemo-resistance in these cells is still unclear. In the current study, immunohistochemical analysis of LSL-KrasG12D; LSL-Trp53R172H; PdxCre (KPC) murine tumors indicated that hypoxic regions developed through tumor progression. This hypoxic “niche” correlated with increased CD133+ population that had an increased HIF1A activity. Consistent with this observation, CD133+ cells had increased glucose uptake and activity of glycolytic pathway enzymes compared to CD133− cells. Mass spectrometric analysis (UPLC-TQD) following metabolic labeling of CD133+ cells with [13C]-U6 glucose confirmed this observation. Furthermore, although both populations had functionally active mitochondria, CD133+ cells had low mitochondrial complex I and complex IV activity and lesser accumulation of ROS in response to standard chemotherapeutic compounds like paclitaxel, 5FU and gemcitabine. CD133+ cells also showed increased resistance to all three chemotherapeutic compounds and treatment with Glut1 inhibitor (STF31) reversed this resistance, promoting apoptotic death in these cells similar to CD133− cells. Our study indicates that the altered metabolic profile of CD133+ pancreatic TIC protects them against apoptosis, by reducing accumulation of ROS induced by standard chemotherapeutic agents, thereby confering chemoresistance. Since resistance to existing chemotherapy contributes to the poor prognosis in pancreatic cancer, our study paves the way for identifying novel therapeutic targets for managing chemoresistance and tumor recurrence in pancreatic cancer. PMID:27472388

  18. Recent insertion/deletion (reINDEL) mutations: increasing awareness to boost molecular-based research in ecology and evolution

    PubMed Central

    Schlick-Steiner, Birgit C; Arthofer, Wolfgang; Moder, Karl; Steiner, Florian M

    2015-01-01

    Today, the comparative analysis of DNA molecules mainly uses information inferred from nucleotide substitutions. Insertion/deletion (INDEL) mutations, in contrast, are largely considered uninformative and discarded, due to our lacking knowledge on their evolution. However, including rather than discarding INDELs would be relevant to any research area in ecology and evolution that uses molecular data. As a practical approach to better understanding INDEL evolution in general, we propose the study of recent INDEL (reINDEL) mutationsmutations where both ancestral and derived state are seen in the sample. The precondition for reINDEL identification is knowledge about the pedigree of the individuals sampled. Sound reINDEL knowledge will allow the improved modeling needed for including INDELs in the downstream analysis of molecular data. Both microsatellites, currently still the predominant marker system in the analysis of populations, and sequences generated by next-generation sequencing, a promising and rapidly developing range of technologies, offer the opportunity for reINDEL identification. However, a 2013 sample of animal microsatellite studies contained unexpectedly few reINDELs identified. As most likely explanation, we hypothesize that reINDELs are underreported rather than absent and that this underreporting stems from common reINDEL unawareness. If our hypothesis applies, increased reINDEL awareness should allow gathering data rapidly. We recommend the routine reporting of either the absence or presence of reINDELs together with standardized key information on the nature of mutations when they are detected and the use of the keyword “reINDEL” to increase visibility in both instances of successful and unsuccessful search. PMID:25628861

  19. Antibiotic Resistance in Pseudomonas aeruginosa Strains with Increased Mutation Frequency Due to Inactivation of the DNA Oxidative Repair System▿

    PubMed Central

    Mandsberg, L. F.; Ciofu, O.; Kirkby, N.; Christiansen, L. E.; Poulsen, H. E.; Høiby, N.

    2009-01-01

    The chronic Pseudomonas aeruginosa infection of the lungs of cystic fibrosis (CF) patients is characterized by the biofilm mode of growth and chronic inflammation dominated by polymorphonuclear leukocytes (PMNs). A high percentage of P. aeruginosa strains show high frequencies of mutations (hypermutators [HP]). P. aeruginosa is exposed to oxygen radicals, both those generated by its own metabolism and especially those released by a large number of PMNs in response to the chronic CF lung infection. Our work therefore focused on the role of the DNA oxidative repair system in the development of HP and antibiotic resistance. We have constructed and characterized mutT, mutY, and mutM mutants in P. aeruginosa strain PAO1. The mutT and mutY mutants showed 28- and 7.5-fold increases in mutation frequencies, respectively, over that for PAO1. These mutators had more oxidative DNA damage (higher levels of 7,8-dihydro-8-oxodeoxyguanosine) than PAO1 after exposure to PMNs, and they developed resistance to antibiotics more frequently. The mechanisms of resistance were increased β-lactamase production and overexpression of the MexCD-OprJ efflux-pump. Mutations in either the mutT or the mutY gene were found in resistant HP clinical isolates from patients with CF, and complementation with wild-type genes reverted the phenotype. In conclusion, oxidative stress might be involved in the development of resistance to antibiotics. We therefore suggest the possible use of antioxidants for CF patients to prevent the development of antibiotic resistance. PMID:19332676

  20. Mast cells control insulitis and increase Treg cells to confer protection against STZ-induced type 1 diabetes in mice.

    PubMed

    Carlos, Daniela; Yaochite, Juliana N U; Rocha, Fernanda A; Toso, Vanina D; Malmegrim, Kelen C R; Ramos, Simone G; Jamur, Maria C; Oliver, Constance; Camara, Niels O; Andrade, Marcus V M; Cunha, Fernando Q; Silva, João S

    2015-10-01

    Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-β, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

  1. Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat.

    PubMed

    Hazard, Brittany; Zhang, Xiaoqin; Colasuonno, Pasqualina; Uauy, Cristobal; Beckles, Diane M; Dubcovsky, Jorge

    2012-01-01

    Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat.

  2. Induced mutations in the starch branching enzyme II (SBEII) genes increase amylose and resistant starch content in durum wheat

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Colasuonno, Pasqualina; Uauy, Cristobal; Beckles, Diane M.; Dubcovsky, Jorge

    2016-01-01

    Starch is the largest component of the wheat (Triticum aestivum L.) grain and consists of approximately 70-80% amylopectin and 20-30% amylose. Amylopectin is a highly-branched, readily digested polysaccharide, whereas amylose has few branches and forms complexes that resist digestion and mimic dietary fiber (resistant starch). Down-regulation of the starch branching enzyme II (SBEII) gene by RNA interference (RNAi) was previously shown to increase amylose content in both hexaploid and tetraploid wheat. We generated ethyl methane sulphonate (EMS) mutants for the SBEIIa-A and SBEIIa-B homoeologs in the tetraploid durum wheat variety Kronos (T. turgidum ssp. durum L.). Single-gene mutants showed non-significant increases in amylose and resistant starch content, but a double mutant combining a SBEIIa-A knock-out mutation with a SBEIIa-B splice-site mutation showed a 22% increase in amylose content (P<0.0001) and a 115% increase in resistant starch content (P<0.0001). In addition, we obtained mutants for the A and B genome copies of the paralogous SBEIIb gene, mapped them 1-2 cM from SBEIIa, and generated double SBEIIa-SBEIIb mutants to study the effect of the SBEIIb gene in the absence of SBEIIa. These mutants are available to those interested in increasing amylose content and resistant starch in durum wheat. PMID:26924849

  3. Mice with mutations in Fas and Fas ligand demonstrate increased herpetic stromal keratitis following corneal infection with HSV-1

    PubMed Central

    Morris, Jessica E.; Zobell, Stephanie; Yin, Xiao-Tang; Zakaria, Hamideh; Summers, Bretton C.; Leib, David A.; Stuart, Patrick M.

    2011-01-01

    Herpes simplex virus-1 (HSV) infection of the cornea leads to a potentially blinding immuno-inflammatory lesion of the cornea that is termed herpetic stromal keratitis (HSK). It has also been demonstrated that one of the factors that limits inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study the role that FasL expression in the cornea plays following acute infection with HSV-1 was determined. HSV-1 infection of both BALB/c and C57BL/6 (B6) mice were compared to their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas antigen (lpr) displayed most severe disease while the FasL defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone-marrow derived cells. Interestingly, while virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Furthermore, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than wild-type B6 mice. Thus neither disease nor mortality correlated with viral replication in these mice. Collectively, these findings indicate that the presence of FasL on the cornea restricts the entry of Fas+ bone marrow-derived inflammatory cells and thus reduces the severity of HSK. PMID:22156346

  4. Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ɛ-cleavage site

    NASA Astrophysics Data System (ADS)

    Chen, Wen; Gamache, Eric; Rosenman, David J.; Xie, Jian; Lopez, Maria M.; Li, Yue-Ming; Wang, Chunyu

    2014-01-01

    The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer’s disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ɛ-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ɛ-cleavage, and consequently shift cleavage preference towards Aβ42.

  5. Increasing prevalence of ciprofloxacin-resistant food-borne Salmonella strains harboring multiple PMQR elements but not target gene mutations

    PubMed Central

    Lin, Dachuan; Chen, Kaichao; Wai-Chi Chan, Edward; Chen, Sheng

    2015-01-01

    Fluoroquinolone resistance in Salmonella has become increasingly prevalent in recent years. To probe the molecular basis of this phenomenon, the genetic and phenotypic features of fluoroquinolone resistant Salmonella strains isolated from food samples were characterized. Among the 82 Salmonella strains tested, resistance rate of the three front line antibiotics of ceftriaxone, ciprofloxacin and azithromycin was 10%, 39% and 25% respectively, which is significantly higher than that reported in other countries. Ciprofloxacin resistant strains typically exhibited cross-resistance to multiple antibiotics including ceftriaxone, primarily due to the presence of multiple PMQR genes and the blaCTX-M-65, blaCTX-M-55 blaCMY-2 and blaCMY-72 elements. The prevalence rate of the oqxAB and aac(6’)-Ib-cr genes were 91% and 75% respectively, followed by qnrS (66%), qnrB (16%) and qnrD (3%). The most common PMQR combination observable was aac(6’)-Ib-cr-oqxAB-qnrS2, which accounted for 50% of the ciprofloxacin resistant strains. Interestingly, such isolates contained either no target mutations or only a single gyrA mutation. Conjugation and hybridization experiments suggested that most PMQR genes were located either in the chromosome or a non-transferrable plasmid. To summarize, findings in this work suggested that PMQRs greatly facilitate development of fluoroquinolone resistance in Salmonella by abolishing the requirement of target gene mutations. PMID:26435519

  6. Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein-protein interactions.

    PubMed

    Hawver, Lisa A; Tehrani, Mohammad; Antczak, Nicole M; Kania, Danielle; Muser, Stephanie; Sefcikova, Jana; Beuning, Penny J

    2015-10-01

    Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD', form UmuD'2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine-thymine dimers, whereas DinB is specialized to copy N(2)-dG adducts, such as N(2)-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N(2)-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9V, H282P, and T412I, to bypass N(2)-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9V to render cells UV-mutable is dependent on the proper length of the arm of UmuD'. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD', which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.

  7. A Single Mutation in the Gene Responsible for the Mucoid Phenotype of Bifidobacterium animalis subsp. lactis Confers Surface and Functional Characteristics

    PubMed Central

    Hidalgo-Cantabrana, Claudio; Sánchez, Borja; Álvarez-Martín, Pablo; López, Patricia; Martínez-Álvarez, Noelia; Delley, Michele; Martí, Marc; Varela, Encarna; Suárez, Ana; Antolín, María; Guarner, Francisco; Berger, Bernard

    2015-01-01

    Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells. PMID:26362981

  8. The tip of the iceberg: quinolone-resistance conferred by mutations in gyrA gene in non-typhoidal Salmonella strains.

    PubMed

    Năşcuţiu, Alexandra-Maria

    2012-01-01

    Food-borne infections due to Salmonella spp. seldom require antimicrobial therapy, but this is compulsory in systemic salmonellosis. Salmonella resistance to a large panel of antibiotics has been described worldwide. Since the introduction of nalidixic acid in therapy, Salmonella spp. have steadily developed resistance, especially over the last three decades. The source of quinolone resistance is thought to be the selective pressure determined by the use of quinolones in both human and veterinary practices. Resistance acquisition of Salmonella strains is a stepwise process. Several mechanisms are described, which can lead to the development of quinolone resistance. The main mechanism is considered to be linked with mutations in the quinolone-resistance determining region (QRDR) of the target genes (gyrA and gyrB encoding DNA gyrase, and parC and parE encoding topoisomerase IV). This first step in mutational resistance usually determines a rise in the nalidixic acid minimal inhibitory concentration (MIC). The most common amino acid substitutions in the GyrA subunit, resulting in varied degrees of quinolone resistance, occur at codons Ser83 and Asp87. Higher levels of resistance may occur by further mutational steps, with amino acid changes in the same or a different target enzyme. Other mechanisms are as well involved, like increased efflux or plasmid-mediated resistance. Acknowledgement of the epidemiology and the onset mechanisms of quinolone resistance in Salmonella spp. is compulsory, and surveillance for resistant bacteria among human, animal and food sources remains critical.

  9. Mutation of cysteine 46 in IKK-beta increases inflammatory responses

    PubMed Central

    Jiang, Zhi Hong; Jiang, Shui Ping; Liu, Yan; Wang, Ting Yu; Yao, Xiao Jun; Su, Xiao Hui; Yan, Feng Gen; Liu, Juan; Leung, Elaine Lai-Han; Yi, Xiao Qin; Wong, Yuen Fan; Zhou, Hua; Liu, Liang

    2015-01-01

    Activation of IκB kinase β (IKK-β) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-β−NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-β. However, mutations in IKK-β have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-β inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-β kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-βC46A transgenic mouse model. We show that a novel IKK-β inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-βC46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-β. PMID:26378659

  10. Do Mutations Causing Low HDL-C Promote Increased Carotid Intima-Media Thickness?

    PubMed Central

    Miller, Michael; Rhyne, Jeffrey; Hong, Seung Ho; Friel, Gina; Dolinar, Christina; Riley, Ward

    2007-01-01

    Background Although observational data support an inverse relationship between high-density lipoprotein (HDL) cholesterol and coronary heart disease (CHD), genetic HDL deficiency states often do not correlate with premature CHD. Methods Carotid intima-media thickness (cIMT) measurements were obtained in cases comprising 10 different mutations in LCAT, ABCA1 and APOA1 to further evaluate the relationship between low HDL resulting from genetic variation and early atherosclerosis. Results In a 1:2 case-control study of sex and age-related (± 5 y) subjects (n=114), cIMT was nearly identical between cases (0.66 ± 0.17 cm) and controls (0.65 ± 0.18 cm) despite significantly lower HDL cholesterol (0.67 vs 1.58 mmol/l) and apolipoprotein A-I levels (96.7 vs. 151.4 mg/dl) (P < 0.05). Conclusions Genetic variants identified in the present study may be insufficient to promote early carotid atherosclerosis. PMID:17113061

  11. Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance.

    PubMed

    Liu, Simu; Bartnikas, Lisa M; Volko, Sigrid M; Ausubel, Frederick M; Tang, Dingzhong

    2016-01-01

    Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

  12. Characterization of pbt genes conferring increased Pb2+ and Cd2+ tolerance upon Achromobacter xylosoxidans A8.

    PubMed

    Hložková, Kateřina; Suman, Jáchym; Strnad, Hynek; Ruml, Tomas; Paces, Vaclav; Kotrba, Pavel

    2013-12-01

    The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb(2+) and Cd(2+). Predicted pbtTRABC products share strong similarities with Pb(2+) uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb(2+)-, Cd(2+)- and Zn(2+)-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb(2+), but not of Cd(2+) or Zn(2+), was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb(2+)-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb(2+), Cd(2+) and Zn(2+) exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  13. msbB deletion confers acute sensitivity to CO2 in Salmonella enterica serovar Typhimurium that can be suppressed by a loss-of-function mutation in zwf.

    PubMed

    Karsten, Verena; Murray, Sean R; Pike, Jeremy; Troy, Kimberly; Ittensohn, Martina; Kondradzhyan, Manvel; Low, K Brooks; Bermudes, David

    2009-08-18

    Pathogens tolerate stress conditions that include low pH, oxidative stress, high salt and high temperature in order to survive inside and outside their hosts. Lipopolysaccharide (LPS), which forms the outer-leaflet of the outer membrane in Gram-negative bacteria, acts as a permeability barrier. The lipid A moiety of LPS anchors it to the outer membrane bilayer. The MsbB enzyme myristoylates the lipid A precursor and loss of this enzyme, in Salmonella, is correlated with reduced virulence and severe growth defects that can both be compensated with extragenic suppressor mutations. We report here that msbB (or msbB somA) Salmonella are highly sensitive to physiological CO2 (5%), resulting in a 3-log reduction in plating efficiency. Under these conditions, msbB Salmonella form long filaments, bulge and lyse. These bacteria are also sensitive to acidic pH and high osmolarity. Although CO2 acidifies LB broth media, buffering LB to pH 7.5 did not restore growth of msbB mutants in CO2, indicating that the CO2-induced growth defects are not due to the effect of CO2 on the pH of the media. A transposon insertion in the glucose metabolism gene zwf compensates for the CO2 sensitivity of msbB Salmonella. The msbB zwf mutants grow on agar, or in broth, in the presence of 5% CO2. In addition, msbB zwf strains show improved growth in low pH or high osmolarity media compared to the single msbB mutant. These results demonstrate that msbB confers acute sensitivity to CO2, acidic pH, and high osmolarity. Disruption of zwf in msbB mutants restores growth in 5% CO2 and results in improved growth in acidic media or in media with high osmolarity. These results add to a growing list of phenotypes caused by msbB and mutations that suppress specific growth defects.

  14. Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 beta-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime.

    PubMed Central

    Imtiaz, U; Manavathu, E K; Mobashery, S; Lerner, S A

    1994-01-01

    The mutation of Arg-244 to Ser (Arg-244-->Ser mutation) in the TEM-1 beta-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme. The Arg-164-->Ser mutation in the TEM-1 beta-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D. A. Weber, C. C. Sanders, J. S. Bakken, and J. P. Quinn, J. Infect. Dis. 162:460-465, 1990). The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate. It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate. Thus, the Arg-164-->Ser mutation in the TEM-1 beta-lactamase suppresses the effect of the Arg-244-->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate. PMID:8067751

  15. Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse.

    PubMed

    Ono, T; Ikehata, H; Nakamura, S; Saito, Y; Hosoi, Y; Takai, Y; Yamada, S; Onodera, J; Yamamoto, K

    2000-02-14

    Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.

  16. G206D Mutation of Presenilin-1 Reduces Pen2 Interaction, Increases Aβ42/Aβ40 Ratio and Elevates ER Ca(2+) Accumulation.

    PubMed

    Chen, Wei-Ting; Hsieh, Yi-Fang; Huang, Yan-Jing; Lin, Che-Ching; Lin, Yen-Tung; Liu, Yu-Chao; Lien, Cheng-Chang; Cheng, Irene Han-Juo

    2015-12-01

    Early-onset familial Alzheimer's disease (AD) is most commonly associated with the mutations in presenilin-1 (PS1). PS1 is the catalytic component of the γ-secretase complex, which cleaves amyloid precursor protein to produce amyloid-β (Aβ), the major cause of AD. Presenilin enhancer 2 (Pen2) is critical for activating γ-secretase and exporting PS1 from endoplasmic reticulum (ER). Among all the familial AD-linked PS1 mutations, mutations at the G206 amino acid are the most adjacent position to the Pen2 binding site. Here, we characterized the effect of a familial AD-linked PS1 G206D mutation on the PS1-Pen2 interaction and the accompanied alteration in γ-secretase-dependent and -independent functions. We found that the G206D mutation reduced PS1-Pen2 interaction, but did not abolish γ-secretase formation and PS1 endoproteolysis. For γ-secretase-dependent function, the G206D mutation increased Aβ42 production but not Notch cleavage. For γ-secretase-independent function, this mutation disrupted the ER calcium homeostasis but not lysosomal calcium homeostasis and autophagosome maturation. Impaired ER calcium homeostasis may due to the reduced mutant PS1 level in the ER. Although this mutation did not alter the cell survival under stress, both increased Aβ42 ratio and disturbed ER calcium regulation could be the mechanisms underlying the pathogenesis of the familial AD-linked PS1 G206D mutation.

  17. Novel Barley (1→3,1→4)-β-Glucan Endohydrolase Alleles Confer Increased Enzyme Thermostability.

    PubMed

    Lauer, Juanita C; Yap, Kuok; Cu, Suong; Burton, Rachel A; Eglinton, Jason K

    2017-01-18

    Barley (1→3,1→4)-β-glucan endohydrolases (β-glucanases; EI and EII) are primarily responsible for hydrolyzing high molecular weight (1→3,1→4)-β-glucans (β-glucan) during germination. Incomplete endosperm modification during malting results in residual β-glucan that can contribute to increased wort viscosity and beer chill haze. Four newly identified forms of EI and EII and the reference enzymes EI-a and EII-a were expressed in Escherichia coli, and the recombinant proteins were characterized for enzyme kinetics and thermostability. EI and EII variants that exhibited higher residual β-glucanase activity than EI-a and EII-a after heat treatment also exhibited increased substrate affinity and decreased turnover rates. The novel EII-l form exhibited significantly increased thermostability compared with the reference EII-a when activity was measured at elevated temperature. EII-l exhibited a T50 value, which indicates the temperature at which 50% of β-glucanase activity remains, 1.3 °C higher than that of EII-a. The irreversible thermal inactivation difference between EII-a and EII-l after 5 min of heat treatment at 56 °C was 11.9%. The functional significance of the three amino acid differences between EII-a and EII-l was examined by making combinatorial mutations in EII-a using site-directed mutagenesis. The S20G and D284E amino acid substitutions were shown to be responsible for the increase in EII-1 thermostability.

  18. Vitamin E confers cytoprotective effects on cardiomyocytes under conditions of heat stress by increasing the expression of metallothionein.

    PubMed

    Wang, Xiaowu; Dong, Wenpeng; Yuan, Binbin; Yang, Yongchao; Yang, Dongpeng; Lin, Xi; Chen, Changfu; Zhang, Weida

    2016-05-01

    Heat stress (HS) is commonly used to refer to the heat load that an individual is subjected to due to either metabolic heat, or environmental factors, including high temperatures and high humidity levels. HS has been reported to affect and even damage the functioning of various organs; overexposure to high temperatures and high humidity may lead to accidental deaths. It has been suggested that the cardiovascular system is primarily targeted by exposure to HS conditions; the HS-induced dysfunction of cardiomyocytes, which is characterized by mitochondrial dysfunction, may result in the development of cardiovascular diseases. The excessive production of reactive oxygen species (ROS) also participates in mitochondrial dysfunction. However, effective methods for the prevention and treatment of mitochondrial and cardiovascular dysfunction induced by exposure to HS are lacking. In the present study, we hypothesized that vitamin E (VE), an antioxidant, is capable of preventing oxidative stress and mitochondrial injury in cardiomyocytes induced by exposure to HS. The results revealed that pre‑treatment with VE increased the expression of metallothionein (MT), which has previously been reported to confer cytoprotective effects, particularly on the cardiovascular system. Pre-treatment with VE restored mitochondrial function in cardiomyocytes under conditions of HS by increasing the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), and by increasing adenosine triphosphate (ATP) levels. Furthermore, pre-treatment with VE decreased the production of ROS, which was induced by exposure to HS and thus exerted antioxidant effects. In addition, pre-treatment with VE attenuated oxidative stress induced by exposure to HS, as demonstrated by the increased levels of antioxidant enzymes [superoxide dismutase (SOD) and glutathione (GSH)], and by the decreased levels

  19. Increased rate of missense/in-frame mutations in individuals with NF1-related pulmonary stenosis: a novel genotype-phenotype correlation.

    PubMed

    Ben-Shachar, Shay; Constantini, Shlomi; Hallevi, Hen; Sach, Emma K; Upadhyaya, Meena; Evans, Gareth D; Huson, Susan M

    2013-05-01

    Neurofibromatosis type 1 (NF1) and its related disorders (NF1-Noonan syndrome (NFNS) and Watson syndrome (WS)) are caused by heterozygous mutations in the NF1 gene. Pulmonary stenosis (PS) occurs more commonly in NF1 and its related disorders than in the general population. This study investigated whether PS is associated with specific types of NF1 gene mutations in NF1, NFNS and WS. The frequency of different NF1 mutation types in a cohort of published and unpublished cases with NF1/NFNS/WS and PS was examined. Compared with NF1 in general, NFNS patients had higher rates of PS (9/35=26% vs 25/2322=1.1%, P value<0.001). Stratification according to mutation type showed that the increased PS rate appears to be driven by the NFNS group with non-truncating mutations. Eight of twelve (66.7%) NFNS cases with non-truncating mutations had PS compared with a 1.1% PS frequency in NF1 in general (P<0.001); there was no increase in the frequency of PS in NFNS patients with truncating mutations. Eight out of eleven (73%) individuals with NF1 and PS, were found to have non-truncating mutations, a much higher frequency than the 19% reported in NF1 cohorts (P<0.015). Only three cases of WS have been published with intragenic mutations, two of three had non-truncating mutations. Therefore, PS in NF1 and its related disorders is clearly associated with non-truncating mutations in the NF1 gene providing a new genotype-phenotype correlation. The data indicate a specific role of non-truncating mutations on the NF1 cardiac phenotype.

  20. Microbial gut overgrowth guarantees increased spontaneous mutation leading to polyclonality and antibiotic resistance in the critically ill.

    PubMed

    van Saene, H K F; Taylor, N; Damjanovic, V; Sarginson, R E

    2008-05-01

    Polyclonality is defined as the occurrence of different genotypes of a bacterial species. We are of the opinion that these different clones originate within the patient. When infections and outbreaks occur, the terms of polyclonal infections and polyclonal outbreaks have been used, respectively. The origin of polyclonality has never been reported, although some authors suggest the acquisition of different clones from different animate and inanimate sources. We think that the gut of the critically ill patient with microbial overgrowth is the ideal site for the de-novo development of new clones, following increased spontaneous mutation.

  1. Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation*

    PubMed Central

    Bryant, Kirsten L.; Antonyak, Marc A.; Cerione, Richard A.; Baird, Barbara; Holowka, David

    2013-01-01

    Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling. PMID:24142702

  2. Polymorphism of FGFR4 Gly388Arg does not confer an increased risk to breast cancer development.

    PubMed

    Naidu, R; Har, Y C; Taib, N A

    2009-01-01

    The genotype analysis of the Gly and Arg allele at codon 388 of fibroblast growth factor receptor-4 (FGFR4) gene was evaluated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in a hospital-based Malaysian population. Peripheral blood samples were collected from 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy. The aim of the present study was to evaluate the association between the FGFR4 Gly388Arg polymorphism and breast cancer risk as well as clinicopathological parameters of the patients. The Gly/Gly, Gly/Arg, Arg/Arg, and Arg allele genotypes were detected in 46.3%, 44.4%, 9.3%, and 53.7% of breast cancer cases, respectively. The distribution of genotype (p = 0.204) and allele (p = 0.086) frequencies of FGFR4 polymorphism were not significantly different between the breast cancer cases and normal individuals. Women who were Arg/ Arg homozygotes (OR = 1.714, 95% CI 0.896-3.278), Gly/Arg heterozygotes (OR = 1.205, 95% CI 0.863-1.683), carriers of Arg allele genotype (OR = 1.269, 95% CI 0.921-1.750), or Arg allele (OR = 1.246, 95% CI 0.970-1.602) were not associated with breast cancer risk. The Arg allele genotype was significantly associated with lymph node metastases (p = 0.001) but not with other clinicopathological parameters. Our findings suggest that the polymorphic variant at codon 388 of FGFR4 gene does not confer increased risk to breast cancer development but it may be a potential genetic marker for tumor prognosis.

  3. The serotonin transporter gene polymorphism STin2 VNTR confers an increased risk of inconsistent response to triptans in migraine patients.

    PubMed

    Terrazzino, Salvatore; Viana, Michele; Floriddia, Elisa; Monaco, Francesco; Mittino, Daniela; Sances, Grazia; Tassorelli, Cristina; Nappi, Giuseppe; Rinaldi, Maurizio; Canonico, Pier Luigi; Genazzani, Armando A

    2010-09-01

    The aim of the present observational study was to assess the value of the C825T polymorphism of the beta-3 subunit of G proteins (GNB3) as well as of variants in the SLC6A4 gene (5HTTLPR and STin2 VNTR) and DRD2 gene (TaqI A and NcoI) as predictive markers for consistency in headache response to triptans in migraine patients. Consistent responders to triptans were defined as the migraineurs who experienced a > or =2 point reduction in a 4-point scale intensity of pain from 3 (severe) to 0 (absent) 2h after triptan administration, in at least two attacks out of the three. Genotyping was performed by PCR and PCR-RFLP on genomic DNA extracted from peripheral blood. The impact of clinical and biological variables on consistency status of headache response to triptans was evaluated by using a binary logistic regression model with stepwise selection. Forty-three (33%) of the 130 migraine patients included in the study did not consistently respond to triptan administration. In a binary logistic regression model, STin 2.12/12 genotype (OR=3.363, 95% CI: 1.262-8.966, P=0.005) and non-use of migraine prophylactic medications (OR=2.848, 95% CI: 1.019-7.959, P=0.010) were found as significant factors increasing the odds of achieving inconsistent response to triptans. The analysis of classificatory power of the model showed moderate values of sensitivity (0.56), high specificity (0.87), and an overall prediction correctness (0.77). These results support the role of STin2 VNTR polymorphism of serotonin transporter gene as a relevant genetic factor conferring a higher risk of inconsistent response to triptans in migraine patients. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  4. Increased muscle coenzyme Q10 in riboflavin responsive MADD with ETFDH gene mutations due to secondary mitochondrial proliferation.

    PubMed

    Wen, Bing; Li, Duoling; Shan, Jingli; Liu, Shuping; Li, Wei; Zhao, Yuying; Lin, Pengfei; Zheng, Jinfan; Li, Danian; Gong, Yaoqin; Yan, Chuanzhu

    2013-06-01

    Multiple acyl-coenzyme A dehydrogenation deficiency (MADD) has a wide range of phenotypic variation ranging from a neonatal lethal form to a mild late-onset form. Our previous data showed that in a group of Chinese patients, a mild type of MADD characterized by myopathy with clinically no other systemic involvement was caused by mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, which encodes electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO). Coenzyme Q10 (CoQ10), a downstream electron receptor of ETF:QO was first reported deficient in muscle of MADD patients with ETFDH gene mutations. Nevertheless, this result was not confirmed in a recently published study. Therefore to elucidate muscle CoQ10 level in a large group of MADD patients may provide further insight into the pathomechanism and therapeutic strategies. In this study, we found that 34 riboflavin responsive patients with ETFDH gene mutations had an elevated CoQ10 pool in muscle by high performance liquid chromatography (HPLC). However, when CoQ10 levels were normalized to citrate synthase, a marker of mitochondrial mass, there was no significant difference between patients and normal controls. Meanwhile, the increased mitochondrial DNA copy number in muscle also supported that the elevated CoQ10 pool was mainly due to mitochondrial mass proliferation. The expression of CoQ10 biosynthesis genes showed no significant changes whereas genes involved in lipid metabolism, such as PPARα, were marked up regulated. Our results suggested that CoQ10 seems not to be a primary factor in riboflavin responsive MADD and the apparent increase in CoQ10 may be secondary to mitochondrial proliferation. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Using polarization-sensitive optical coherence tomography to identify tumor stromal fibrosis and increase tumor biopsy yield (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hariri, Lida P.; Adams, David C.; Miller, Alyssa J.; Mino-Kenudson, Mari; Suter, Melissa J.

    2016-03-01

    Tissue biopsy is the principal method used to diagnose tumors in a variety of organ systems. It is essential to maximize tumor yield in biopsy specimens for both clinical diagnostic and research purposes. This is particularly important in tumors where additional tissue is needed for molecular analysis to identify patients who would benefit from mutation-specific targeted therapy, such as in lung carcinomas. Inadvertent sampling of fibrotic stroma within tumor nodules contaminates biopsies, decreases tumor yield, and can impede diagnosis. The ability to assess tumor composition and guide biopsy site selection in real time is likely to improve diagnostic yield. Polarization sensitive OCT (PS-OCT) measures birefringence in organized tissues, such as collagen, and could be used to distinguish tumor from fibrosis. In this study, PS-OCT was obtained in 65 lung nodule samples from surgical resection specimens containing varying ratios of tumor and fibrosis. PS-OCT was obtained with either a custom-built helical scanning catheter (0.8 or 1.6mm in diameter) or a dual-axis bench top scanner. Strong birefringence was observed in nodules containing dense fibrosis, with no birefringence in adjacent regions of tumor. Tumors admixed with early, loosely-organized collagen demonstrated mild-to-moderate birefringence, and tumors with little collagen content showed little to no birefringent signal. PS-OCT provides significant insights into tumor nodule composition, and has potential to differentiate tumor from stromal fibrosis during biopsy site selection to increase diagnostic tumor yield.

  6. Amplification of the Gp41 gene for detection of mutations conferring resistance to HIV-1 fusion inhibitors on genotypic assay

    NASA Astrophysics Data System (ADS)

    Tanumihardja, J.; Bela, B.

    2017-08-01

    Fusion inhibitors have potential for future use in HIV control programs in Indonesia, so the capacity to test resistance to such drugs needs to be developed. Resistance-detection with a genotypic assay began with amplification of the target gene, gp41. Based on the sequence of the two most common HIV subtypes in Indonesia, AE and B, a primer pair was designed. Plasma samples containing both subtypes were extracted to obtain HIV RNA. Using PCR, the primer pair was used to produce the amplification product, the identity of which was checked based on length under electrophoresis. Eleven plasma samples were included in this study. One-step PCR using the primer pair was able to amplify gp41 from 54.5% of the samples, and an unspecific amplification product was seen in 1.1% of the samples. Amplification failed in 36.4% of the samples, which may be due to an inappropriate primer sequence. It was also found that the optimal annealing temperature for producing the single expected band was 57.2 °C. With one-step PCR, the designed primer pair amplified the HIV-1 gp41 gene from subtypes AE and B. However, further research should be done to determine the conditions that will increase the sensitivity and specificity of the amplification process.

  7. Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin.

    PubMed

    Adhikari, Arjun S; Kooiker, Kristina B; Sarkar, Saswata S; Liu, Chao; Bernstein, Daniel; Spudich, James A; Ruppel, Kathleen M

    2016-12-13

    Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

  8. Asn194Lys mutation in RVG29 peptide increases GFP transgene delivery by endocytosis to neuroblastoma and astrocyte cells.

    PubMed

    Villa-Cedillo, Sheila Adela; Rodríguez-Rocha, Humberto; Zavala-Flores, Laura Mireya; Montes-de-Oca-Luna, Roberto; García-García, Aracely; Loera-Arias, Maria de Jesus; Saucedo-Cárdenas, Odila

    2017-10-01

    A cell-penetrating peptide-based delivery system could target specific types of cells for therapeutic genes delivery. To increase the gene delivery efficiency into neuronal phenotype cells, we introduced an Asn194Lys mutation to RVG29 peptide derived from rabies virus glycoprotein and added a nuclear localization signal to enhance its nuclear import. Mutant RVG or wild-type RVG peptide, a karyophilic peptide (KP) and a plasmid encoding green fluorescent protein (pGL) were bound by electrostatic charges to form four different kinds of RVG complexes. Immunofluorescence was used to assess the gene transfection efficiency into astrocytes, oligodendrocyte precursor cells (OPCs), SH-SY5Y, HeLa and NIH/3T3 cells. The cellular uptake mechanism of RVG29 complexes was examined using endocytosis inhibitors. The mRVG29 peptide has the ability to enhance the nuclear import of plasmids. The Asn194Lys mutation in RVG29 peptide of the pGL-mRVG29 complex and the addition of KP to the pGL-RVG29-KP complex increased the capacity to deliver DNA by endocytosis in astrocytes and SH-SY5Y cells. The complexes pGL-mRVG29 and pGL-RVG29-KP have specificity for transfecting astrocytes and SH-SY5Y cells. The karyophilic capacity of this new mRVG peptide render it promising candidate to act as gene delivery vector into the brain cells. © 2017 Royal Pharmaceutical Society.

  9. A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice

    PubMed Central

    Huang, Hong Ming; Bauer, Denis C.; Lelliott, Patrick M.; Greth, Andreas; McMorran, Brendan J.; Foote, Simon J.; Burgio, Gaetan

    2016-01-01

    Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1(MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1(MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1(MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1(MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance. PMID:27848995

  10. A rare mutation in UNC5C predisposes to Alzheimer’s disease and increases neuronal cell death

    PubMed Central

    Wetzel-Smith, MK; Hunkapiller, J; Bhangale, TR; Srinivasan, K; Maloney, JA; Atwal, JK; Sa, SM; Yaylaoglu, MB; Foreman, O; Ortmann, W; Rathore, N; Hansen, DV; Tessier-Lavigne, M; Mayeux, R; Pericak-Vance, M; Haines, J; Farrer, LA; Schellenberg, GD; Goate, A; Behrens, TW

    2015-01-01

    We have identified a rare coding mutation, T835M (rs137875858), in the Netrin receptor UNC5C that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer’s disease (LOAD), and was associated with disease across four large case/control cohorts (OR = 2.15, Pmeta= 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in several cell types, including neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to multiple neurodegenerative stimuli, including β-Amyloid (Aβ). Based on these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer’s disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer’s brain. PMID:25419706

  11. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers.

    PubMed

    Alquézar, Carolina; Esteras, Noemí; de la Encarnación, Ana; Alzualde, Ainhoa; Moreno, Fermín; López de Munain, Adolfo; Martín-Requero, Angeles

    2014-04-01

    Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Previously, we reported cell cycle-related alterations in lymphoblasts from FTLD-TDP patients, carrying the c.709-1G>A null PGRN mutation, suggesting aberrant cell cycle activation in affected neurons. Here we report that PGRN haploinsufficiency activates the extracellular signal-regulated protein kinases 1 and 2 pathway in a Ca(2+), protein kinase C-dependent, and pertussis toxin-sensitive manner. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that noncanonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II. Moreover, treatment of control cells with exogenous Wingless-type 5a (Wnt5a)-activated Ca(2+)/calmodulin kinase II (CaMKII), increased extracellular signal-regulated protein kinases 1 and 2 activity and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLD-TDP pathology and suggest a novel target for therapeutic intervention.

  12. Increased expression of pro-angiogenic factors and vascularization in thyroid hyperfunctioning adenomas with and without TSH receptor activating mutations.

    PubMed

    Celano, Marilena; Sponziello, Marialuisa; Tallini, Giovanni; Maggisano, Valentina; Bruno, Rocco; Dima, Mariavittoria; Di Oto, Enrico; Redler, Adriano; Durante, Cosimo; Sacco, Rosario; Filetti, Sebastiano; Russo, Diego

    2013-02-01

    Autonomously functioning thyroid nodules (AFTN) are known to receive an increased blood influx necessary to sustain their high rate of growth and hormone production. Here, we investigated the expression of hematic and lymphatic vases in a series of 20 AFTN compared with the contralateral non-tumor tissues of the same patients, and the transcript levels of proteins involved in the control of vascular proliferation, including the vascular endothelial growth factor (VEGF) and platelet-derived growth factors (PDGF) and their receptors and the endothelial nitric oxide synthase (eNOS). In parallel, the expression of the differentiation markers sodium/iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (Tg), and TSH receptor (TSHR) was also investigated. The data were further analyzed comparing subgroups of tumors with or without mutations in the TSHR gene. Analysis by means of CD31 and D2-40 immunostaining showed in AFTN an increased number of hematic, but not lymphatic, vessels in parallel with an enhanced proliferation rate shown by increased Ki67 staining. Quantitative RT-PCR analysis revealed an increase of VEGF, VEGFR1 and 2, PDGF-A, PDGF-B, and eNOS expression in tumor versus normal tissues. Also, higher transcript levels of NIS, TPO, and Tg were detected. Comparison of the two subgroups of samples revealed only few differences in the expression of the genes examined. In conclusion, these data demonstrate an increased expression of angiogenesis-related factors associated with an enhanced proliferation of hematic, but not lymphatic, vessels in AFTNs. In this context, the presence of TSHR mutations may only slightly influence the expression of pro-angiogenic growth factors.

  13. A protective antigen mutation increases the pH threshold of anthrax toxin receptor 2-mediated pore formation.

    PubMed

    Dennis, Melissa K; Mogridge, Jeremy

    2014-04-08

    Anthrax toxin protective antigen (PA) binds cellular receptors and self-assembles into oligomeric prepores. A prepore converts to a protein translocating pore after it has been transported to an endosome where the low pH triggers formation of a membrane-spanning β-barrel channel. Formation of this channel occurs after some PA-receptor contacts are broken to allow pore formation, while others are retained to preserve receptor association. The interaction between PA and anthrax toxin receptor 1 (ANTXR1) is weaker than its interaction with ANTXR2 such that the pH threshold of ANTXR1-mediated pore formation is higher by 1 pH unit. Here we examine receptor-specific differences in toxin binding and pore formation by mutating PA residue G342 that selectively abuts ANTXR2. Mutation of G342 to valine, leucine, isoleucine, or tryptophan increased the amount of PA bound to ANTXR1-expressing cells and decreased the amount of PA bound to ANTXR2-expressing cells. The more conservative G342A mutation did not affect the level of binding to ANTXR2, but ANTXR2-bound PA-G342A prepores exhibited a pH threshold higher than that of wild-type prepores. Mixtures of wild-type PA and PA-G342A were functional in toxicity assays, and the pH threshold of ANTXR2-mediated pore formation was dictated by the relative amounts of the two proteins in the hetero-oligomers. These results suggest that PA subunits within an oligomer do not have to be triggered simultaneously for a productive membrane insertion event to occur.

  14. Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation.

    PubMed

    Bogdanova, Anna; Goede, Jeroen S; Weiss, Erwin; Bogdanov, Nikolay; Bennekou, Poul; Bernhardt, Ingolf; Lutz, Hans U

    2010-02-01

    Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient's erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient's erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. In the cold, the cryohydrocytosis patient's erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO(3)-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na(+)/K(+) exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K(+) uptake. Unidirectional K(+) influx measurements showed that the patient's cells have abnormally high activities of the cation-proton exchanger and the K(+),Cl(-) co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient's erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.

  15. Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

    PubMed Central

    Bogdanova, Anna; Goede, Jeroen S.; Weiss, Erwin; Bogdanov, Nikolay; Bennekou, Poul; Bernhardt, Ingolf; Lutz, Hans U.

    2010-01-01

    Background Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. Design and Methods Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. Results In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO3-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na+/K+ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K+ uptake. Unidirectional K+ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K+,Cl− co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors. Conclusions These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis. PMID:20015879

  16. Increased uracil insertion in DNA is cytotoxic and increases the frequency of mutation, double strand break formation and VSG switching in Trypanosoma brucei.

    PubMed

    Castillo-Acosta, Víctor M; Aguilar-Pereyra, Fernando; Bart, Jean-Mathieu; Navarro, Miguel; Ruiz-Pérez, Luis M; Vidal, Antonio E; González-Pacanowska, Dolores

    2012-12-01

    Deoxyuridine 5'-triphosphate pyrophosphatase (dUTPase) and uracil-DNA glycosylase (UNG) are key enzymes involved in the control of the presence of uracil in DNA. While dUTPase prevents uracil misincorporation by removing dUTP from the deoxynucleotide pool, UNG excises uracil from DNA as a first step of the base excision repair pathway (BER). Here, we report that strong down-regulation of dUTPase in UNG-deficient Trypanosoma brucei cells greatly impairs cell viability in both bloodstream and procyclic forms, underscoring the extreme sensitivity of trypanosomes to uracil in DNA. Depletion of dUTPase activity in the absence of UNG provoked cell cycle alterations, massive dUTP misincorporation into DNA and chromosomal fragmentation. Overall, trypanosomatid cells that lack dUTPase and UNG activities exhibited greater proliferation defects and DNA damage than cells deficient in only one of these activities. To determine the mutagenic consequences of uracil in DNA, mutation rates and spectra were analyzed in dUTPase-depleted cells in the presence of UNG activity. These cells displayed a spontaneous mutation rate 9-fold higher than the parental cell line. Base substitutions at A:T base pairs and deletion frequencies were both significantly enhanced which is consistent with the generation of mutagenic AP sites and DNA strand breaks. The increase in strand breaks conveyed a concomitant increase in VSG switching in vitro. The low tolerance of T. brucei to uracil in DNA emphasizes the importance of uracil removal and regulation of intracellular dUTP pool levels in cell viability and genetic stability and suggests potential strategies to compromise parasite survival.

  17. Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

    PubMed

    Dunn, Cory D

    2011-10-01

    Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan.

  18. p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines.

    PubMed

    Servomaa, K; Kiuru, A; Grénman, R; Pekkola-Heino, K; Pulkkinen, J O; Rytömaa, T

    1996-05-01

    The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity. In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants alpha and beta in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4-10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4 +/- 0.1 to 2.6 +/- 0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly (P < 0.01) more radioresistant (mean inactivation dose 2.23 +/- 0.15 Gy) than cell lines which lacked a wild-type gene (1.82 +/- 0.24 Gy). Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.

  19. MDE heteroduplex analysis of PCR products spanning each exon of the fibrillin (FBN1) gene greatly increases the efficiency of mutation detection in the Marfan syndrome

    SciTech Connect

    Nijbroek, G.; Dietz, H.C.; Pereira, L.; Ramirz, F.

    1994-09-01

    Defects in fibrillin (FNB1) cause the Marfan syndrome (MFS). Classic Marfan phenotype cosegregates with intragenic and/or flanking marker alleles in all families tested and a significant number of FBN1 mutations have been identified in affected individuals. Using a standard method of mutation detection, SSCP analysis of overlapping RT-PCR amplimers that span the entire coding sequence, the general experience has been a low yield of identifiable mutations, ranging from 10-20%. Possible explanations included low sensitivity of mutation screening procedures, under-representation of mutant transcript in patient samples either due to deletions or mutant alleles containing premature termination codons, clustering of mutations in yet uncharacterized regions of the gene, including regulatory elements, or genetic heterogeneity. In order to compensate for a potential reduced mutant transcript stability, we have devised a method to screen directly from genomic DNA. The intronic boundaries flanking each of the 65 FBN1 exons were characterized and primer pairs were fashioned such that all splice junctions would be included in the resultant amplimers. The entire gene was screened for a panel of 9 probands with classic Marfan syndrome using mutation detection enhancement (MDE) gel heteroduplex analysis. A mutation was identified in 5/9 (55%) of patient samples. All were either missense mutations involving a cysteine residue or small deletions that did not create a frame shift. In addition, 10 novel polymorphisms were found. We conclude that the majority of mutations causing Marfan syndrome reside in the FBN1 gene and that mutations creating premature termination codons are not the predominant cause of inefficient mutation detection using RT-PCR. We are currently modifying screening methods to increase sensitivity and targeting putative FBN1 gene promoter sequences for study.

  20. Picoliter-Droplet Digital Polymerase Chain Reaction-Based Analysis of Cell-Free Plasma DNA to Assess EGFR Mutations in Lung Adenocarcinoma That Confer Resistance to Tyrosine-Kinase Inhibitors.

    PubMed

    Seki, Yoshitaka; Fujiwara, Yutaka; Kohno, Takashi; Takai, Erina; Sunami, Kuniko; Goto, Yasushi; Horinouchi, Hidehito; Kanda, Shintaro; Nokihara, Hiroshi; Watanabe, Shun-ichi; Ichikawa, Hitoshi; Yamamoto, Noboru; Kuwano, Kazuyoshi; Ohe, Yuichiro

    2016-02-01

    The objective of this study was to evaluate the utility of analyzing cell-free plasma DNA (cfDNA) by picoliter-droplet digital polymerase chain reaction (ddPCR) to detect EGFR mutations that confer resistance to tyrosine-kinase inhibitors (TKIs) used for treatment of lung adenocarcinoma (LADC). Thirty-five LADC patients who received epidermal growth factor receptor (EGFR)-TKI therapy, including ten who received tumor rebiopsy after development of resistance, were subjected to picoliter-ddPCR-cfDNA analysis to determine the fraction of cfDNA with TKI-sensitive (L858R and inflame exon 19 deletions) and -resistant (i.e., T790M) mutations, as well as their concordance with mutation status in rebiopsied tumor tissues. cfDNA samples from 15 (94%) of 16 patients who acquired resistance were positive for TKI-sensitive mutations. Also, 7 (44%) were positive for the T790M mutation, with fractions of T790M (+) cfDNA ranging from 7.4% to 97%. T790M positivity in cfDNA was consistent in eight of ten patients for whom rebiopsied tumor tissues were analyzed, whereas the remaining cases were negative in cfDNA and positive in rebiopsied tumors. Prior to EGFR-TKI therapy, cfDNAs from 9 (38%) and 0 of 24 patients were positive for TKI-sensitive and T790M mutations, respectively. Next-generation sequencing of cfDNA from one patient who exhibited innate resistance to TKI despite a high fraction of TKI-sensitive mutations and the absence of the T790M mutation in his cfDNA revealed the presence of the L747P mutation, a known driver of TKI resistance. Picoliter-ddPCR examination of cfDNA, supported by next-generation sequencing analysis, enables noninvasive assessment of EGFR mutations that confer resistance to TKIs. Noninvasive monitoring of the predominance of tumors harboring the secondary T790M mutation in the activating mutation in EGFR gene is necessary for precise and effective treatment of lung adenocarcinoma. Because cells harboring the T790M mutation are resistant to epidermal

  1. Patient Participation at Health Care Conferences: Engaged Patients Increase Information Flow, Expand Propagation, and Deepen Engagement in the Conversation of Tweets Compared to Physicians or Researchers

    PubMed Central

    2017-01-01

    Background Health care conferences present a unique opportunity to network, spark innovation, and disseminate novel information to a large audience, but the dissemination of information typically stays within very specific networks. Social network analysis can be adopted to understand the flow of information between virtual social communities and the role of patients within the network. Objective The purpose of this study is to examine the impact engaged patients bring to health care conference social media information flow and how they expand dissemination and distribution of tweets compared to other health care conference stakeholders such as physicians and researchers. Methods From January 2014 through December 2016, 7,644,549 tweets were analyzed from 1672 health care conferences with at least 1000 tweets who had registered in Symplur’s Health Care Hashtag Project from 2014 to 2016. The tweet content was analyzed to create a list of the top 100 influencers by mention from each conference, who were then subsequently categorized by stakeholder group. Multivariate linear regression models were created using stepwise function building to identify factors explaining variability as predictor variables for the model in which conference tweets were taken as the dependent variable. Results Inclusion of engaged patients in health care conference social media was low compared to that of physicians and has not significantly changed over the last 3 years. When engaged patient voices are included in health care conferences, they greatly increase information flow as measured by total tweet volume (beta=301.6) compared to physicians (beta=137.3, P<.001), expand propagation of information tweeted during a conference as measured by social media impressions created (beta=1,700,000) compared to physicians (beta=270,000, P<.001), and deepen engagement in the tweet conversation as measured by replies to their tweets (beta=24.4) compared to physicians (beta=5.5, P<.001). Social

  2. Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.

    PubMed Central

    Macek, M; Mackova, A; Hamosh, A; Hilman, B C; Selden, R F; Lucotte, G; Friedman, K J; Knowles, M R; Rosenstein, B J; Cutting, G R

    1997-01-01

    Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans. PMID:9150159

  3. Chronic Ethanol Consumption Increases Myocardial Mitochondrial DNA Mutations: A Potential Contribution by Mitochondrial Topoisomerases

    PubMed Central

    Laurent, D.; Mathew, J.E.; Mitry, M.; Taft, M.; Force, A.; Edwards, J.G.

    2014-01-01

    Aims: Alcoholic cardiomyopathy (ACM) presents as decreased myocardial contractility, arrhythmias and secondary non-ischemic dilated cardiomyopathy leading to heart failure. Mitochondrial dysfunction is known to have a significant role in the development and complications of ACM. This study investigated if chronic ethanol feeding promoted myocardial mitochondrial topoisomerase dysfunction as one underlying cause of mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in ACM. Methods: The impact of chronic ethanol exposure on the myocardial mitochondria was examined in both neonatal cardiomyocytes using 50 mM ethanol for 6 days and in rats assigned to control or ethanol feeding groups for 4 months. Results: Chronic ethanol feeding led to significant (P < 0.05) decreases in M-mode Fractional Shortening, ejection fraction, and the cardiac output index as well as increases in Tau. Ethanol feeding promoted mitochondrial dysfunction as evidenced by significantly decreased left ventricle cytochrome oxidase activity and decreases in mitochondrial protein content. Both in rats and in cultured cardiomyocytes, chronic ethanol presentation significantly increased mtDNA damage. Using isolated myocardial mitochondria, both mitochondrial topoisomerase-dependent DNA cleavage and DNA relaxation were significantly altered by ethanol feeding. Conclusion: Chronic ethanol feeding compromised cardiovascular and mitochondrial function as a result of a decline in mtDNA integrity that was in part the consequence of mitochondrial topoisomerase dysfunction. Understanding the regulation of the mitochondrial topoisomerases is critical for protection of mtDNA, not only for the management of alcoholic cardiomyopathy, but also for the many other clinical treatments that targets the topoisomerases in the alcoholic patient. PMID:24852753

  4. A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

    PubMed Central

    Veits, Jutta; Tauscher, Kerstin; Ziller, Mario; Teifke, Jens P.; Stech, Jürgen; Mettenleiter, Thomas C.

    2015-01-01

    ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding

  5. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.

    PubMed

    Jakubowski, Hieronim; Boers, Godfried H J; Strauss, Kevin A

    2008-12-01

    Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.

  6. Identifying potential functional impact of mutations and polymorphisms: linking heart failure, increased risk of arrhythmias and sudden cardiac death

    PubMed Central

    Jagu, Benoît; Charpentier, Flavien; Toumaniantz, Gilles

    2013-01-01

    Researchers and clinicians have discovered several important concepts regarding the mechanisms responsible for increased risk of arrhythmias, heart failure, and sudden cardiac death. One major step in defining the molecular basis of normal and abnormal cardiac electrical behavior has been the identification of single mutations that greatly increase the risk for arrhythmias and sudden cardiac death by changing channel-gating characteristics. Indeed, mutations in several genes encoding ion channels, such as SCN5A, which encodes the major cardiac Na+ channel, have emerged as the basis for a variety of inherited cardiac arrhythmias such as long QT syndrome, Brugada syndrome, progressive cardiac conduction disorder, sinus node dysfunction, or sudden infant death syndrome. In addition, genes encoding ion channel accessory proteins, like anchoring or chaperone proteins, which modify the expression, the regulation of endocytosis, and the degradation of ion channel a-subunits have also been reported as susceptibility genes for arrhythmic syndromes. The regulation of ion channel protein expression also depends on a fine-tuned balance among different other mechanisms, such as gene transcription, RNA processing, post-transcriptional control of gene expression by miRNA, protein synthesis, assembly and post-translational modification and trafficking. The aim of this review is to inventory, through the description of few representative examples, the role of these different biogenic mechanisms in arrhythmogenesis, HF and SCD in order to help the researcher to identify all the processes that could lead to arrhythmias. Identification of novel targets for drug intervention should result from further understanding of these fundamental mechanisms. PMID:24065925

  7. Deletion of p66Shc in mice increases the frequency of size-change mutations in the lacZ transgene.

    PubMed

    Beltrami, Elena; Ruggiero, Antonella; Busuttil, Rita; Migliaccio, Enrica; Pelicci, Pier Giuseppe; Vijg, Jan; Giorgio, Marco

    2013-04-01

    Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2 O2 . These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism.

  8. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts

    PubMed Central

    Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A.; Verma, Amit; Boultwood, Jacqueline

    2015-01-01

    Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival. PMID:26623729

  9. ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

    PubMed

    Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A; Verma, Amit; Boultwood, Jacqueline

    2015-12-29

    Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

  10. Amyloid-beta (Aβ) D7H mutation increases oligomeric Aβ42 and alters properties of Aβ-zinc/copper assemblies.

    PubMed

    Chen, Wei-Ting; Hong, Chen-Jee; Lin, Ya-Tzu; Chang, Wen-Han; Huang, He-Ting; Liao, Jhih-Ying; Chang, Yu-Jen; Hsieh, Yi-Fang; Cheng, Chih-Ya; Liu, Hsiu-Chih; Chen, Yun-Ru; Cheng, Irene H

    2012-01-01

    Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (Aβ(D7H)), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn(2+) or Cu(2+), Aβ(D7H) aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn(2+) and Cu(2+) in the etiology of AD.

  11. The fitness costs of antibiotic resistance mutations

    PubMed Central

    Melnyk, Anita H; Wong, Alex; Kassen, Rees

    2015-01-01

    Antibiotic resistance is increasing in pathogenic microbial populations and is thus a major threat to public health. The fate of a resistance mutation in pathogen populations is determined in part by its fitness. Mutations that suffer little or no fitness cost are more likely to persist in the absence of antibiotic treatment. In this review, we performed a meta-analysis to investigate the fitness costs associated with single mutational events that confer resistance. Generally, these mutations were costly, although several drug classes and species of bacteria on average did not show a cost. Further investigations into the rate and fitness values of compensatory mutations that alleviate the costs of resistance will help us to better understand both the emergence and management of antibiotic resistance in clinical settings. PMID:25861385

  12. Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.

    PubMed

    Liu, Xia; Zheng, Hong; Li, Xiaobo; Wang, Siying; Meyerson, Howard J; Yang, Wentian; Neel, Benjamin G; Qu, Cheng-Kui

    2016-01-26

    Gain-of-function (GOF) mutations of protein tyrosine phosphatase nonreceptor type 11 Ptpn11 (Shp2), a protein tyrosine phosphatase implicated in multiple cell signaling pathways, are associated with childhood leukemias and solid tumors. The underlying mechanisms are not fully understood. Here, we report that Ptpn11 GOF mutations disturb mitosis and cytokinesis, causing chromosomal instability and greatly increased susceptibility to DNA damage-induced malignancies. We find that Shp2 is distributed to the kinetochore, centrosome, spindle midzone, and midbody, all of which are known to play critical roles in chromosome segregation and cytokinesis. Mouse embryonic fibroblasts with Ptpn11 GOF mutations show a compromised mitotic checkpoint. Centrosome amplification and aberrant mitosis with misaligned or lagging chromosomes are significantly increased in Ptpn11-mutated mouse and patient cells. Abnormal cytokinesis is also markedly increased in these cells. Further mechanistic analyses reveal that GOF mutant Shp2 hyperactivates the Polo-like kinase 1 (Plk1) kinase by enhancing c-Src kinase-mediated tyrosine phosphorylation of Plk1. This study provides novel insights into the tumorigenesis associated with Ptpn11 GOF mutations and cautions that DNA-damaging treatments in Noonan syndrome patients with germ-line Ptpn11 GOF mutations could increase the risk of therapy-induced malignancies.

  13. Follow #eHealth2011: Measuring the Role and Effectiveness of Online and Social Media in Increasing the Outreach of a Scientific Conference.

    PubMed

    Winandy, Marcel; Kostkova, Patty; de Quincey, Ed; St Louis, Connie; Szomszor, Martin

    2016-07-19

    Social media promotion is increasingly adopted by organizers of industry and academic events; however, the success of social media strategies is rarely questioned or the real impact scientifically analyzed. We propose a framework that defines and analyses the impact, outreach, and effectiveness of social media for event promotion and research dissemination to participants of a scientific event as well as to the virtual audience through the Web. Online communication channels Twitter, Facebook, Flickr, and a Liveblog were trialed and their impact measured on outreach during five phases of an eHealth conference: the setup, active and last-minute promotion phases before the conference, the actual event, and after the conference. Planned outreach through online channels and social media before and during the event reached an audience several magnitudes larger in size than would have been possible using traditional means. In the particular case of eHealth 2011, the outreach using traditional means would have been 74 attendees plus 23 extra as sold proceedings and the number of downloaded articles from the online proceedings (4107 until October 2013). The audience for the conference reached via online channels and social media was estimated at more than 5300 in total during the event. The role of Twitter for promotion before the event was complemented by an increased usage of the website and Facebook during the event followed by a sharp increase of views of posters on Flickr after the event. Although our case study is focused on a particular audience around eHealth 2011, our framework provides a template for redefining "audience" and outreach of events, merging traditional physical and virtual communities and providing an outline on how these could be successfully reached in clearly defined event phases.

  14. Follow #eHealth2011: Measuring the Role and Effectiveness of Online and Social Media in Increasing the Outreach of a Scientific Conference

    PubMed Central

    Winandy, Marcel; St Louis, Connie; Szomszor, Martin

    2016-01-01

    Background Social media promotion is increasingly adopted by organizers of industry and academic events; however, the success of social media strategies is rarely questioned or the real impact scientifically analyzed. Objective We propose a framework that defines and analyses the impact, outreach, and effectiveness of social media for event promotion and research dissemination to participants of a scientific event as well as to the virtual audience through the Web. Methods Online communication channels Twitter, Facebook, Flickr, and a Liveblog were trialed and their impact measured on outreach during five phases of an eHealth conference: the setup, active and last-minute promotion phases before the conference, the actual event, and after the conference. Results Planned outreach through online channels and social media before and during the event reached an audience several magnitudes larger in size than would have been possible using traditional means. In the particular case of eHealth 2011, the outreach using traditional means would have been 74 attendees plus 23 extra as sold proceedings and the number of downloaded articles from the online proceedings (4107 until October 2013). The audience for the conference reached via online channels and social media was estimated at more than 5300 in total during the event. The role of Twitter for promotion before the event was complemented by an increased usage of the website and Facebook during the event followed by a sharp increase of views of posters on Flickr after the event. Conclusions Although our case study is focused on a particular audience around eHealth 2011, our framework provides a template for redefining “audience” and outreach of events, merging traditional physical and virtual communities and providing an outline on how these could be successfully reached in clearly defined event phases. PMID:27436012