Kinetics of mutation induction by ultraviolet light in excision-deficient yeast.

Science.gov (United States)

Eckardt, F; Haynes, R H

1977-02-01

We have measured the frequency of UV-induced reversions (locus plus suppressor) for the ochre alleles ade2-1 and lys2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad2-20). For very low UV doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm2 (25% survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm2 (4.4% survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a "single event" Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25% less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29% greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth.

Kinetics of mutation induction by ultraviolet light in excision-deficient yeast

International Nuclear Information System (INIS)

Eckardt, F.; Haynes, R.H.

1977-01-01

We have measured the frequency of uv-induced reversions (locus plus suppressor) for the ochre alleles ade 2-1 and lys 2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad 2-20). For very low uv doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm 2 (25 percent survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm 2 (4.4 percent survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a ''single event'' Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25 percent less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29 percent greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth

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

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S. Domenice

2004-01-01

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

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

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Elisabeth Humphris-Narayanan

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

Hidden Randomness between Fitness Landscapes Limits Reverse Evolution

Science.gov (United States)

Tan, Longzhi; Serene, Stephen; Xiao Chao, Hui; Gore, Jeff

2012-02-01

Natural populations must constantly adapt to the ever-changing environment. A fundamental question in evolutionary biology is whether adaptations can be reversed by returning the population to its ancestral environment. Traditionally, reverse evolution is defined as restoring an ancestral phenotype (physical characteristics such as body size), and the classic Dollo's Law has hypothesized the impossibility of reversing complex adaptations. However, this ``law'' remains ambiguous unless reverse evolution can be studied at the level of genotypes (the underlying genome sequence). We measured the fitness landscapes of a bacterial antibiotic-resistance gene and analyzed the reversibility of evolution as a global, statistical feature of the landscapes. In both experiments and simulations, we find that an adaptation's reversibility declines as the number of mutations it involves increases, suggesting a probabilistic form of Dollo's Law at the molecular level. We also show computationally that slowly switching between environments facilitates reverse evolution in small populations, where clonal interference is negligible or moderate. This is an analogy to thermodynamics, where the reversibility of a physical process is maximized when conditions are modified infinitely slowly.

Ultraviolet-induced reversion of cyc1 alleles in radiation-sensitive strains of yeast. III. rev 3 mutant strains

International Nuclear Information System (INIS)

Lawrence, C.W.; Crhistensen, R.B.

1979-01-01

The role of rev3 gene function in uv-induced mutagenesis in the yeast Saccharomyces cerevisiae has been examined by determining the reversion of 12 well-defined cyc1 mutations in diploid strains homozygous for the rev3-1 or rev3-3 allale. The 12 cyc1 alleles include one ochre, one amber, four initiation, two proline missense, and four frameshift mutations. We find that the rev3 mutations reduce the frequency of UV-induced reversion of all of the cyc1 alleles, though different classes of alleles respond to a different extent. These results imply that the rev3 gene function is required for the production of a wide variety of mutational events, though probably not all, and show that each of the three rev loci have different mutational phenotypes. Such diverse phenotypes are not predicted by the unitary model for bacterial mutagenes, suggesting that this is at best an incomplete description of eukaryotic mutagenesis

Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames Test

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Eliana Aparecida Varanda

2012-05-01

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

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

NARCIS (Netherlands)

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

2013-01-01

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

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

International Nuclear Information System (INIS)

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

1988-01-01

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

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

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Erich Heidenreich

2016-12-01

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

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

Science.gov (United States)

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

2013-11-01

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

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

Science.gov (United States)

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

2016-09-15

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

A novel ion-beam-mutation effect application in identification of gene involved in bacterial antagonism to fungal infection of ornamental crops

Energy Technology Data Exchange (ETDEWEB)

Mahadtanapuk, S. [Faculty of Agriculture and Natural Resources, University of Phayao, Maeka, Muang, Phayao 56000 (Thailand); Teraarusiri, W. [Central Laboratory, University of Phayao, Maeka, Muang, Phayao 56000 (Thailand); Nanakorn, W. [The Crown Property Bureau, 173 Nakhonratchasrima Road, Dusit, Bangkok 10300 (Thailand); Yu, L.D., E-mail: yuld@thep-center.org [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Thongkumkoon, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S., E-mail: soanu.1@gmail.com [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

2014-05-01

Highlights: • Ion beam bombardment induced mutation in bacterial B. licheniformis. • A mutant lost antifungal activity. • DNA fingerprint of the mutant was analyzed. • The lost gene was indentified to code for TrxR gene. • TrxR gene from B. licheniformis expressed the flower antagonism to fungi. - Abstract: This work is on a novel application of ion beam effect on biological mutation. Bacillus licheniformis (B. licheniformis) is a common soil bacterium with an antagonistic effect on Curcuma alismatifolia Gagnep. and Chrysanthemum indicum Linn. In an attempt to control fungal diseases of local crops by utilizing B. licheniformis, we carried out gene analysis of the bacterium to understand the bacterial antagonistic mechanism. The bacterial cells were bombarded to induce mutations using nitrogen ion beam. After ion bombardment, DNA analysis revealed that the modified polymorphism fragment present in the wild type was missing in a bacterial mutant which lost the antifungal activity. The fragments conserved in the wild type but lost in the mutant bacteria was identified to code for the thioredoxin reductase (TrxR) gene. The gene analysis showed that the TrxR gene from B. licheniformis had the expression of the antagonism to fungi in a synchronous time evolution with the fungus inhibition when the bacteria were co-cultivated with the fungi. The collective results indicate the TrxR gene responsible for the antagonism of bacteria B. licheniformis to fungal infection.

A novel ion-beam-mutation effect application in identification of gene involved in bacterial antagonism to fungal infection of ornamental crops

International Nuclear Information System (INIS)

Mahadtanapuk, S.; Teraarusiri, W.; Nanakorn, W.; Yu, L.D.; Thongkumkoon, P.; Anuntalabhochai, S.

2014-01-01

Highlights: • Ion beam bombardment induced mutation in bacterial B. licheniformis. • A mutant lost antifungal activity. • DNA fingerprint of the mutant was analyzed. • The lost gene was indentified to code for TrxR gene. • TrxR gene from B. licheniformis expressed the flower antagonism to fungi. - Abstract: This work is on a novel application of ion beam effect on biological mutation. Bacillus licheniformis (B. licheniformis) is a common soil bacterium with an antagonistic effect on Curcuma alismatifolia Gagnep. and Chrysanthemum indicum Linn. In an attempt to control fungal diseases of local crops by utilizing B. licheniformis, we carried out gene analysis of the bacterium to understand the bacterial antagonistic mechanism. The bacterial cells were bombarded to induce mutations using nitrogen ion beam. After ion bombardment, DNA analysis revealed that the modified polymorphism fragment present in the wild type was missing in a bacterial mutant which lost the antifungal activity. The fragments conserved in the wild type but lost in the mutant bacteria was identified to code for the thioredoxin reductase (TrxR) gene. The gene analysis showed that the TrxR gene from B. licheniformis had the expression of the antagonism to fungi in a synchronous time evolution with the fungus inhibition when the bacteria were co-cultivated with the fungi. The collective results indicate the TrxR gene responsible for the antagonism of bacteria B. licheniformis to fungal infection

Bacterial lipoprotein-induced tolerance is reversed by overexpression of IRAK-1.

LENUS (Irish Health Repository)

Li, Chong Hui

2012-03-01

Tolerance to bacterial cell wall components including bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Reduced Toll-like receptor 2 (TLR2) and IL-1 receptor-associated kinase 1 (IRAK-1) expression is a characteristic of the downregulated TLR signaling pathway observed in BLP-tolerised cells. In this study, we attempted to clarify whether TLR2 and\\/or IRAK-1 are the key molecules responsible for BLP-induced tolerance. Transfection of HEK293 cells and THP-1 cells with the plasmid encoding TLR2 affected neither BLP tolerisation-induced NF-κB deactivation nor BLP tolerisation-attenuated pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) production, indicating that BLP tolerance develops despite overexpression of TLR2 in these cells. In contrast, overexpression of IRAK-1 reversed BLP-induced tolerance, as transfection of IRAK-1 expressing vector resulted in a dose-dependent NF-κB activation and TNF-α release in BLP-tolerised cells. Furthermore, BLP-tolerised cells exhibited markedly repressed NF-κB p65 phosphorylation and impaired binding of p65 to several pro-inflammatory cytokine gene promoters including TNF-α and interleukin-6 (IL-6). Overexpression of IRAK-1 restored the nuclear transactivation of p65 at both TNF-α and IL-6 promoters. These results indicate a crucial role for IRAK-1 in BLP-induced tolerance, and suggest IRAK-1 as a potential target for manipulation of the TLR-mediated inflammatory response during microbial sepsis.

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli.

Science.gov (United States)

Qiu, Haixiang; Gong, Jiansen; Butaye, Patrick; Lu, Guangwu; Huang, Ke; Zhu, Guoqiang; Zhang, Jilei; Hathcock, Terri; Cheng, Darong; Wang, Chengming

2018-05-14

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Auto-production of biosurfactants reverses the coffee ring effect in a bacterial system

Science.gov (United States)

Sempels, Wouter; de Dier, Raf; Mizuno, Hideaki; Hofkens, Johan; Vermant, Jan

2013-04-01

The deposition of material at the edge of evaporating droplets, known as the ‘coffee ring effect’, is caused by a radially outward capillary flow. This phenomenon is common to a wide array of systems including colloidal and bacterial systems. The role of surfactants in counteracting these coffee ring depositions is related to the occurrence of local vortices known as Marangoni eddies. Here we show that these swirling flows are universal, and not only lead to a uniform deposition of colloids but also occur in living bacterial systems. Experiments on Pseudomonas aeruginosa suggest that the auto-production of biosurfactants has an essential role in creating a homogeneous deposition of the bacteria upon drying. Moreover, at biologically relevant conditions, intricate time-dependent flows are observed in addition to the vortex regime, which are also effective in reversing the coffee ring effect at even lower surfactant concentrations.

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

Science.gov (United States)

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

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

International Nuclear Information System (INIS)

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

1995-01-01

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

Quantitative analysis of replication-related mutation and selection pressures in bacterial chromosomes and plasmids using generalised GC skew index

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Suzuki Haruo

2009-12-01

Full Text Available Abstract Background Due to their bi-directional replication machinery starting from a single finite origin, bacterial genomes show characteristic nucleotide compositional bias between the two replichores, which can be visualised through GC skew or (C-G/(C+G. Although this polarisation is used for computational prediction of replication origins in many bacterial genomes, the degree of GC skew visibility varies widely among different species, necessitating a quantitative measurement of GC skew strength in order to provide confidence measures for GC skew-based predictions of replication origins. Results Here we discuss a quantitative index for the measurement of GC skew strength, named the generalised GC skew index (gGCSI, which is applicable to genomes of any length, including bacterial chromosomes and plasmids. We demonstrate that gGCSI is independent of the window size and can thus be used to compare genomes with different sizes, such as bacterial chromosomes and plasmids. It can suggest the existence of different replication mechanisms in archaea and of rolling-circle replication in plasmids. Correlation of gGCSI values between plasmids and their corresponding host chromosomes suggests that within the same strain, these replicons have reproduced using the same replication machinery and thus exhibit similar strengths of replication strand skew. Conclusions gGCSI can be applied to genomes of any length and thus allows comparative study of replication-related mutation and selection pressures in genomes of different lengths such as bacterial chromosomes and plasmids. Using gGCSI, we showed that replication-related mutation or selection pressure is similar for replicons with similar machinery.

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

Energy Technology Data Exchange (ETDEWEB)

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

1995-08-01

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

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

Science.gov (United States)

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

2015-01-01

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

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

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Mark A Winters

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

A novel ion-beam-mutation effect application in identification of gene involved in bacterial antagonism to fungal infection of ornamental crops

Science.gov (United States)

Mahadtanapuk, S.; Teraarusiri, W.; Nanakorn, W.; Yu, L. D.; Thongkumkoon, P.; Anuntalabhochai, S.

2014-05-01

This work is on a novel application of ion beam effect on biological mutation. Bacillus licheniformis (B. licheniformis) is a common soil bacterium with an antagonistic effect on Curcuma alismatifolia Gagnep. and Chrysanthemum indicum Linn. In an attempt to control fungal diseases of local crops by utilizing B. licheniformis, we carried out gene analysis of the bacterium to understand the bacterial antagonistic mechanism. The bacterial cells were bombarded to induce mutations using nitrogen ion beam. After ion bombardment, DNA analysis revealed that the modified polymorphism fragment present in the wild type was missing in a bacterial mutant which lost the antifungal activity. The fragments conserved in the wild type but lost in the mutant bacteria was identified to code for the thioredoxin reductase (TrxR) gene. The gene analysis showed that the TrxR gene from B. licheniformis had the expression of the antagonism to fungi in a synchronous time evolution with the fungus inhibition when the bacteria were co-cultivated with the fungi. The collective results indicate the TrxR gene responsible for the antagonism of bacteria B. licheniformis to fungal infection.

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

Science.gov (United States)

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

2013-07-01

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

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

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

2017-12-15

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

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

Science.gov (United States)

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

2012-05-01

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

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

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

2014-04-01

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

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

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Su X

2016-11-01

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

Over-additive increase of bacterial mutations by combined action of ultraviolet light and alkylation

International Nuclear Information System (INIS)

Herrmann, S.; Hotz, G.; Herrlich, P.

1982-05-01

Mutagenic agents added in combination may contribute to an overall biological effect in proportion to the effect they would have if given individually. The combined effect may, however, not just be additive but rather result in a response above or below expectation if the two mutagenic pathways interacted at some level. We report here on one such example. Ultraviolet light and subsequent treatment with the alkylating agent ethylmethane-sulfonate (EMS) led to an over-additive increase of bacterial mutations. This is interesting with respect to unravelling the level mutagens interact. In addition such data may relate to the human situation which is only in the process of being assessed. (orig.) [de

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

Indian Academy of Sciences (India)

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

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

Science.gov (United States)

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

2011-12-01

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

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

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Yasushi Ogawa

2014-05-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1982-01-01

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

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

Science.gov (United States)

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

2016-05-01

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

A new experimental system for study on adaptive mutations

Institute of Scientific and Technical Information of China (English)

Lü; Zhong; (

2001-01-01

［1］Luria, S. E., Delbrück, M., Mutation of bacteria from virus sensitivity to virus resistance, Genetics, 1943, 28: 491.［2］Lederberg, J., Lederberg, E. M., Replica plating and indirect selection of bacteria mutants, J. Bacteriol., 1952, 63: 399.［3］Carins, J., Overbaugh, J., Miller, S., The origin of mutants, Nature, 1988, 355: 142.［4］Foster, P. L., Adaptive mutation: the uses of adversity, Annu. Rev. Microbiol., 1993, 47: 467.［5］Hall, B. G., Adaptive mutagenesis: a process that generates almost exclusively beneficient mutations, Genetica, 1998, 102/103: 109.［6］Kasak, L., Horak, R., Kivisaar, M., Promotor-creating mutations in Psuedmonas putida: A model system for the study of mutation in starving bacteria, PNAS, 1997, 94: 3134.［7］Steele, D. F., Jinks-Robertson, An examination of adaptive reversion in Saccharomyces cerevisiae, Genetics, 1992, 132: 9.［8］Davis, R. W., Botstein, Roth, J. R., Advanced Bacterial Genetics--A Manual for Genetic Engineering, New York: Cold Spring Harbor Laboratory Press, 1980, 13.［9］Miller, J. H., Experiments in Molecular Genetics, New York: Cold Spring Harbor Laboratory Press, 1972, 352.［10］ Lea, D. E., Coulson, C. A., The distribution of the numbers of mutants in bacterial populations, J. Genetics, 1949, 49: 264.［11］ Hughes, K. T., Roth, J. R., Transitory cis complementation: a method for provided transposition function to defective transposons, Genetics, 1988, 119: 9.［12］ Sanderson, K. E., Roth J., Linkage map of Salmonella typhimurium, Microbiol. Rev., 1988, 52: 485.［13］ He, B., Shiau, A., Choi, K. Y., Genes of the E. coli pur region are negatively controlled by a repressor-operator interaction, J. Bacteriol., 1990, 172: 4555.［14］ Liu, B., Huang, Y., Wang, A. Q., Regulation of purine biosynthetic genes expression in Salmonella typhimurium (IV)--Oc mutation site of purG and its function analysis, Science in China, Ser. C, 1997, 40(3): 238.［15］ Tang, H., Qin, J. C., Wang, A. Q

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

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

2017-11-08

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

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

Science.gov (United States)

2016-09-07

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

Generation of mutation hotspots in ageing bacterial colonies

DEFF Research Database (Denmark)

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

2016-01-01

: most mutations were located in just a few hotspots in the genome, and over time, mutations increasingly were consistent with the involvement of 8-oxo-guanosine, formed exclusively on the transcribed strand. This work provides strong support for retromutagenesis as a general process creating adaptive...

Mutator activity in Schizophyllum commune

Energy Technology Data Exchange (ETDEWEB)

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

1983-01-01

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

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

Directory of Open Access Journals (Sweden)

Jie Xu

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

Modification of mutation frequency in Saccharomyces Cerevisiae

International Nuclear Information System (INIS)

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

1976-01-01

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

The use of a cloned bacterial gene to study mutation in mammalian cells

International Nuclear Information System (INIS)

Thacker, J.; Debenham, P.G.; Stretch, A.; Webb, M.B.T.

1983-01-01

The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. (orig./AJ)

Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

DEFF Research Database (Denmark)

Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

2015-01-01

Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types...

A case of reversible posterior leukoencephalopathy syndrome which developed during chemoradiotherapy for head and neck cancer. The involvement of bacterial translocation was considered

International Nuclear Information System (INIS)

Tachibana, Shinya; Terao, Hajime; Sanbe, Takeyuki; Katsuno, Masahiro; Takemura, Hideki

2007-01-01

Combination therapy such as chemotherapy and radiotherapy is often chosen, depending on the case, for head and neck cancer in view of the preservation of potency. However, on the other hand, it is necessary to note the onset of therapeutic side effects. The patient was a 35-year-old woman. During chemoradiotherapy for mesopharyngeal carcinoma, she suddenly developed shock and multiple organ failure, requiring intensive treatment. She also developed reversible central nerve symptoms during the course. The involvement of bacterial translocation was thought to be the cause of shock, and the reversible central nerve symptoms were considered to be a pathological condition, known as reversible posterior leukoencephalopathy syndrome. We discuss these conditions on the basis of the clinical features, and the process that led to diagnosis in this case. (author)

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

Science.gov (United States)

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

2015-06-01

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

Adaptive mutation: has the unicorn landed?

Science.gov (United States)

Foster, P L

1998-04-01

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

Adaptive mutation: has the unicorn landed?

Science.gov (United States)

Foster, P L

1998-01-01

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

Predictable Phenotypes of Antibiotic Resistance Mutations.

Science.gov (United States)

Knopp, M; Andersson, D I

2018-05-15

Antibiotic-resistant bacteria represent a major threat to our ability to treat bacterial infections. Two factors that determine the evolutionary success of antibiotic resistance mutations are their impact on resistance level and the fitness cost. Recent studies suggest that resistance mutations commonly show epistatic interactions, which would complicate predictions of their stability in bacterial populations. We analyzed 13 different chromosomal resistance mutations and 10 host strains of Salmonella enterica and Escherichia coli to address two main questions. (i) Are there epistatic interactions between different chromosomal resistance mutations? (ii) How does the strain background and genetic distance influence the effect of chromosomal resistance mutations on resistance and fitness? Our results show that the effects of combined resistance mutations on resistance and fitness are largely predictable and that epistasis remains rare even when up to four mutations were combined. Furthermore, a majority of the mutations, especially target alteration mutations, demonstrate strain-independent phenotypes across different species. This study extends our understanding of epistasis among resistance mutations and shows that interactions between different resistance mutations are often predictable from the characteristics of the individual mutations. IMPORTANCE The spread of antibiotic-resistant bacteria imposes an urgent threat to public health. The ability to forecast the evolutionary success of resistant mutants would help to combat dissemination of antibiotic resistance. Previous studies have shown that the phenotypic effects (fitness and resistance level) of resistance mutations can vary substantially depending on the genetic context in which they occur. We conducted a broad screen using many different resistance mutations and host strains to identify potential epistatic interactions between various types of resistance mutations and to determine the effect of strain

Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis.

Science.gov (United States)

Be'er, Avraham; Florin, E-L; Fisher, Carolyn R; Swinney, Harry L; Payne, Shelley M

2011-01-01

Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting

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

Science.gov (United States)

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

2017-11-22

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

Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription-PCR in temperature gradient gel electrophoresis fingerprints.

Science.gov (United States)

Felske, A; Akkermans, A D; De Vos, W M

1998-11-01

A novel approach was developed to quantify rRNA sequences in complex bacterial communities. The main bacterial 16S rRNAs in Drentse A grassland soils (The Netherlands) were amplified by reverse transcription (RT)-PCR with bacterium-specific primers and were separated by temperature gradient gel electrophoresis (TGGE). The primer pair used (primers U968-GC and L1401) was found to amplify with the same efficiency 16S rRNAs from bacterial cultures containing different taxa and cloned 16S ribosomal DNA amplicons from uncultured soil bacteria. The sequence-specific efficiency of amplification was determined by monitoring the amplification kinetics by kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs resulted in identical amplicon yields. The sequence-specific detection system used for competitive amplifications was TGGE, which also has been found to be suitable for simultaneous quantification of more than one sequence. We demonstrate that this approach can be applied to TGGE fingerprints of soil bacteria to estimate the ratios of the bacterial 16S rRNAs.

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

Science.gov (United States)

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

2015-01-01

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

A Mutator Phenotype Promoting the Emergence of Spontaneous Oxidative Stress-Resistant Mutants in Campylobacter jejuni.

Science.gov (United States)

Dai, Lei; Sahin, Orhan; Tang, Yizhi; Zhang, Qijing

2017-12-15

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress-resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OX R ) in C. jejuni We further demonstrated that MutY malfunction did not directly contribute to the OX R phenotype but increased the spontaneous mutation rate in the peroxide regulator gene perR , which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OX R phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OX R mutants in bacterial organisms. IMPORTANCE Although a mutator phenotype has been shown to promote antibiotic resistance in many bacterial species, little is known about its contribution to the emergence of OX R mutants. This work describes the link between a mutator phenotype and the enhanced emergence of OX R mutants as well as its underlying mechanism involving DNA repair and mutations in PerR. Since DNA repair systems and PerR are well conserved in many bacterial species, especially in Gram positives, the same mechanism may operate in multiple bacterial species. Additionally, we developed a novel method that allows for rapid quantification of spontaneous OX R mutants in a bacterial population. This method represents a technical

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

Science.gov (United States)

Li, Jianwei; Handler, Alfred M

2017-09-28

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

An alternative derivation of the stationary distribution of the multivariate neutral Wright-Fisher model for low mutation rates with a view to mutation rate estimation from site frequency data.

Science.gov (United States)

Schrempf, Dominik; Hobolth, Asger

2017-04-01

Recently, Burden and Tang (2016) provided an analytical expression for the stationary distribution of the multivariate neutral Wright-Fisher model with low mutation rates. In this paper we present a simple, alternative derivation that illustrates the approximation. Our proof is based on the discrete multivariate boundary mutation model which has three key ingredients. First, the decoupled Moran model is used to describe genetic drift. Second, low mutation rates are assumed by limiting mutations to monomorphic states. Third, the mutation rate matrix is separated into a time-reversible part and a flux part, as suggested by Burden and Tang (2016). An application of our result to data from several great apes reveals that the assumption of stationarity may be inadequate or that other evolutionary forces like selection or biased gene conversion are acting. Furthermore we find that the model with a reversible mutation rate matrix provides a reasonably good fit to the data compared to the one with a non-reversible mutation rate matrix. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2011-11-01

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

The perturbation of tryptophan fluorescence by phenylalanine to alanine mutations identifies the hydrophobic core in a subset of bacterial Ig-like domains.

Science.gov (United States)

Raman, Rajeev; Ptak, Christopher P; Hsieh, Ching-Lin; Oswald, Robert E; Chang, Yung-Fu; Sharma, Yogendra

2013-07-09

Many host-parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin.

Reversals and collisions optimize protein exchange in bacterial swarms

Energy Technology Data Exchange (ETDEWEB)

Amiri, Aboutaleb; Harvey, Cameron; Buchmann, Amy; Christley, Scott; Shrout, Joshua D.; Aranson, Igor S.; Alber, Mark

2017-03-01

Swarming groups of bacteria coordinate their behavior by self-organizing as a population to move over surfaces in search of nutrients and optimal niches for colonization. Many open questions remain about the cues used by swarming bacteria to achieve this self-organization. While chemical cue signaling known as quorum sensing is well-described, swarming bacteria often act and coordinate on time scales that could not be achieved via these extracellular quorum sensing cues. Here, cell-cell contact-dependent protein exchange is explored as amechanism of intercellular signaling for the bacterium Myxococcus xanthus. A detailed biologically calibrated computational model is used to study how M. xanthus optimizes the connection rate between cells and maximizes the spread of an extracellular protein within the population. The maximum rate of protein spreading is observed for cells that reverse direction optimally for swarming. Cells that reverse too slowly or too fast fail to spread extracellular protein efficiently. In particular, a specific range of cell reversal frequencies was observed to maximize the cell-cell connection rate and minimize the time of protein spreading. Furthermore, our findings suggest that predesigned motion reversal can be employed to enhance the collective behavior of biological synthetic active systems.

Induction of spontaneous and UV-induced mutations during commitment to meiosis in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Machida, I.; Nakai, S.

1980-01-01

Inductions of reversions of nonsense, missense and frameshift-type mutations were investigated in a diploid cell population of Saccharomyces cerevisiae during commitment to meiosis, by using the medium-transfer technique from sporulation medium to vegetative medium. The yields of spontaneous reverse mutations obtained from the cells that were committed to different stages during meiosis were rather constant irrespective of the alleles tested, although the yields of both intergenic and intragenic recombinations markedly increased. The susceptibilities to UV-induced reverse mutations examined during commitment to meiosis were not changed appreciably. It is concluded that induction of base-change-type mutations in meiosis is not essentially different from that in mitosis. (orig.)

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

International Nuclear Information System (INIS)

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

2004-01-01

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

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

Directory of Open Access Journals (Sweden)

Jessica H Brehm

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

Is low-energy-ion bombardment generated X-ray emission a secondary mutational source to ion-beam-induced genetic mutation?

Energy Technology Data Exchange (ETDEWEB)

Thongkumkoon, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Thopan, P.; Yaopromsiri, C. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Suwannakachorn, D. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

2013-07-15

Highlights: ► Detected X-ray emission from metal, plastic and biological samples. ► Characteristic X-ray emission was detected from metal but not from non-metals. ► Low-energy ion bombarded bacteria held in different sample holders. ► Bacteria held in metal holder had higher mutation rate than in plastic holder. ► Ion-beam-induced X-ray from biological sample is not a basic mutation source. -- Abstract: Low-energy ion beam biotechnology has achieved tremendous successes in inducing crop mutation and gene transfer. However, mechanisms involved in the related processes are not yet well understood. In ion-beam-induced mutation, ion-bombardment-produced X-ray has been proposed to be one of the secondary mutation sources, but the speculation has not yet been experimentally tested. We carried out this investigation to test whether the low-energy ion-beam-produced X-ray was a source of ion-beam-induced mutation. In the investigation, X-ray emission from 29-keV nitrogen- or argon- ion beam bombarded bacterial Escherichia coli (E. coli) cells held in a metal or plastic sample holder was in situ detected using a highly sensitive X-ray detector. The ion beam bombarded bacterial cells held in different material holders were observed for mutation induction. The results led to a conclusion that secondary X-ray emitted from ion-beam-bombarded biological living materials themselves was not a, or at least a negligible, mutational source, but the ion-beam-induced X-ray emission from the metal that made the sample holder could be a source of mutation.

Is low-energy-ion bombardment generated X-ray emission a secondary mutational source to ion-beam-induced genetic mutation?

International Nuclear Information System (INIS)

Thongkumkoon, P.; Prakrajang, K.; Thopan, P.; Yaopromsiri, C.; Suwannakachorn, D.; Yu, L.D.

2013-01-01

Highlights: ► Detected X-ray emission from metal, plastic and biological samples. ► Characteristic X-ray emission was detected from metal but not from non-metals. ► Low-energy ion bombarded bacteria held in different sample holders. ► Bacteria held in metal holder had higher mutation rate than in plastic holder. ► Ion-beam-induced X-ray from biological sample is not a basic mutation source. -- Abstract: Low-energy ion beam biotechnology has achieved tremendous successes in inducing crop mutation and gene transfer. However, mechanisms involved in the related processes are not yet well understood. In ion-beam-induced mutation, ion-bombardment-produced X-ray has been proposed to be one of the secondary mutation sources, but the speculation has not yet been experimentally tested. We carried out this investigation to test whether the low-energy ion-beam-produced X-ray was a source of ion-beam-induced mutation. In the investigation, X-ray emission from 29-keV nitrogen- or argon- ion beam bombarded bacterial Escherichia coli (E. coli) cells held in a metal or plastic sample holder was in situ detected using a highly sensitive X-ray detector. The ion beam bombarded bacterial cells held in different material holders were observed for mutation induction. The results led to a conclusion that secondary X-ray emitted from ion-beam-bombarded biological living materials themselves was not a, or at least a negligible, mutational source, but the ion-beam-induced X-ray emission from the metal that made the sample holder could be a source of mutation

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Science.gov (United States)

2010-07-01

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

The neglected intrinsic resistome of bacterial pathogens.

Directory of Open Access Journals (Sweden)

Alicia Fajardo

Full Text Available Bacteria with intrinsic resistance to antibiotics are a worrisome health problem. It is widely believed that intrinsic antibiotic resistance of bacterial pathogens is mainly the consequence of cellular impermeability and activity of efflux pumps. However, the analysis of transposon-tagged Pseudomonas aeruginosa mutants presented in this article shows that this phenotype emerges from the action of numerous proteins from all functional categories. Mutations in some genes make P. aeruginosa more susceptible to antibiotics and thereby represent new targets. Mutations in other genes make P. aeruginosa more resistant and therefore define novel mechanisms for mutation-driven acquisition of antibiotic resistance, opening a new research field based in the prediction of resistance before it emerges in clinical environments. Antibiotics are not just weapons against bacterial competitors, but also natural signalling molecules. Our results demonstrate that antibiotic resistance genes are not merely protective shields and offer a more comprehensive view of the role of antibiotic resistance genes in the clinic and in nature.

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

NARCIS (Netherlands)

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

2017-01-01

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

Mutation induction in Haemophilus influenzae by ICR-191. Pt. 1

International Nuclear Information System (INIS)

Perdue, S.W.; Kimball, R.F.; McGray, P.C.; Tennessee Univ., Oak Ridge

1981-01-01

The investigation of mutagenic mechanisms in Haemophilus influenzae has been confined until now to mutagens that normally produce mainly base pair substitutions. This paper describes the development of a system suitable for detecting frameshift mutations induced by ICR-191. The system involves reversions from thymidine dependence to thymidine independence. Evidence is presented from a comparison of the responses to ICR-191 and to N-methyl-N'-nitro-N-nitrosoguanidine that the system is specific for frameshift mutations. The genetic recombination involved in transformation leads to a marked increase in spontaneous reversion of the frameshift mutations but not of the base substitution mutations. Presumably, this is a consequence of mispairing, with consequent change in the number of bases, during the recombination. (orig.)

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

Science.gov (United States)

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

2018-05-23

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

Repair-resistant mutation in Neurospora

International Nuclear Information System (INIS)

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

1987-01-01

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

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

Indian Academy of Sciences (India)

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

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

Directory of Open Access Journals (Sweden)

Mohammad A. Alqahtani

2015-01-01

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

Reversible optic neuropathy with OPA1 exon 5b mutation

DEFF Research Database (Denmark)

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

2008-01-01

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

Functional recovery of biofilm bacterial communities after copper exposure

International Nuclear Information System (INIS)

Boivin, Marie-Elene Y.; Massieux, Boris; Breure, Anton M.; Greve, Gerdit D.; Rutgers, Michiel; Admiraal, Wim

2006-01-01

Potential of bacterial communities in biofilms to recover after copper exposure was investigated. Biofilms grown outdoor in shallow water on glass dishes were exposed in the laboratory to 0.6, 2.1, 6.8 μmol/l copper amended surface water and a reference and subsequently to un-amended surface water. Transitions of bacterial communities were characterised with denaturing gradient gel electrophoresis (DGGE) and community-level physiological profiles (CLPP). Exposure to 6.8 μmol/l copper provoked distinct changes in DGGE profiles of bacterial consortia, which did not reverse upon copper depuration. Exposure to 2.1 and 6.8 μmol/l copper was found to induce marked changes in CLPP of bacterial communities that proved to be reversible during copper depuration. Furthermore, copper exposure induced the development of copper-tolerance, which was partially lost during depuration. It is concluded that bacterial communities exposed to copper contaminated water for a period of 26 days are capable to restore their metabolic attributes after introduction of unpolluted water in aquaria for 28 days. - Genetically different bacterial communities can have similar functions and tolerance to copper

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

International Nuclear Information System (INIS)

Kusaba, Makoto

2007-01-01

Ionizing radiation has been used for inducing mutations and improving crops since the discovery by STADLER (1928) that X-rays could induce mutations in barley. At the end of 2004, the whole genome sequence of rice was determined (INTERNATIONAL RICE GENOME SEQUENCING PROJECT, 2005). What can γ-ray-induced mutations contribute now that this has been achieved? One answer could be the elucidation of the functions of the numerous genes revealed by the complete sequence of the rice genome. This includes identification of mutants through reverse genetics and the isolation of genes containing mutations through forward genetics using molecular markers and sequence information. Another answer could be mutation breeding using reverse genetics. But first we must know what kind of DNA lesions are caused by γ-rays. In this article, I describe the production of DNA lesions, and then discuss how γ-ray-induced mutations can contribute to the elucidation of gene function and to mutation breeding. (author)

Generation of mutation hotspots in ageing bacterial colonies

DEFF Research Database (Denmark)

Sekowska, Agnieszka; Wendel, Sofie; Nørholm, Morten

How do ageing bacterial colonies generate adaptive mutants? Over a period of two months, we isolated on ageing colonies outgrowing mutants able to use a new carbon source, and sequenced their genomes. This allowed us to uncover exquisite details on the molecular mechanism behind their adaptation:...

TERT promoter mutations are highly recurrent in SHH subgroup medulloblastoma

NARCIS (Netherlands)

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

2013-01-01

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

Assessment of the extent of bacterial growth in reverse osmosis system for improving drinking water quality.

Science.gov (United States)

Park, Se-keun; Hu, Jiang Yong

2010-01-01

This study was carried out to assess reverse osmosis (RO) treatment efficacy of drinking water in terms of biological stability in the distribution system. Two flat-sheet RO membranes were used in this study. Experiments were designed to investigate the growth of biofilm and bulk phase bacteria for the RO-treated water flowing through a model distribution system under controlled conditions without disinfectants. RO membranes improved the water quality of drinking water in terms of inorganic, organic and bacterial contents. Organic matter including the fraction available for microbes was efficiently removed by the RO membranes tested. More than 99% of bacterial cells in the tap water was retained by the RO membranes, leaving water. In spite of the low nutrient contents and few cells in the RO permeates, monitoring of the model distribution systems receiving the RO permeates showed that remarkable biofilm accumulation and bulk cell growth occurred in the RO permeate water. In quasi-steady state, the total cell numbers in the biofilm and bulk water were of order 10(3) cells/cm(2) and 10(3) cells/mL, respectively, which were about 2 orders of magnitude lower than those grown in the tap water produced from conventional water treatment. The culturable heterotrophic bacteria constituted a significant part of the total cells (20.7-32.1% in biofilms and 21.3-46.3% in bulk waters). Biofilm maximum density and production rate were of the order 10(4) cells/cm(2) and 10(2) cells/cm(2)/day, respectively. The specific cell growth rate of bacteria in the biofilms was found to be much lower than those in the bulk waters (0.04-0.05 day(-1) versus 0.28-0.36 day(-1)). The overall specific cell growth rate which indicates the growth potential in the whole system was calculated as 0.07-0.08 day(-1), representing a doubling time of 9.1-10.1 days. These observations can be indicative of possibilities for bacterial growth in the RO permeate water with easily assimilable organic carbon

Seryl-tRNA Synthetases from Methanogenic Archaea: Suppression of Bacterial Amber Mutation and Heterologous Toxicity

Directory of Open Access Journals (Sweden)

Drasko Boko

2010-01-01

Full Text Available Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS, evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.

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

DEFF Research Database (Denmark)

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

2015-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-04-10

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

Rare beneficial mutations can halt Muller's ratchet

Science.gov (United States)

Balick, Daniel; Goyal, Sidhartha; Jerison, Elizabeth; Neher, Richard; Shraiman, Boris; Desai, Michael

2012-02-01

In viral, bacterial, and other asexual populations, the vast majority of non-neutral mutations are deleterious. This motivates the application of models without beneficial mutations. Here we show that the presence of surprisingly few compensatory mutations halts fitness decay in these models. Production of deleterious mutations is balanced by purifying selection, stabilizing the fitness distribution. However, stochastic vanishing of fitness classes can lead to slow fitness decay (i.e. Muller's ratchet). For weakly deleterious mutations, production overwhelms purification, rapidly decreasing population fitness. We show that when beneficial mutations are introduced, a stable steady state emerges in the form of a dynamic mutation-selection balance. We argue this state is generic for all mutation rates and population sizes, and is reached as an end state as genomes become saturated by either beneficial or deleterious mutations. Assuming all mutations have the same magnitude selective effect, we calculate the fraction of beneficial mutations necessary to maintain the dynamic balance. This may explain the unexpected maintenance of asexual genomes, as in mitochondria, in the presence of selection. This will affect in the statistics of genetic diversity in these populations.

Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

International Nuclear Information System (INIS)

Kato, T.; Nakano, E.

1981-01-01

The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

Bacterial infection increases risk of carcinogenesis by targeting mitochondria

DEFF Research Database (Denmark)

Strickertsson, Jesper A.B.; Desler, Claus; Rasmussen, Lene Juel

2017-01-01

pathways, and compares the impact of the bacterial alteration of mitochondrial function to that of cancer. Bacterial virulence factors have been demonstrated to induce mutations of mitochondrial DNA (mtDNA) and to modulate DNA repair pathways of the mitochondria. Furthermore, virulence factors can induce...... or impair the intrinsic apoptotic pathway. The effect of bacterial targeting of mitochondria is analogous to behavior of mitochondria in a wide array of tumours, and this strongly suggests that mitochondrial targeting of bacteria is a risk factor for carcinogenesis....

CHARACTERIZATION OF ENU-INDUCED MUTATIONS IN RED BLOOD CELL STRUCTURAL PROTEINS

Directory of Open Access Journals (Sweden)

Katrina Kildey

2013-03-01

Full Text Available Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b demonstrated increased RBC count, haemoglobin (Hb and haematocrit (HCT. The third Spnb1 mutation (spectrin-1β c and mutation in Epb4.1 (band 4.1 did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of

76 FR 65385 - Testing of Certain High Production Volume Chemicals; Third Group of Chemicals

Science.gov (United States)

2011-10-21

... Mutations: Bacterial Reverse Mutation Test (in vitro): 40 CFR 799.9510. b. Chromosomal Damage: In Vitro... alternate test. 6. Mammalian Toxicity--Repeated Dose/Reproduction/Developmental--a. Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test: 40 CFR 799.9365. b...

77 FR 28340 - Revocation of TSCA Section 4 Testing Requirements for One High Production Volume Chemical Substance

Science.gov (United States)

2012-05-14

... data on C.I. Pigment Blue 61 found the study on mammalian acute toxicity and the bacterial mutation..., water solubility, biodegradation, fish acute toxicity, mammalian acute toxicity, bacterial reverse... toxicity with a reproduction/developmental toxicity screen. Studies responding to those test requirements...

Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators

DEFF Research Database (Denmark)

Marvig, Rasmus Lykke; Johansen, Helle Krogh; Molin, Søren

2013-01-01

Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic...... targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long...... likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain...

Geomagnetic polarity reversals as a mechanism for the punctuated equilibrium model of biological evolution

International Nuclear Information System (INIS)

Welsh, J.S.; Welsh, A.L.; Welsh, W.F.

2003-01-01

In contrast to what is predicted by classical Darwinian theory (phyletic gradualism), the fossil record typically displays a pattern of relatively sudden, dramatic changes as detailed by Eldregde and Gould's model of punctuated equilibrium. Evolutionary biologists have been at a loss to explain the ultimate source of the new mutations that drive evolution. One hypothesis holds that the abrupt speciation seen in the punctuated equilibrium model is secondary to an increased mutation rate resulting from periodically increased levels of ionizing radiation on the Earth's surface. Sporadic geomagnetic pole reversals, occurring every few million years on the average, are accompanied by alterations in the strength of the Earth's magnetic field and magnetosphere. This diminution may allow charged cosmic radiation to bombard Earth with less attenuation, thereby resulting in increased mutation rates. This episodic fluctuation in the magnetosphere is an attractive mechanism for the observed fossil record. Selected periods and epochs of geologic history for which data was available were reviewed for both geomagnetic pole reversal history and fossil record. Anomalies in either were scrutinized in greater depth and correlations were made. A 35 million year span (118-83 Ma) was identified during the Early/Middle Cretaceous period that was devoid of geomagnetic polarity reversals(the Cretaceous normal superchron). Examination of the fossil record (including several invertebrate and vertebrate taxons) during the Cretaceous normal superchron does not reveal any significant gap or slowing of speciation. Although increased terrestrial radiation exposure due to a diminution of the Earth's magnetosphere caused by a reversal of geomagnetic polarity is an attractive explanation for the mechanism of punctuated equilibrium, our investigation suggests that such polarity reversals cannot fully provide the driving force behind biological evolution. Further research is required to determine if

BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

LENUS (Irish Health Repository)

Stordal, Britta

2013-06-01

Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

Mutation induction in spores of Bacillus subtilis by accelerated very heavy ions

International Nuclear Information System (INIS)

Baltschukat, K.; Horneck, G.; Buecker, H.; Facius, R.; Schaefer, M.

1986-01-01

Mutation induction (resistance to sodium azide) in spores of Bacillus subtilis was investigated after irradiation with heavy ions from Neon to Uranium with specific particle energies between 0.17 and 18.6 MeV/u. A strong dependence of the mutation induction cross section on particle charge and energy was observed. From the results it was concluded that mutation induction in bacterial spores by very heavy ions is mainly caused by secondary electrons. (orig.)

Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli

International Nuclear Information System (INIS)

Hu, S.C.; Court, D.L.; Zweig, M.; Levin, J.G.

1986-01-01

The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated and 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s)

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

International Nuclear Information System (INIS)

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

1999-01-01

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

Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance

DEFF Research Database (Denmark)

Klitgaard, Rasmus N; Ntokou, Eleni; Nørgaard, Katrine

2015-01-01

-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3...

Aphid thermal tolerance is governed by a point mutation in bacterial symbionts.

Directory of Open Access Journals (Sweden)

Helen E Dunbar

2007-05-01

Full Text Available Symbiosis is a ubiquitous phenomenon generating biological complexity, affecting adaptation, and expanding ecological capabilities. However, symbionts, which can be subject to genetic limitations such as clonality and genomic degradation, also impose constraints on hosts. A model of obligate symbiosis is that between aphids and the bacterium Buchnera aphidicola, which supplies essential nutrients. We report a mutation in Buchnera of the aphid Acyrthosiphon pisum that recurs in laboratory lines and occurs in field populations. This single nucleotide deletion affects a homopolymeric run within the heat-shock transcriptional promoter for ibpA, encoding a small heat-shock protein. This Buchnera mutation virtually eliminates the transcriptional response of ibpA to heat stress and lowers its expression even at cool or moderate temperatures. Furthermore, this symbiont mutation dramatically affects host fitness in a manner dependent on thermal environment. Following a short heat exposure as juveniles, aphids bearing short-allele symbionts produced few or no progeny and contained almost no Buchnera, in contrast to aphids bearing symbionts without the deletion. Conversely, under constant cool conditions, aphids containing symbionts with the short allele reproduced earlier and maintained higher reproductive rates. The short allele has appreciable frequencies in field populations (up to 20%, further supporting the view that lowering of ibpA expression improves host fitness under some conditions. This recurring Buchnera mutation governs thermal tolerance of aphid hosts. Other cases in which symbiont microevolution has a major effect on host ecological tolerance are likely to be widespread because of the high mutation rates of symbiotic bacteria and their crucial roles in host metabolism and development.

40 CFR 798.5265 - The salmonella typhimurium reverse mutation assay.

Science.gov (United States)

2010-07-01

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

Impact of bacterial infections on aging and cancer

DEFF Research Database (Denmark)

Strickertsson, Jesper A B; Madsen, Claus Desler; Rasmussen, Lene Juel

2014-01-01

DNA repair subunits of major DNA repair pathways and increase production of reactive oxygen species (ROS). Defects in DNA repair cause mutations and genomic instability and are found in several cancers as well as in progeroid syndromes. This review describes our contemporary view on how bacterial...

Experimental evolution and the dynamics of genomic mutation rate modifiers.

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Raynes, Y; Sniegowski, P D

2014-11-01

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

Quinolone Resistance Reversion by Targeting the SOS Response.

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

2017-10-10

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

IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity

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Chiang, Sarah; Weigelt, Britta; Wen, Huei-Chi; Pareja, Fresia; Raghavendra, Ashwini; Martelotto, Luciano G.; Burke, Kathleen A.; Basili, Thais; Li, Anqi; Geyer, Felipe C.; Piscuoglio, Salvatore; Ng, Charlotte K.Y.; Jungbluth, Achim A.; Balss, Jörg; Pusch, Stefan; Baker, Gabrielle M.; Cole, Kimberly S.; von Deimling, Andreas; Batten, Julie M.; Marotti, Jonathan D.; Soh, Hwei-Choo; McCalip, Benjamin L.; Serrano, Jonathan; Lim, Raymond S.; Siziopikou, Kalliopi P.; Lu, Song; Liu, Xiaolong; Hammour, Tarek; Brogi, Edi; Snuderl, Matija; Iafrate, A. John; Reis-Filho, Jorge S.; Schnitt, Stuart J.

2017-01-01

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. PMID:27913435

Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

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Alessandro Pandini

Full Text Available Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM domains (amino-terminal (FliGN, middle (FliGM and FliGC as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6. FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM

Impact of mutations in Toll-like receptor pathway genes on esophageal carcinogenesis.

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Daffolyn Rachael Fels Elliott

2017-05-01

Full Text Available Esophageal adenocarcinoma (EAC develops in an inflammatory microenvironment with reduced microbial diversity, but mechanisms for these influences remain poorly characterized. We hypothesized that mutations targeting the Toll-like receptor (TLR pathway could disrupt innate immune signaling and promote a microenvironment that favors tumorigenesis. Through interrogating whole genome sequencing data from 171 EAC patients, we showed that non-synonymous mutations collectively affect the TLR pathway in 25/171 (14.6%, PathScan p = 8.7x10-5 tumors. TLR mutant cases were associated with more proximal tumors and metastatic disease, indicating possible clinical significance of these mutations. Only rare mutations were identified in adjacent Barrett's esophagus samples. We validated our findings in an external EAC dataset with non-synonymous TLR pathway mutations in 33/149 (22.1%, PathScan p = 0.05 tumors, and in other solid tumor types exposed to microbiomes in the COSMIC database (10,318 samples, including uterine endometrioid carcinoma (188/320, 58.8%, cutaneous melanoma (377/988, 38.2%, colorectal adenocarcinoma (402/1519, 26.5%, and stomach adenocarcinoma (151/579, 26.1%. TLR4 was the most frequently mutated gene with eleven mutations in 10/171 (5.8% of EAC tumors. The TLR4 mutants E439G, S570I, F703C and R787H were confirmed to have impaired reactivity to bacterial lipopolysaccharide with marked reductions in signaling by luciferase reporter assays. Overall, our findings show that TLR pathway genes are recurrently mutated in EAC, and TLR4 mutations have decreased responsiveness to bacterial lipopolysaccharide and may play a role in disease pathogenesis in a subset of patients.

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

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Shi-qi An

2014-10-01

Full Text Available Bis-(3',5' cyclic di-guanylate (cyclic di-GMP is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc. This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d∼2 µM. Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

Clinical Outcomes of Lung Transplantation in Patients with Telomerase Mutations

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Tokman, Sofya; Singer, Jonathan P.; Devine, Megan S.; Westall, Glen P.; Aubert, John-David; Tamm, Michael; Snell, Gregory I.; Lee, Joyce S.; Goldberg, Hilary J.; Kukreja, Jasleen; Golden, Jeffrey A.; Leard, Lorriana E.; Garcia, Christine K.; Hays, Steven R.

2017-01-01

Background Successful lung transplantation (LT) for patients with pulmonary fibrosis from telomerase mutations is limited by systemic complications of telomerase dysfunction including myelosuppression, cirrhosis, and malignancy. We describe clinical outcomes among 14 LT recipients with telomerase mutations. Methods Subjects underwent LT between February 2005 and April 2014 at 5 LT centers. We abstracted data from medical records, focusing on outcomes reflecting post-LT treatment effects likely to be complicated by telomerase mutations. Results The median age of subjects was 60.5 years (IQR 52.0–62.0), 64.3% were male, and the mean post-LT observation time was 3.2 years (SD ±2.9). Eleven subjects had a mutation in telomerase reverse transcriptase, 2 in telomerase RNA component, and 1 had an uncharacterized mutation. Ten subjects were leukopenic post-LT; leukopenia prompted cessation of mycophenolate mofetil in 5 and treatment with filgrastim in 4. Six subjects had recurrent lower respiratory tract infections (LRTI), 7 had acute cellular rejection (ACR) (A1), and 4 developed chronic lung allograft dysfunction (CLAD). Ten LT recipients developed chronic renal insufficiency and 8 experienced acute, reversible renal failure. Three developed cancer, none had cirrhosis. Thirteen subjects were alive at data censorship. Conclusions The clinical course for LT recipients with telomerase mutations is complicated by renal disease, leukopenia prompting a change in the immunosuppressive regimen, and recurrent LTRI. In contrast, cirrhosis was absent, ACR was mild, and development of CLAD was comparable to other LT populations. While posing challenges, lung transplantation may be feasible for patients with pulmonary fibrosis due to telomerase mutations. PMID:26169663

Clavibacter michiganensis ssp. michiganensis: bacterial canker of tomato, molecular interactions and disease management.

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Nandi, Munmun; Macdonald, Jacqueline; Liu, Peng; Weselowski, Brian; Yuan, Ze-Chun

2018-03-12

Bacterial canker disease is considered to be one of the most destructive diseases of tomato (Solanum lycopersicum), and is caused by the seed-borne Gram-positive bacterium Clavibacter michiganensis ssp. michiganensis (Cmm). This vascular pathogen generally invades and proliferates in the xylem through natural openings or wounds, causing wilt and canker symptoms. The incidence of symptomless latent infections and the invasion of tomato seeds by Cmm are widespread. Pathogenicity is mediated by virulence factors and transcriptional regulators encoded by the chromosome and two natural plasmids. The virulence factors include serine proteases, cell wall-degrading enzymes (cellulases, xylanases, pectinases) and others. Mutational analyses of these genes and gene expression profiling (via quantitative reverse transcription-polymerase chain reaction, transcriptomics and proteomics) have begun to shed light on their roles in colonization and virulence, whereas the expression of tomato genes in response to Cmm infection suggests plant factors involved in the defence response. These findings may aid in the generation of target-specific bactericides or new resistant varieties of tomato. Meanwhile, various chemical and biological controls have been researched to control Cmm. This review presents a detailed investigation regarding the pathogen Cmm, bacterial canker infection, molecular interactions between Cmm and tomato, and current perspectives on improved disease management. © 2018 AGRICULTURE AND AGRI-FOOD CANADA. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

Modeling physiological resistance in bacterial biofilms.

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Cogan, N G; Cortez, Ricardo; Fauci, Lisa

2005-07-01

A mathematical model of the action of antimicrobial agents on bacterial biofilms is presented. The model includes the fluid dynamics in and around the biofilm, advective and diffusive transport of two chemical constituents and the mechanism of physiological resistance. Although the mathematical model applies in three dimensions, we present two-dimensional simulations for arbitrary biofilm domains and various dosing strategies. The model allows the prediction of the spatial evolution of bacterial population and chemical constituents as well as different dosing strategies based on the fluid motion. We find that the interaction between the nutrient and the antimicrobial agent can reproduce survival curves which are comparable to other model predictions as well as experimental results. The model predicts that exposing the biofilm to low concentration doses of antimicrobial agent for longer time is more effective than short time dosing with high antimicrobial agent concentration. The effects of flow reversal and the roughness of the fluid/biofilm are also investigated. We find that reversing the flow increases the effectiveness of dosing. In addition, we show that overall survival decreases with increasing surface roughness.

Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.

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Nagaraj, V; Skillman, L; Li, D; Xie, Z; Ho, G

2017-07-01

Control of biofouling on seawater reverse osmosis (SWRO) membranes is a major challenge as treatments can be expensive, damage the membrane material and often biocides do not remove the polymers in which bacteria are embedded. Biological control has been largely ignored for biofouling control. The objective of this study was to demonstrate the effectiveness of xanthine oxidase enzyme against complex fouling communities and then identify naturally occurring bacterial strains that produce the free radical generating enzyme. Initially, 64 bacterial strains were isolated from different locations of the Perth Seawater Desalination Plant. In our preceding study, 25/64 isolates were selected from the culture collection as models for biofouling studies, based on their prevalence in comparison to the genomic bacterial community. In this study, screening of these model strains was performed using a nitroblue tetrazolium assay in the presence of hypoxanthine as substrate. Enzyme activity was measured by absorbance. Nine of 25 strains tested positive for xanthine oxidase production, of which Exiguobacterium from sand filters and Microbacterium from RO membranes exhibited significant levels of enzyme production. Other genera that produced xanthine oxidase were Marinomonas, Pseudomonas, Bacillus, Pseudoalteromonas and Staphylococcus. Strain variations were observed between members of the genera Microbacterium and Bacillus. Xanthine oxidase, an oxidoreductase enzyme that generates reactive oxygen species, is endogenously produced by many bacterial species. In this study, production of the enzyme by bacterial isolates from a full-scale desalination plant was investigated for potential use as biological control of membrane fouling in seawater desalination. We have previously demonstrated that free radicals generated by a commercially available xanthine oxidase in the presence of a hypoxanthine substrate, effectively dispersed biofilm polysaccharides on industrially fouled membranes

Mutation exposed: a neutral explanation for extreme base composition of an endosymbiont genome.

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Wernegreen, Jennifer J; Funk, Daniel J

2004-12-01

The influence of neutral mutation pressure versus selection on base composition evolution is a subject of considerable controversy. Yet the present study represents the first explicit population genetic analysis of this issue in prokaryotes, the group in which base composition variation is most dramatic. Here, we explore the impact of mutation and selection on the dynamics of synonymous changes in Buchnera aphidicola, the AT-rich bacterial endosymbiont of aphids. Specifically, we evaluated three forms of evidence. (i) We compared the frequencies of directional base changes (AT-->GC vs. GC-->AT) at synonymous sites within and between Buchnera species, to test for selective preference versus effective neutrality of these mutational categories. Reconstructed mutational changes across a robust intraspecific phylogeny showed a nearly 1:1 AT-->GC:GC-->AT ratio. Likewise, stationarity of base composition among Buchnera species indicated equal rates of AT-->GC and GC-->AT substitutions. The similarity of these patterns within and between species supported the neutral model. (ii) We observed an equivalence of relative per-site AT mutation rate and current AT content at synonymous sites, indicating that base composition is at mutational equilibrium. (iii) We demonstrated statistically greater equality in the frequency of mutational categories in Buchnera than in parallel mammalian studies that documented selection on synonymous sites. Our results indicate that effectively neutral mutational pressure, rather than selection, represents the major force driving base composition evolution in Buchnera. Thus they further corroborate recent evidence for the critical role of reduced N(e) in the molecular evolution of bacterial endosymbionts.

Prevalence of drug resistance mutations and non-B subtypes in newly diagnosed HIV-1 patients in Denmark

DEFF Research Database (Denmark)

Jørgensen, Louise B; Christensen, Marianne B; Gerstoft, Jan

2003-01-01

The aim of this study was to monitor the prevalence of drug resistance mutations in newly diagnosed HIV-1 positive individuals in Denmark. In addition we assessed the prevalence of non-B subtypes based on phylogenetic analysis of the pol gene. Plasma samples from 104 newly diagnosed HIV-1 positive...... patients were obtained in the year 2000. The entire protease gene and 320 amino acids of the reverse transcriptase gene were genotyped. Sequences were obtained from 97 patients. No subjects displayed primary resistance mutations in the protease gene, whereas all carried 1 or more secondary mutations....... Resistance mutations in the RT-gene associated with NRTI-resistance were found in 1 patient, who was infected with zidovudine resistant HIV-1 harbouring the M41L mutation in combination with T215S and L210S. The T215S mutation has been showed to be associated with reversion of zidovudine resistance. The T215...

Mutations in 23S rRNA at the Peptidyl Transferase Center and Their Relationship to Linezolid Binding and Cross-Resistance

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Long, Katherine; Munck, Christian

2010-01-01

The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations...... at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. The largest decrease in linezolid susceptibility for any of the introduced single mutations was observed with the G2576U mutation at a position that is 7.9 Å from linezolid....... Smaller decreases were observed with the A2503G, U2504G, and G2505A mutations at nucleotides proximal to linezolid, showing that the degree of resistance conferred is not simply inversely proportional to the nucleotide-drug distance. The double mutations G2032A-C2499A, G2032A-U2504G, C2055A-U2504G, and C...

Abundance of bacterial and diatom fouling on various surfaces

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PrabhaDevi

Abundance of bacterial and diatom fouling on aluminium, fibreglass and stainless steel were studied from Dona Paula waters of the Zuari Estuary. Both these forms were reversibly attached in large numbers to surfaces during the initial 24 hr...

Simulation Study for Transfer of Antibiotic Resistance via Mutator Subpopulation

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Philipsen, Kirsten Riber; Christiansen, Lasse Engbo; Aarestrup, Frank Møller

Evolution of antibiotic resistance in bacterial populations is an increasing problem having fatal consequences for treatment of diseases. Therefore it is very important to understand this evolution. Traditionally evolution is considered to happen by single point mutations, where each mutant must...

A BRCA2 mutation incorrectly mapped in the original BRCA2 reference sequence, is a common West Danish founder mutation disrupting mRNA splicing

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Thomassen, Mads; Pedersen, Inge Søkilde; Vogel, Ida

2011-01-01

Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose carriers to breast and ovarian cancer. The authors have identified a mutation in BRCA2, 7845+1G>A (c.7617+1G>A), not previously regarded as deleterious because of incorrect mapping of the splice junction in the originally...... published genomic reference sequence. This reference sequence is generally used in many laboratories and it maps the mutation 16 base pairs inside intron 15. However, according to the recent reference sequences the mutation is located in the consensus donor splice sequence. By reverse transcriptase analysis......, loss of exon 15 in the final transcript interrupting the open reading frame was demonstrated. Furthermore, the mutation segregates with a cancer phenotype in 18 Danish families. By genetic analysis of more than 3,500 Danish breast/ovarian cancer risk families, the mutation was identified as the most...

Where antibiotic resistance mutations meet quorum-sensing

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Rok Krašovec

2014-06-01

Full Text Available We do not need to rehearse the grim story of the global rise of antibiotic resistant microbes. But what if it were possible to control the rate with which antibiotic resistance evolves by de novo mutation? It seems that some bacteria may already do exactly that: they modify the rate at which they mutate to antibiotic resistance dependent on their biological environment. In our recent study [Krašovec, et al. Nat. Commun. (2014, 5, 3742] we find that this modification depends on the density of the bacterial population and cell-cell interactions (rather than, for instance, the level of stress. Specifically, the wild-type strains of Escherichia coli we used will, in minimal glucose media, modify their rate of mutation to rifampicin resistance according to the density of wild-type cells. Intriguingly, the higher the density, the lower the mutation rate (Figure 1. Why this novel density-dependent ‘mutation rate plasticity’ (DD-MRP occurs is a question at several levels. Answers are currently fragmentary, but involve the quorum-sensing gene luxS and its role in the activated methyl cycle.

New mutations affecting induced mutagenesis in yeast.

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

1985-01-01

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

Effect of genes controlling radiation sensitivity on chemically induced mutations in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, L.

1976-01-01

The effect of 16 different genes (rad) conferring radiation sensitivity on chemically induced reversion in the yeast Saccharomyces cerevisiae was determined. The site of reversion used was a well-defined chain initiation mutant mapping in the structural gene coding for iso-1-cytochrome c. High doses of EMS and HNO 2 resulted in decreased reversion of cyc1-131 in rad6, rad9 and rad15 strains compared to the normal RAD + strains. In addition, rad52 greatly decreased EMS reversion of cyc1-131 but had no effect on HNO 2 -induced reversion; rad18, on the other hand, increased HNO 2 -induced reversion but did not alter EMS-induced reversion. When NQO was used as the mutagen, every rad gene tested, except for rad18, had an effect on reversion; rad6, rad9, rad15, rad17, rad18, rad22, rev1, rev2, and rev3 lowered NQO reversion while rad1, rad2, rad3, rad4, rad10, rad12, and rad16 increased it compared to the RAD + strain. The effect of rad genes on chemical mutagenesis is discussed in terms of their effect on uv mutagenesis. It is concluded that although the nature of the repair pathways may differ for uv- and chemically-induced mutations in yeast, a functional repair system is required for the induction of mutation by the chemical agents NQO, EMS, and HNO 2

Reversible infantile mitochondrial diseases.

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Boczonadi, Veronika; Bansagi, Boglarka; Horvath, Rita

2015-05-01

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

Novel mutations underlying argininosuccinic aciduria in Saudi Arabia

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Rashed Mohamed S

2010-03-01

Full Text Available Abstract Background Argininosuccinic aciduria (ASAuria is an autosomal recessive disorder of the urea cycle relatively common in Saudi Arabia as a consequence of extensive consanguinity. It is the most common urea cycle disorder identified in the Saudi population, which therefore prioritizes the need to delineate the underlying molecular defects leading to disease. Findings We utilized Whole Genome Amplification (WGA, PCR and direct sequencing to identify mutations underlying ASAuria cases diagnosed by our institution. A missense mutation that accounts for 50% of Saudi ASAuria patients was recently reported by our laboratory. In this study we report a further six novel mutations (and one previously reported found in Saudi patients with ASAuria. The novel four missense, one nonsense and one splice-site mutation were confirmed by their absence in >300 chromosomes from the normal population. Pathogenicity of the novel splice-site mutation was also confirmed using reverse transcriptase-PCR analysis. Cross species amino acid conservation at the substituted residues described were observed in some but not all instances. Conclusions Together, the eight mutations described by our laboratory, encompass >90% of ASAuria patients in Saudi Arabia and add to about 45 other ASAuria mutations reported worldwide.

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

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Dunham Stephen

2009-11-01

Full Text Available Abstract Background Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions. Results A nitroreductase- encoding gene was identified in the N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of ~3 × 10-6 - 8 × 10-8 among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene. Conclusion We found that nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.

An approximate stationary solution for multi-allele neutral diffusion with low mutation rates.

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Burden, Conrad J; Tang, Yurong

2016-12-01

We address the problem of determining the stationary distribution of the multi-allelic, neutral-evolution Wright-Fisher model in the diffusion limit. A full solution to this problem for an arbitrary K×K mutation rate matrix involves solving for the stationary solution of a forward Kolmogorov equation over a (K-1)-dimensional simplex, and remains intractable. In most practical situations mutations rates are slow on the scale of the diffusion limit and the solution is heavily concentrated on the corners and edges of the simplex. In this paper we present a practical approximate solution for slow mutation rates in the form of a set of line densities along the edges of the simplex. The method of solution relies on parameterising the general non-reversible rate matrix as the sum of a reversible part and a set of (K-1)(K-2)/2 independent terms corresponding to fluxes of probability along closed paths around faces of the simplex. The solution is potentially a first step in estimating non-reversible evolutionary rate matrices from observed allele frequency spectra. Copyright © 2016 Elsevier Inc. All rights reserved.

Lateral Gene Transfer Dynamics in the Ancient Bacterial Genus Streptomyces

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Bradon R. McDonald

2017-06-01

Full Text Available Lateral gene transfer (LGT profoundly shapes the evolution of bacterial lineages. LGT across disparate phylogenetic groups and genome content diversity between related organisms suggest a model of bacterial evolution that views LGT as rampant and promiscuous. It has even driven the argument that species concepts and tree-based phylogenetics cannot be applied to bacteria. Here, we show that acquisition and retention of genes through LGT are surprisingly rare in the ubiquitous and biomedically important bacterial genus Streptomyces. Using a molecular clock, we estimate that the Streptomyces bacteria are ~380 million years old, indicating that this bacterial genus is as ancient as land vertebrates. Calibrating LGT rate to this geologic time span, we find that on average only 10 genes per million years were acquired and subsequently maintained. Over that same time span, Streptomyces accumulated thousands of point mutations. By explicitly incorporating evolutionary timescale into our analyses, we provide a dramatically different view on the dynamics of LGT and its impact on bacterial evolution.

The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations

International Nuclear Information System (INIS)

Flores-Rozas, H.; Kolodner, R.D.

1998-01-01

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of NLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex

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

LENUS (Irish Health Repository)

Bansode, Vijay B

2011-10-13

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

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

DEFF Research Database (Denmark)

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

2017-01-01

H mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial...... adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study...... mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel...

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

International Nuclear Information System (INIS)

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

2008-01-01

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

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

International Nuclear Information System (INIS)

Tyrrell, R.M.

1980-01-01

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

Teaching the fluctuation test in silico by using mutate: a program to distinguish between the adaptive and spontaneous mutation hypotheses.

Science.gov (United States)

Carvajal-Rodríguez, Antonio

2012-07-01

Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary aspects of current genetics such as sequence databases, DNA mutations, and hypothesis testing, while introducing the fluctuation test. This seminal experiment was originally performed studying Escherichia coli resistance to the infection by bacteriophage T1. The fluctuation test initiated the modern bacterial genetics that 25 years later ushered in the era of the recombinant DNA. Nowadays we know that some deletions in fhuA, the gene responsible for E. coli membrane receptor of T1, could cause the E. coli resistance to this phage. For the sake of simplicity, we will introduce the assumption that a single mutation generates the resistance to T1. During the practical, the students use the program to download some fhuA gene sequences, manually introduce some stop codon mutations, and design a fluctuation test to obtain data for distinguishing between preadaptative (spontaneous) and induced (adaptive) mutation hypotheses. The program can be launched from a browser or, if preferred, its executable file can be downloaded from http://webs.uvigo.es/acraaj/MutateWeb/Mutate.html. It requires the Java 5.0 (or higher) Runtime Environment (freely available at http://www.java.com). Copyright © 2012 Wiley Periodicals, Inc.

Elongation factor P mediates a novel post-transcriptional regulatory pathway critical for bacterial virulence

DEFF Research Database (Denmark)

Zou, S Betty; Roy, Hervé; Ibba, Michael

2012-01-01

Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability of the path......Bacterial pathogens detect and integrate multiple environmental signals to coordinate appropriate changes in gene expression including the selective expression of virulence factors, changes to metabolism and the activation of stress response systems. Mutations that abolish the ability...... our laboratory and others now suggests that EF-P, previously thought to be essential, instead plays an ancillary role in translation by regulating the synthesis of a relatively limited subset of proteins. Other observations suggest that the eukaryotic homolog of EF-P, eIF5A, may illicit similar...

Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers

Directory of Open Access Journals (Sweden)

Céline Brochier-Armanet

2006-01-01

Full Text Available Reverse gyrase, an enzyme of uncertain funtion, is present in all hyperthermophilic archaea and bacteria. Previous phylogenetic studies have suggested that the gene for reverse gyrase has an archaeal origin and was transferred laterally (LGT to the ancestors of the two bacterial hyperthermophilic phyla, Thermotogales and Aquificales. Here, we performed an in-depth analysis of the evolutionary history of reverse gyrase in light of genomic progress. We found genes coding for reverse gyrase in the genomes of several thermophilic bacteria that belong to phyla other than Aquificales and Thermotogales. Several of these bacteria are not, strictly speaking, hyperthermophiles because their reported optimal growth temperatures are below 80 °C. Furthermore, we detected a reverse gyrase gene in the sequence of the large plasmid of Thermus thermophilus strain HB8, suggesting a possible mechanism of transfer to the T. thermophilus strain HB8 involving plasmids and transposases. The archaeal part of the reverse gyrase tree is congruent with recent phylogenies of the archaeal domain based on ribosomal proteins or RNA polymerase subunits. Although poorly resolved, the complete reverse gyrase phylogeny suggests an ancient acquisition of the gene by bacteria via one or two LGT events, followed by its secondary distribution by LGT within bacteria. Finally, several genes of archaeal origin located in proximity to the reverse gyrase gene in bacterial genomes have bacterial homologues mostly in thermophiles or hyperthermophiles, raising the possibility that they were co-transferred with the reverse gyrase gene. Our new analysis of the reverse gyrase history strengthens the hypothesis that the acquisition of reverse gyrase may have been a crucial evolutionary step in the adaptation of bacteria to high-temperature environments. However, it also questions the role of this enzyme in thermophilic bacteria and the selective advantage its presence could provide.

Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers.

Science.gov (United States)

Brochier-Armanet, Céline; Forterre, Patrick

2007-05-01

Reverse gyrase, an enzyme of uncertain funtion, is present in all hyperthermophilic archaea and bacteria. Previous phylogenetic studies have suggested that the gene for reverse gyrase has an archaeal origin and was transferred laterally (LGT) to the ancestors of the two bacterial hyperthermophilic phyla, Thermotogales and Aquificales. Here, we performed an in-depth analysis of the evolutionary history of reverse gyrase in light of genomic progress. We found genes coding for reverse gyrase in the genomes of several thermophilic bacteria that belong to phyla other than Aquificales and Thermotogales. Several of these bacteria are not, strictly speaking, hyperthermophiles because their reported optimal growth temperatures are below 80 degrees C. Furthermore, we detected a reverse gyrase gene in the sequence of the large plasmid of Thermus thermophilus strain HB8, suggesting a possible mechanism of transfer to the T. thermophilus strain HB8 involving plasmids and transposases. The archaeal part of the reverse gyrase tree is congruent with recent phylogenies of the archaeal domain based on ribosomal proteins or RNA polymerase subunits. Although poorly resolved, the complete reverse gyrase phylogeny suggests an ancient acquisition of the gene by bacteria via one or two LGT events, followed by its secondary distribution by LGT within bacteria. Finally, several genes of archaeal origin located in proximity to the reverse gyrase gene in bacterial genomes have bacterial homologues mostly in thermophiles or hyperthermophiles, raising the possibility that they were co-transferred with the reverse gyrase gene. Our new analysis of the reverse gyrase history strengthens the hypothesis that the acquisition of reverse gyrase may have been a crucial evolutionary step in the adaptation of bacteria to high-temperature environments. However, it also questions the role of this enzyme in thermophilic bacteria and the selective advantage its presence could provide.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.

Science.gov (United States)

Hamilton, P J; Campbell, N G; Sharma, S; Erreger, K; Herborg Hansen, F; Saunders, C; Belovich, A N; Sahai, M A; Cook, E H; Gether, U; McHaourab, H S; Matthies, H J G; Sutcliffe, J S; Galli, A

2013-12-01

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Diploid yeast cells yield homozygous spontaneous mutations

Science.gov (United States)

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

1993-01-01

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

Mutation breeding for tomato bacterial with disease resistance through in vitro techniques

International Nuclear Information System (INIS)

Keoboonrueng, S.; Charaensatapon, R.; Panichsukpatana, C.; Jatisation, J.

1994-01-01

Cotyledons of half month and one month-old seedlings of tomato varieties Sida, SVRDC 4, Sidatip 2 and VF 137 irradiated with gamma rays at the dose of 10 Gy were cultured on most suitable medium found, MS supplemented with 0.01 mg/1 NAA and 2.0 mg/1 BAP. Sometimes calli and multiple shoots derived from normal seedlings were irradiated with gamma rays at the doses of 5 and 8 Gy, respectively. Plantlets from in vitro culture were screened in the greenhouse by soaking roots in bacterial suspension at transplanting time or by pouring bacterial suspension on injured roots at 2-3 wk. after transplant. The concentration of bacterial suspension was 10 5 -10 7 cells/ml. Total of 2541 tomato plantlets were screened and only 11 plants survived. They were, 5 plants from non-irradiated Sida, 1 and 2 plants from 5 and 10 Gy Sida, respectively, 2 plants from 5 Gy Sidatip 2 and single plant from non-irradiated VF 137. Most of the surviving plants were susceptible to tomato yellow leaf curl virus and only fruits from one Sida plant irradiated with 10 Gy could be harvested. Plants from these seeds will be further selected

Finding Relational Associations in HIV Resistance Mutation Data

Science.gov (United States)

Richter, Lothar; Augustin, Regina; Kramer, Stefan

HIV therapy optimization is a hard task due to rapidly evolving mutations leading to drug resistance. Over the past five years, several machine learning approaches have been developed for decision support, mostly to predict therapy failure from the genotypic sequence of viral proteins and additional factors. In this paper, we define a relational representation for an important part of the data, namely the sequences of a viral protein (reverse transcriptase), their mutations, and the drug resistance(s) associated with those mutations. The data were retrieved from the Los Alamos National Laboratories' (LANL) HIV databases. In contrast to existing work in this area, we do not aim directly for predictive modeling, but take one step back and apply descriptive mining methods to develop a better understanding of the correlations and associations between mutations and resistances. In our particular application, we use the Warmr algorithm to detect non-trivial patterns connecting mutations and resistances. Our findings suggest that well-known facts can be rediscovered, but also hint at the potential of discovering yet unknown associations.

Mutations and epimutations in the origin of cancer

Energy Technology Data Exchange (ETDEWEB)

Peltomaeki, Paeivi, E-mail: Paivi.Peltomaki@Helsinki.Fi

2012-02-15

Cancer is traditionally viewed as a disease of abnormal cell proliferation controlled by a series of mutations. Mutations typically affect oncogenes or tumor suppressor genes thereby conferring growth advantage. Genomic instability facilitates mutation accumulation. Recent findings demonstrate that activation of oncogenes and inactivation of tumor suppressor genes, as well as genomic instability, can be achieved by epigenetic mechanisms as well. Unlike genetic mutations, epimutations do not change the base sequence of DNA and are potentially reversible. Similar to genetic mutations, epimutations are associated with specific patterns of gene expression that are heritable through cell divisions. Knudson's hypothesis postulates that inactivation of tumor suppressor genes requires two hits, with the first hit occurring either in somatic cells (sporadic cancer) or in the germline (hereditary cancer) and the second one always being somatic. Studies on hereditary and sporadic forms of colorectal carcinoma have made it evident that, apart from genetic mutations, epimutations may serve as either hit or both. Furthermore, recent next-generation sequencing studies show that epigenetic genes, such as those encoding histone modifying enzymes and subunits for chromatin remodeling systems, are themselves frequent targets of somatic mutations in cancer and can act like tumor suppressor genes or oncogenes. This review discusses genetic vs. epigenetic origin of cancer, including cancer susceptibility, in light of recent discoveries. Situations in which mutations and epimutations occur to serve analogous purposes are highlighted.

Amorphous structure of iron oxide of bacterial origin

Energy Technology Data Exchange (ETDEWEB)

Hashimoto, Hideki; Fujii, Tatsuo [Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530 (Japan); Kohara, Shinji [Japan Synchrotron Radiation Research Institute, Sayo, Hyogo 679-5198 (Japan); Asaoka, Hiroshi [Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530 (Japan); Kusano, Yoshihiro [Department of Fine and Applied Arts, Kurashiki University of Science and the Arts, Kurashiki, Okayama 712-8505 (Japan); Ikeda, Yasunori [Research Institute for Production Development, Sakyo-ku, Kyoto 606-0805 (Japan); Nakanishi, Makoto [Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530 (Japan); Benino, Yasuhiko; Nanba, Tokuro [Graduate School of Environmental Science, Okayama University, Okayama 700-8530 (Japan); Takada, Jun, E-mail: jtakada@cc.okayama-u.ac.jp [Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530 (Japan); JST, CREST, Okayama 700-8530 (Japan)

2012-12-14

In nature, there are various iron oxides produced by the water-habitant bacterial group called 'iron-oxidizing bacteria'. These iron oxides have been studied mainly from biological and geochemical perspectives. Today, attempts are made to use such iron oxides as novel functional materials in several applications. However, their quantitative structural characteristics are still unclear. We studied the structure of iron oxide of microtubular form consisting of amorphous nanoparticles formed by an iron-oxidizing bacterium, Leptothrix ochracea, using a combination of high-energy X-ray diffraction and reverse Monte Carlo simulation. We found that its structure consists of a framework of corner- and edge-sharing distorted FeO{sub 6} octahedral units, while SiO{sub 4} tetrahedral units are isolated in the framework. The results reveal the atomic arrangement of iron oxide of bacterial origin, which is essential for investigating its potential as a functional material. -- Highlights: Black-Right-Pointing-Pointer The amorphous structure of bacterial iron oxide was investigated. Black-Right-Pointing-Pointer The structure was simulated by high-energy X-ray diffraction and reverse Monte Carlo simulation. Black-Right-Pointing-Pointer The structure was constructed of a framework of corner- and edge-sharing distorted FeO{sub 6} octahedral units. Black-Right-Pointing-Pointer SiO{sub 4} tetrahedral units were distributed isolatedly in the framework of FeO{sub 6} octahedral units.

Economic and agricultural impact of mutation breeding in fruit trees

International Nuclear Information System (INIS)

Spiegel Roy, P.

1990-01-01

Constraints of conventional cross breeding in fruit trees, wide market acceptance of definite cultivars, especially in apple, pear, citrus and wine grape, and the increased impact of natural mutants provide incentives for mutation breeding. Only few induced mutants in fruit trees have been commercialized and are being planted on a large scale. The main method followed in mutation breeding of tree fruit has been acute irradiation of meristematic multicellular buds but, Chimera formation and reversion present a serious problem. 87 refs, 4 tabs

The latest progress of TILLING technique and its prospects in irradiation mutation breeding

International Nuclear Information System (INIS)

Du Yan; Yu Lixia; Liu Qingfang; Zhou Libin; Li Wenjian

2011-01-01

TILLING (Targeting Induced Local Lesions IN Genomes) is a novel, high-throughput and low-cost reverse genetics technique. In recent years, with innovation of the mutation screening techniques, TILLING platform has become more diversified, which makes the operation of TILLING technique more simple and rapid. For this reason, it is widely used in crop breeding research. In this paper, we summarized the latest progress of TILLING technique, meanwhile, we also discussed the prospect of combining irradiation mutation with the high-throughput TILLING technique in mutation breeding. (authors)

Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens.

Directory of Open Access Journals (Sweden)

Christoph B Geier

Full Text Available Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency.

Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

Science.gov (United States)

Badran, Ahmed H; Liu, David R

2015-10-07

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

In vitro and in vivo safety evaluation of Acer tegmentosum.

Science.gov (United States)

Hwang, Youn-Hwan; Park, Hwayong; Ma, Jin Yeul

2013-06-21

Acer tegmentosum, which contains salidroside and tyrosol, has been used for the treatment of hepatic disorders in eastern Asia. However, little is known about its safety. To determine the safety of Acer tegmentosum, we evaluated its acute oral toxicity and genotoxicity profiles. Salidroside and tyrosol present in Acer tegmentosum were quantified using high-performance liquid chromatography. Acute oral toxicity testing of Acer tegmentosum was performed in rats. Genotoxicity of Acer tegmentosum was assessed by bacterial reverse mutation, chromosomal aberration, and bone marrow micronucleus tests. All the tests were conducted in accordance with the good laboratory practices. The amounts of salidroside and tyrosol in Acer tegmentosum were found to be 85.01±1.21mg/g and 3.12±0.04mg/g, respectively. In the bacterial reverse mutation test, Acer tegmentosum increased the number of revertant Salmonella typhimurium TA98 colonies, regardless of metabolic activation by S9 mixture. In contrast, Acer tegmentosum application did not significantly increase the number of chromosomal aberrations in Chinese hamster ovary (CHO)-K1 cells and micronucleated polychromatic erythrocytes in mice. In the acute oral toxicity test, the median lethal dose (LD50) of Acer tegmentosum was found to be >2000mg/kg in rats. Take together, Acer tegmentosum exhibits mutagenicity, which was evident from the bacterial reverse mutation test. Further studies are needed to identify the components responsible for such an effect and the underlying mechanisms. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

Science.gov (United States)

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

2017-01-01

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

Screening for duplications, deletions and a common intronic mutation detects 35% of second mutations in patients with USH2A monoallelic mutations on Sanger sequencing.

Science.gov (United States)

Steele-Stallard, Heather B; Le Quesne Stabej, Polona; Lenassi, Eva; Luxon, Linda M; Claustres, Mireille; Roux, Anne-Francoise; Webster, Andrew R; Bitner-Glindzicz, Maria

2013-08-08

Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G. Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints. Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome. We found that 8 of

Genotoxicity of extracts of Japanese traditional herbal medicines (Kampo)

OpenAIRE

Makoto, Katami; Haruo, Kuboniwa; Shunichi, Maemura; Toshihiko, Yanagisawa; New Drug Discovery Laboratory, R & D Division, TSUMURA & Co.; New Drug Discovery Laboratory, R & D Division, TSUMURA & Co.; New Drug Discovery Laboratory, R & D Division, TSUMURA & Co.; New Drug Discovery Laboratory, R & D Division, TSUMURA & Co.

2002-01-01

The possible genotoxicity potential of 128 Japanese traditional herbal medicines (Kampo) was investigated using a bacterial reverse mutation test (the Ames test), an in vivo micronucleus test (MN test) in mouse bone marrow cells and an unscheduled DNA synthesis test (UDS test) in rat hepatocytes. Of 128 Kampo extracts examined, 98 did not induce mutations in bacteria while 30 induced mutations weakly in Salmonella typhimurium TA1537. Extracts of Scutellariae Radix, a common herbal drug, and i...

Gene mutation in ATM/PI3K region of nasopharyngeal carcinoma cell lines

International Nuclear Information System (INIS)

Wang Hongmei; Wu Xinyao; Xia Yunfei

2002-01-01

Objective: To define the correlation between nasopharyngeal carcinoma (NPC) cell radiosensitivity and gene mutation in the ATM/PI3K coding region. Methods: The gene mutation in the ATM/PI3K region of nasopharyngeal carcinoma cell lines which vary in radiosensitivity, was monitored by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-marked ddNTP cycle sequencing technique. Results: No gene mutation was detected in the ATM/PI3K region of either CNE1 or CNE2. Conclusion: Disparity in intrinsic radiosensitivity between different NPC cell lines depends on some other factors and mechanism without being related to ATM/PI3K mutations

Environmental stresses can alleviate the average deleterious effect of mutations

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Leibler Stanislas

2003-05-01

Full Text Available Abstract Background Fundamental questions in evolutionary genetics, including the possible advantage of sexual reproduction, depend critically on the effects of deleterious mutations on fitness. Limited existing experimental evidence suggests that, on average, such effects tend to be aggravated under environmental stresses, consistent with the perception that stress diminishes the organism's ability to tolerate deleterious mutations. Here, we ask whether there are also stresses with the opposite influence, under which the organism becomes more tolerant to mutations. Results We developed a technique, based on bioluminescence, which allows accurate automated measurements of bacterial growth rates at very low cell densities. Using this system, we measured growth rates of Escherichia coli mutants under a diverse set of environmental stresses. In contrast to the perception that stress always reduces the organism's ability to tolerate mutations, our measurements identified stresses that do the opposite – that is, despite decreasing wild-type growth, they alleviate, on average, the effect of deleterious mutations. Conclusions Our results show a qualitative difference between various environmental stresses ranging from alleviation to aggravation of the average effect of mutations. We further show how the existence of stresses that are biased towards alleviation of the effects of mutations may imply the existence of average epistatic interactions between mutations. The results thus offer a connection between the two main factors controlling the effects of deleterious mutations: environmental conditions and epistatic interactions.

Ancient genes establish stress-induced mutation as a hallmark of cancer.

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Cisneros, Luis; Bussey, Kimberly J; Orr, Adam J; Miočević, Milica; Lineweaver, Charles H; Davies, Paul

2017-01-01

Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms should have evolved with or after the emergence of multicellularity. This leads to two related, but distinct hypotheses: 1) Somatic mutations in cancer will occur in genes that are younger than the emergence of multicellularity (1000 million years [MY]); and 2) genes that are frequently mutated in cancer and whose mutations are functionally important for the emergence of the cancer phenotype evolved within the past 1000 million years, and thus would exhibit an age distribution that is skewed to younger genes. In order to investigate these hypotheses we estimated the evolutionary ages of all human genes and then studied the probability of mutation and their biological function in relation to their age and genomic location for both normal germline and cancer contexts. We observed that under a model of uniform random mutation across the genome, controlled for gene size, genes less than 500 MY were more frequently mutated in both cases. Paradoxically, causal genes, defined in the COSMIC Cancer Gene Census, were depleted in this age group. When we used functional enrichment analysis to explain this unexpected result we discovered that COSMIC genes with recessive disease phenotypes were enriched for DNA repair and cell cycle control. The non-mutated genes in these pathways are orthologous to those underlying stress-induced mutation in bacteria, which results in the clustering of single nucleotide variations. COSMIC genes were less common in regions where the probability of observing mutational clusters is high, although they are approximately 2-fold more likely to harbor mutational clusters compared to other human genes. Our results suggest this ancient mutational response to

Ela2 mutations and clinical manifestations in familial congenital neutropenia.

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Shiohara, Masaaki; Shigemura, Tomonari; Saito, Shoji; Tanaka, Miyuki; Yanagisawa, Ryu; Sakashita, Kazuo; Asada, Hiroshi; Ishii, Eizaburo; Koike, Kazutoshi; Chin, Motoaki; Kobayashi, Masao; Koike, Kenichi

2009-05-01

Three familial cases of each of severe congenital neutropenia (SCN) and cyclic neutropenia (CN) in addition to 3 sporadic cases of SCN were analyzed for neutrophil elastase (Ela2) gene mutation. The contents of the neutrophil-specific granule proteins cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were also analyzed in SCN. Genomic DNA was extracted from the patients' peripheral blood or bone marrow, and the coding sequence of the Ela2 gene was amplified by polymerase chain reaction and subjected to direct sequencing. The contents of antimicrobial peptides were analyzed by flow cytometry. Three cases of familial SCN (P13L, R52P, and S97L), 2 of familial CN (W212stop and P110L), and 1 of sporadic SCN (V72M) were shown to have heterozygous mutations in the Ela2 gene. W212stop found in a familial CN case was a novel mutation of Ela2. Prophylactic treatment for growth factors or antibiotic prophylaxis against bacterial infection was useful for lowering the frequency of infectious episodes. Adult patients tended to have less frequent infections compared with minors in the same family. The contents of both cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were significantly reduced in SCN compared with healthy controls. Prophylaxis by growth factor or antibiotics is useful for decreasing risks of bacterial infections in SCN and CN. Adults were likely to have less frequent infections than children in familial cases of SCN and CN with the same mutation of Ela2.

Bioremoval of heavy metals by bacterial biomass.

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Aryal, Mahendra; Liakopoulou-Kyriakides, Maria

2015-01-01

Heavy metals are among the most common pollutants found in the environment. Health problems due to the heavy metal pollution become a major concern throughout the world, and therefore, various treatment technologies such as reverse osmosis, ion exchange, solvent extraction, chemical precipitation, and adsorption are adopted to reduce or eliminate their concentration in the environment. Biosorption is a cost-effective and environmental friendly technique, and it can be used for detoxification of heavy metals in industrial effluents as an alternative treatment technology. Biosorption characteristics of various bacterial species are reviewed here with respect to the results reported so far. The role of physical, chemical, and biological modification of bacterial cells for heavy metal removal is presented. The paper evaluates the different kinetic, equilibrium, and thermodynamic models used in bacterial sorption of heavy metals. Biomass characterization and sorption mechanisms as well as elution of metal ions and regeneration of biomass are also discussed.

Common mutations of hepatitis B virus and their clinical significance

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HU Airong

2016-06-01

Full Text Available Hepatitis B virus (HBV tends to mutate easily due to its special structure and life cycle. Mutation changes the biological behavior of HBV and its sensitivity to antiviral drugs and even affects therapeutic effect and accelerate disease progression. The point mutations are commonly see in the pre-S/S open reading frame (ORF, which may be associated with immune escape and occult HBV infection. The G1896A mutation is often observed in the pre-C/C-ORF and is associated with the development of HBeAg-negative chronic hepatitis B (CHB, hepatocellular carcinoma (HCC, and severe chronic hepatitis (liver failure. The mutations in P-ORF mainly occur in the reverse transcriptase (RT domain and are closely related to the resistance to nucleos(tide analogues. The A1762T and G1764A mutations occur in the basal core promoter (BCP, which overlaps with X-ORF, and may be associated with HBeAg-negative CHB, HCC, and severe chronic hepatitis (liver failure. Clarification of the association between these mutations and diseases helps to develop tailor-made diagnostic and therapeutic regimens for patients with HBV infection.

A precise, efficient radiometric assay for bacterial growth

International Nuclear Information System (INIS)

Boonkitticharoen, V.; Ehrhardt, C.; Kirchner, P.T.

1984-01-01

The two-compartment radiometric assay for bacterial growth promised major advantages over systems in clinical use, but poor reproducibility and counting efficiency limited its application. In this method, 14-CO/sub 2/ produced by bacterial metabolism of C-14-glucose is trapped and counted on filter paper impregnated with NaOH and fluors. The authors sought to improve assay efficiency and precision through a systematic study of relevant physical and chemical factors. Improvements in efficiency (88% vs. 10%) and in precision (relative S.D. 5% vs. 40%) were produced by a) reversing growth medium and scintillator chambers to permit vigorous agitation, b) increasing NaOH quantity and using a supersaturated PPO solution and c) adding detergent to improve uniformity of NaOH-PPO mixture. Inoculum size, substrate concentration and O/sub 2/ transfer rate affected assay sensitivity but not bacterial growth rate. The authors' assay reliably detects bacterial growth for inocula of 10,000 organisms in 1 hour and for 25 organisms within 4 1/2 hours, thus surpassing other existing clinical and research methods

Changes in bacterial profiles after periodontal treatment associated with respiratory quality of asthmatic children

OpenAIRE

Wiyarni Pambudi; Imelda Fabiola; Retno Indrawati; Haryono Utomo; Anang Endaryanto; Ariyanto Harsono

2016-01-01

Background Despite the reduction phenomenon of asthma exacerbation after dental plaque control, no scientific report has been found to describe the link between bacterial profiles and respiratory quality in children with asthma. Objective To investigate association between bacterial profiles changes and improvement in respiratory quality after periodontal treatment. Methods Asthmatic children with FEV1 reversibility ~ 12% and dental plaque index ~ 2 who qualified for incl...

Gamma radiation-induced mutant of NSIC RC144 with broad-spectrum resistance to bacterial blight

International Nuclear Information System (INIS)

Alfonso, A.A.; Avellanoza, E.S.; Miranda, R.T.; Espejo, E.O.; Garcia, N.S.

2014-01-01

Mutant lines derived from gamma radiation-treated commercial variety NSIC RC144 were produced and screened for novel resistance to bacterial blight, one of the most serious diseases of rice. Preliminary screening of a bulk M2 population through induced method using race 3 of the pathogen Xanthomonas oryzae pv. oryzae (Xoo) resulted in the selection of 89 resistant plants. Subsequent repeated bacterial blight screenings and generation advance for five seasons resulted in the selection of two highly resistant M7 sister lines whose origin can be traced to a single M2 plant. DNA fingerprinting using 63 genome-wide simple sequence repeat (SSR) markers revealed an identical pattern in these lines. Using the same set of markers, they also exhibited 98% similarity to wild type NSIC RC144 indicating that the resistance is due to mutation and not due to genetic admixture or seed impurity. Two seasons of bacterial blight screening using 14 local isolates representing ten races of Xoo revealed an identical reaction pattern in these lines. The reaction pattern was observed to be unique compared to known patterns in four IRBB isolines (IRBB 4, 5, 7 and 21) with strong resistant reaction to bacterial blight suggesting possible novel resistance. The susceptible reaction in F1 testcrosses using Xoo race 6 and the segregation patterns in two F2 populations that fit with the expected 3 susceptible: 1 resistant ratio (P = 0.4, ns) suggest a single-gene recessive mutation in these lines. These mutants are now being used as resistance donor in the breeding program while further molecular characterization to map and characterize the mutated gene is being pursued

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

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Frenoy, Antoine; Bonhoeffer, Sebastian

2018-05-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the

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

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Crumpacker Clyde S

2011-01-01

Full Text Available Abstract Background The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1 includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses. Methods Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM cells in the absence of selection pressure. Replication capacity (RC of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74. Results Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT virus. Although attenuated in comparison to WT virus

Forward and reverse mutagenesis in C. elegans

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Kutscher, Lena M.; Shaham, Shai

2014-01-01

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

77 FR 75039 - Propiconazole; Pesticide Tolerances

Science.gov (United States)

2012-12-19

... maternally toxic. In the two generation reproduction study in rats, offspring toxicity occurred at a higher... transformation assay, bacterial reverse mutation assay, Chinese hamster bone marrow chromosomal aberration assay... reproduction study in rats, neither quantitative nor qualitative evidence of increased susceptibility of...

A single polymerase (L) mutation in avian metapneumovirus increased virulence and partially maintained virus viability at an elevated temperature.

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Brown, Paul A; Lupini, Caterina; Catelli, Elena; Clubbe, Jayne; Ricchizzi, Enrico; Naylor, Clive J

2011-02-01

Previously, a virulent avian metapneumovirus, farm isolate Italy 309/04, was shown to have been derived from a live vaccine. Virulence due to the five nucleotide mutations associated with the reversion to virulence was investigated by their addition to the genome of the vaccine strain using reverse genetics. Virulence of these recombinant viruses was determined by infection of 1-day-old turkeys. Disease levels resulting from the combined two matrix mutations was indistinguishable from that produced by the recombinant vaccine, whereas the combined three L gene mutations increased disease to a level (P<0.0001) that was indistinguishable from that caused by the revertant Italy 309/04 virus. Testing of the L mutations individually showed that two mutations did not increase virulence, while the third mutation, corresponding to an asparagine to aspartic acid substitution, produced virulence indistinguishable from that caused by Italy 309/04. In contrast to the vaccine, the virulent mutant also showed increased viability at temperatures typical of turkey core tissues. The notion that increased viral virulence resulted from enhanced ability to replicate in tissues away from the cool respiratory tract, cannot be discounted.

New Mutation Identified in the SRY Gene High Mobility Group (HMG

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Feride İffet Şahin

2013-06-01

Full Text Available Mutations in the SRY gene prevent the differentiation of the fetal gonads to testes and cause developing female phenotype, and as a result sex reversal and pure gonadal dysgenesis (Swyer syndrome can be developed. Different types of mutations identified in the SRY gene are responsible for 15% of the gonadal dysgenesis. In this study, we report a new mutation (R132P in the High Mobility Group (HMG region of SRY gene was detected in a patient with primary amenorrhea who has 46,XY karyotype. This mutation leads to replacement of the polar and basic arginine with a nonpolar hydrophobic proline residue at aminoacid 132 in the nuclear localization signal region of the protein. With this case report we want to emphasize the genetic approach to the patients with gonadal dysgenesis. If Y chromosome is detected during cytogenetic analysis, revealing the presence of the SRY gene and identification of mutations in this gene by sequencing analysis is become important in.

p53-dependent manner of persistent activation of the radiation-induced reversion in the pink-eyed unstable mouse embryo

International Nuclear Information System (INIS)

Shiraishi, K.; Yonezawa, M.; Niwa, O.

2003-01-01

Full text: We previously reported that radiation has an ability to induce genomic instability which causes delayed and untargeted mutation. These mutations aren't accounted for by the usual relationship between DNA damages and repair. However, the mechanisms of a long-term memory of DNA damage and the persistence of up-regulated recombination activity have yet to be elucidated. The mouse pink-eyed unstable (pun) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts black to the wild type in germ cells as well as somatic cells. The frequency of reversion was estimated by counting cluster of pigment cells in retinal pigment epithelium. Twice increase of the reversion was observed in F1 mice born to 6Gy irradiated male at spermatozoa stage, but not at other spermatogenesis stages( -tid, -cyte, -gonia ). Trans-genarational effect in F2 mice also didn't observe. Therefore, this phenomenon only occurs under the restricted germ cell stage. Additionally, the reversion frequency of p53 deficient F1 mouse born to irradiated sperm was less than irradiated wild mouse. 5aza-dc chemical agent, which is DNA methylation emzyme inhibitor, also suppressed pun allele recombination in mouse embryo. These data indicate that p53 contributes delayed and untargeted mutation, perhaps, by regulation of DNA metylation status

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.

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Valentina Guerrini

Full Text Available Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.

Ancient Genes Establish Stress-Induced Mutation as a Hallmark of Cancer

NARCIS (Netherlands)

Cisneros, L; Bussey, K; Orr, A; Miočević, M.; Lineweaver, C; Davies, Paul

2017-01-01

Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms

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

Science.gov (United States)

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

2016-07-01

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

Absence of sensitization of reversions in yeast by metronidazole

International Nuclear Information System (INIS)

Krishnan, D.; Singh, D.R.; Mahajan, J.M.; Madhvanath, U.

1977-01-01

Metronidazole (Flagyl; 2-methyl-5-nitroimidazole-1-ethanol) is of potential clinical application as a radiosensitizer. Studies have been made of the effect of 1mM metronidazole on γ-induced heteroallelic recombination (reversion) under euoxic or hypoxic conditions in yeast strain BZ34, and compared with the effect on survival. The o.e.r. for reversions was 2.2, and very close to that for survival (2.3), indicating that oxygen equally sensitized gross cellular events such as killing, and genetic phenomena like reversions. The introduction of metronidazole during irradiation sensitized cells with killing as the end point only in hypoxic condition, whereas with reversion as the end point no sensitization due to the chemical was observed either in euoxic or hypoxic conditions. Since DNA damage is responsible for both cell-killing and mutations, the sensitizer might have been expected to have had an equal effect on both these end-points. The results show that only in a limited sense can metronidazole be considered to be an oxygen mimic. (U.K.)

A/α-specific effect of the mms3 mutation on ultraviolet mutagenesis in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Martin, P.; Prakash, L.; Prakash, S.

1981-01-01

A new gene involved in error-prone repair of ultraviolet (uv) damage has been identified in Saccharomyces cerevisiae by the mms3-1 mutation. Uv-induced reversion is reduced in diploids that are homozygous for mms3-1, only if they are also heterozygous (MATa/MATα) at the mating type locus. The mms3-1 mutation has no effect on uv-induced reversion either in haploids or MATa/MATα or MATα/MATα diploids. The mutation confers sensitivity to uv and methyl methane sulfonate in both haploids and diploids. Even though mutation induction by uv is restored to wild-type levels in MATa/MATa mms3-1/mms3-1 or MATα/MATα mms3-1/mms3-1 diploids, such strains still retain sensitivity to the lethal effects of uv. Survival after uv irradiation in mms3-1 rad double mutant combinations indicates that mms3-1 is epistatic to rad6-1 whereas non-epistatic interactions are observed with rad3 and rad52 mutants. When present in the homozygous state in MATa/MATα his1-1/his1-315 heteroallelic diploids, mms3-1 was found to lower uv-induced mitotic recombination

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

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Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves

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

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Deguchi Tomonori

2010-09-01

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

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

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

1989-08-01

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

Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

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Rattray, Alison; Santoyo, Gustavo; Shafer, Brenda; Strathern, Jeffrey N

2015-01-01

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

Bacmeta: simulator for genomic evolution in bacterial metapopulations.

Science.gov (United States)

Sipola, Aleksi; Marttinen, Pekka; Corander, Jukka

2018-02-20

The advent of genomic data from densely sampled bacterial populations has created a need for flexible simulators by which models and hypotheses can be efficiently investigated in the light of empirical observations. Bacmeta provides fast stochastic simulation of neutral evolution within a large collection of interconnected bacterial populations with completely adjustable connectivity network. Stochastic events of mutations, recombinations, insertions/deletions, migrations and microepidemics can be simulated in discrete non-overlapping generations with a Wright-Fisher model that operates on explicit sequence data of any desired genome length. Each model component, including locus, bacterial strain, population, and ultimately the whole metapopulation, is efficiently simulated using C ++ objects, and detailed metadata from each level can be acquired. The software can be executed in a cluster environment using simple textual input files, enabling, e.g., large-scale simulations and likelihood-free inference. Bacmeta is implemented with C ++ for Linux, Mac and Windows. It is available at https://bitbucket.org/aleksisipola/bacmeta under the BSD 3-clause license. aleksi.sipola@helsinki.fi, jukka.corander@medisin.uio.no. Supplementary data are available at Bioinformatics online.

Lack of chemically induced mutation in repair-deficient mutants of yeast

International Nuclear Information System (INIS)

Prakash, L.

1974-01-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), β-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents. (auth)

Lack of chemically induced mutation in repair-deficient mutants of yeast.

Science.gov (United States)

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Novel TGM5 mutations in acral peeling skin syndrome.

Science.gov (United States)

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

2015-04-01

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

Induced mutation for disease resistance in rice with special reference to blast, bacterial blight and tungro

International Nuclear Information System (INIS)

Mathur, S.C.

1983-01-01

Rice varieties Ratna, Pusa 2-21, Vijaya and Pankaj have been treated with gamma rays, EMS or sodium azide to improve their resistance against blast, bacterial leaf blight or tungro virus. For blast and tungro, mutants with improved resistance were selected. Variation in reaction to bacterial leaf blight has been used in crossbreeding to accumulate genes for resistance. (author)

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

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Barbara Ziegler

2011-11-01

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

75 FR 5546 - Proposed Significant New Use Rule for Multi-walled Carbon Nanotubes

Science.gov (United States)

2010-02-03

... effects of the PMN substance: A bacterial reverse mutation test (OPPTS Harmonized Test Guideline 870.5100) in vitro; a mammalian erythrocyte micronucleus test (OPPTS Harmonized Test Guideline 870.5395 or Organisation for Economic Co-operation and Development (OECD) 474 test guideline) in vivo, in bone marrow, by...

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

International Nuclear Information System (INIS)

Yoshikawa, Isao; Takatsuji, Toshihiro

2004-01-01

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

In vivo somatic mutation systems in the mouse

International Nuclear Information System (INIS)

Russell, L.B.

1979-01-01

In an effort to meet the need for a fast and cheap in vivo prescreen for inherited mammalian point mutations, a somatic forward-mutation method, originally developed in an x-ray experiment, has more recently been tested in work with chemical mutagens. The method makes use of coat-color mutations because the gene product is usually locally expressed, mosaics can be detected with minimal effort, and opportunities for making comparison with induction of germinal point mutations are greatest.--Following treatment of embryos that are heterozygous at specific coat-color loci, various induced genetic changes can result in expression of the recessive (RS) in clones derived from mutant melanocyte precursor cells. However, other events, such as decrease in the number of precursor cells, or disturbed differentiation, can also result in spots, which with careful classification can usually be distinguished from RS's on the basis of their location and color. When this is done, the relative RS frequencies for a series of compounds at least roughly parallel the relative spermatogonial mutation rates. The fact that easily measurable (though low) RS rates are obtained with compounds that have yielded negative results in spermatogonial tests is not surprising in view of the fact that RS's can be caused by several mechanisms besides point mutation.--In spite of the parallelism observed in one laboratory, the usefulness of the in vivo somatic mutation method as a prescreen could come to be doubted because of major discrepancies between results of similar experiments at different laboratories. However, It appears probable that at least some of these discrepancies are due to failure to discriminate between spots that probably resulted from melanocyte insufficiency and spots that resulted from expression of the recessive.--Reverse somatic mutation systems can potentially avoid some of the pitfalls of forward mutation systems. Such system are still in developmental stages

Effectiveness of seawater reverse osmosis (SWRO) pretreatment systems in removing transparent exopolymer particles (TEP) substances

KAUST Repository

Lee, Shang-Tse

2015-01-01

Transparent exopolymer particles (TEP) have been reported as one of the main factors of membrane fouling in seawater reverse osmosis (SWRO) process. Research has been focused on algal TEP so far, overlooking bacterial TEP. This thesis investigated

Germline TERT promoter mutations are rare in familial melanoma

DEFF Research Database (Denmark)

Harland, Mark; Petljak, Mia; Robles-Espinoza, Carla Daniela

2016-01-01

Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP, POT1), have also been implicated in melanomagenesis. Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.-57...... T>G variant) has been reported in one family. We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls. Germline lymphocyte telomere length was estimated in carriers....... The c.-57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls. One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour. The blood leukocyte...

Activating HER2 mutations in HER2 gene amplification negative breast cancer.

Science.gov (United States)

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

2013-02-01

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

Non-homologous end joining dependency of {gamma}-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

Energy Technology Data Exchange (ETDEWEB)

Heidenreich, Erich [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)]. E-mail: erich.heidenreich@meduniwien.ac.at; Eisler, Herfried [Institute of Cancer Research, Division of Molecular Genetics, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna (Austria)

2004-11-22

There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by {gamma}-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the {gamma}-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.

Non-homologous end joining dependency of γ-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

International Nuclear Information System (INIS)

Heidenreich, Erich; Eisler, Herfried

2004-01-01

There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by γ-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the γ-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development

Harnessing CRISPR-Cas systems for bacterial genome editing.

Science.gov (United States)

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Heterozygous RTEL1 mutations are associated with familial pulmonary fibrosis.

Science.gov (United States)

Kannengiesser, Caroline; Borie, Raphael; Ménard, Christelle; Réocreux, Marion; Nitschké, Patrick; Gazal, Steven; Mal, Hervé; Taillé, Camille; Cadranel, Jacques; Nunes, Hilario; Valeyre, Dominique; Cordier, Jean François; Callebaut, Isabelle; Boileau, Catherine; Cottin, Vincent; Grandchamp, Bernard; Revy, Patrick; Crestani, Bruno

2015-08-01

Pulmonary fibrosis is a fatal disease with progressive loss of respiratory function. Defective telomere maintenance leading to telomere shortening is a cause of pulmonary fibrosis, as mutations in the telomerase component genes TERT (reverse transcriptase) and TERC (RNA component) are found in 15% of familial pulmonary fibrosis (FPF) cases. However, so far, about 85% of FPF remain genetically uncharacterised.Here, in order to identify new genetic causes of FPF, we performed whole-exome sequencing, with a candidate-gene approach, of 47 affected subjects from 35 families with FPF without TERT and TERC mutations.We identified heterozygous mutations in regulator of telomere elongation helicase 1 (RTEL1) in four families. RTEL1 is a DNA helicase with roles in DNA replication, genome stability, DNA repair and telomere maintenance. The heterozygous RTEL1 mutations segregated as an autosomal dominant trait in FPF, and were predicted by structural analyses to severely affect the function and/or stability of RTEL1. In agreement with this, RTEL1-mutated patients exhibited short telomeres in comparison with age-matched controls.Our results provide evidence that heterozygous RTEL1 mutations are responsible for FPF and, thereby, extend the clinical spectrum of RTEL1 deficiency. Thus, RTEL1 enlarges the number of telomere-associated genes implicated in FPF. Copyright ©ERS 2015.

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

Directory of Open Access Journals (Sweden)

Alice Sheehan

2018-03-01

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

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

Science.gov (United States)

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

2018-01-01

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

Optimization of lag phase shapes the evolution of a bacterial enzyme.

Science.gov (United States)

Adkar, Bharat V; Manhart, Michael; Bhattacharyya, Sanchari; Tian, Jian; Musharbash, Michael; Shakhnovich, Eugene I

2017-04-28

Mutations provide the variation that drives evolution, yet their effects on fitness remain poorly understood. Here we explore how mutations in the essential enzyme adenylate kinase (Adk) of Escherichia coli affect multiple phases of population growth. We introduce a biophysical fitness landscape for these phases, showing how they depend on molecular and cellular properties of Adk. We find that Adk catalytic capacity in the cell (the product of activity and abundance) is the major determinant of mutational fitness effects. We show that bacterial lag times are at a well-defined optimum with respect to Adk's catalytic capacity, while exponential growth rates are only weakly affected by variation in Adk. Direct pairwise competitions between strains show how environmental conditions modulate the outcome of a competition where growth rates and lag times have a tradeoff, shedding light on the multidimensional nature of fitness and its importance in the evolutionary optimization of enzymes.

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

International Nuclear Information System (INIS)

Mihalatos, Markos; Fountzilas, George; Agnantis, Niki J; Nasioulas, Georgios; Apessos, Angela; Dauwerse, Hans; Velissariou, Voula; Psychias, Aristidis; Koliopanos, Alexander; Petropoulos, Konstantinos; Triantafillidis, John K; Danielidis, Ioannis

2005-01-01

Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

Normoxic Recovery Mimicking Treatment of Sleep Apnea Does Not Reverse Intermittent Hypoxia-Induced Bacterial Dysbiosis and Low-Grade Endotoxemia in Mice.

Science.gov (United States)

Moreno-Indias, Isabel; Torres, Marta; Sanchez-Alcoholado, Lidia; Cardona, Fernando; Almendros, Isaac; Gozal, David; Montserrat, Josep M; Queipo-Ortuño, Maria I; Farré, Ramon

2016-10-01

Intermittent hypoxia (IH) mimicking obstructive sleep apnea (OSA) significantly modifies gut microbiota in mice. However, whether these IH-induced gut microbiome changes are reversible after restoring normal oxygenation (the equivalent of effective OSA therapy) is unknown. The aim of this study was to investigate gut microbiota composition and circulating endotoxemia after a post-IH normoxic period in a mouse model of OSA. Ten mice were subjected to IH (40 sec 21% O2-20 sec 5% O2) for 6 h/day for 6 w and 10 mice breathing normoxic air (NM) were used as controls. After exposures, both groups were subjected to 6 w in normoxia. Microbiome composition of fecal samples was determined by 16S ribosomal RNA (rRNA) pyrosequencing. Bioinformatic analysis was performed by Quantitative Insights into Microbial Ecology. Plasma lipopolysaccharide (LPS) levels were measured by endotoxin assay. After normoxic recovery, the Chao and Shannon indices of each group suggested similar bacterial richness and diversity. 16S rRNA pyrosequencing analysis showed that IH-exposed mice had a significant decrease in the abundance of Bacteroidetes and a significant increase of Firmicutes and Deferribacteres compared to the NM group. After normoxic recovery, circulating LPS concentrations were higher in the IH group (P < 0.009). Moreover, the IH group showed a negative and significant correlation between the abundance of Lactobacillus and Ruminococcus and significant positive correlations between the abundance of Mucispirillum and Desulfovibrio and plasma LPS levels, respectively. Even after prolonged normoxic recovery after IH exposures, gut microbiota and circulating endotoxemia remain negatively altered, suggesting that potential benefits of OSA treatment for reversing OSA-induced changes in gut microbiota may either require a longer period or alternative interventions. © 2016 Associated Professional Sleep Societies, LLC.

Host imprints on bacterial genomes--rapid, divergent evolution in individual patients.

Directory of Open Access Journals (Sweden)

Jaroslaw Zdziarski

Full Text Available Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.

Autonomously responsive pumping by a bacterial flagellar forest: A mean-field approach

Science.gov (United States)

Martindale, James D.; Fu, Henry C.

2017-09-01

This study is motivated by a microfluidic device that imparts a magnetic torque on an array of bacterial flagella. Bacterial flagella can transform their helical geometry autonomously in response to properties of the background fluid, which provides an intriguing mechanism allowing their use as an engineered element for the regulation or transport of chemicals in microscale applications. The synchronization of flagellar phase has been widely studied in biological contexts, but here we examine the synchronization of flagellar tilt, which is necessary for effective pumping. We first examine the effects of helical geometry and tilt on the pumping flows generated by a single rotating flagellum. Next, we explore a mean-field model for an array of helical flagella to understand how collective tilt arises and influences pumping. The mean-field methodology allows us to take into account possible phase differences through a time-averaging procedure and to model an infinite array of flagella. We find array separation distances, magnetic field strengths, and rotation frequencies that produce nontrivial self-consistent pumping solutions. For individual flagella, pumping is reversed when helicity or rotation is reversed; in contrast, when collective effects are included, self-consistent tilted pumping solutions become untilted nonpumping solutions when helicity or rotation is reversed.

Parasites and deleterious mutations: interactions influencing the evolutionary maintenance of sex.

Science.gov (United States)

Park, A W; Jokela, J; Michalakis, Y

2010-05-01

The restrictive assumptions associated with purely genetic and purely ecological mechanisms suggest that neither of the two forces, in isolation, can offer a general explanation for the evolutionary maintenance of sex. Consequently, attention has turned to pluralistic models (i.e. models that apply both ecological and genetic mechanisms). Existing research has shown that combining mutation accumulation and parasitism allows restrictive assumptions about genetic and parasite parameter values to be relaxed while still predicting the maintenance of sex. However, several empirical studies have shown that deleterious mutations and parasitism can reduce fitness to a greater extent than would be expected if the two acted independently. We show how interactions between these genetic and ecological forces can completely reverse predictions about the evolution of reproductive modes. Moreover, we demonstrate that synergistic interactions between infection and deleterious mutations can render sex evolutionarily stable even when there is antagonistic epistasis among deleterious mutations, thereby widening the conditions for the evolutionary maintenance of sex.

Study of hTERT and Histone 3 Mutations in Medulloblastoma.

Science.gov (United States)

Viana-Pereira, Marta; Almeida, Gisele Caravina; Stavale, João Norberto; Malheiro, Susana; Clara, Carlos; Lobo, Patrícia; Pimentel, José; Reis, Rui Manuel

2017-01-01

Hotspot activating mutations of the telomerase reverse transcriptase (hTERT) promoter region were recently described in several tumor types. These mutations lead to enhanced expression of telomerase, being responsible for telomere maintenance and allowing continuous cell division. Additionally, there are alternative telomere maintenance mechanisms, associated with histone H3 mutations, responsible for disrupting the histone code and affecting the regulation of transcription. Here, we investigated the clinical relevance of these mechanistically related molecules in medulloblastoma. Sixty-nine medulloblastomas, formalin fixed and paraffin embedded, from a cohort of patients aged 1.5-70 years, were used to investigate the hotspot mutations of the hTERT promoter region, i.e. H3F3A and HIST1H3B, using Sanger sequencing. We successfully sequenced hTERT in all 69 medulloblastoma samples and identified a total of 19 mutated cases (27.5%). c.-124:G>A and c.-146:G>A mutations were detected, respectively, in 16 and 3 samples. Similar to previous reports, hTERT mutations were more frequent in older patients (p < 0.0001), being found only in 5 patients <20 years of age. In addition, hTERT-mutated tumors were more frequently recurrent (p = 0.026) and hTERT mutations were significantly enriched in tumors located in the right cerebellar hemisphere (p = 0.039). No mutations were found on the H3F3A or HIST1H3B genes. hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. On the other hand, histone 3 mutations do not seem to be present in medulloblastoma. © 2016 S. Karger AG, Basel.

His+ reversions caused in Salmonella typhimurium by different types of ionizing radiation

International Nuclear Information System (INIS)

Roos, H.; Thomas, W.H.; Fitzek, M.; Kellerer, A.M.

1988-01-01

The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co gamma rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV alpha particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/micron. The ratio of the effectiveness of this radiation to gamma rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the alpha particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating

Reversible antibiotic tolerance induced in Staphylococcus aureus by concurrent drug exposure

DEFF Research Database (Denmark)

Haaber, Jakob Krause; Friberg, Cathrine; McCreary, Mark

2015-01-01

UNLABELLED: Resistance of Staphylococcus aureus to beta-lactam antibiotics has led to increasing use of the glycopeptide antibiotic vancomycin as a life-saving treatment for major S. aureus infections. Coinfection by an unrelated bacterial species may necessitate concurrent treatment with a second...... antibiotic that targets the coinfecting pathogen. While investigating factors that affect bacterial antibiotic sensitivity, we discovered that susceptibility of S. aureus to vancomycin is reduced by concurrent exposure to colistin, a cationic peptide antimicrobial employed to treat infections by Gram......-negative pathogens. We show that colistin-induced vancomycin tolerance persists only as long as the inducer is present and is accompanied by gene expression changes similar to those resulting from mutations that produce stably inherited reduction of vancomycin sensitivity (vancomycin-intermediate S. aureus [VISA...

UV-induced tandem double mutations in the trpA gene of E. coli

International Nuclear Information System (INIS)

Piechocki, R.; Langhammer, R.

1980-01-01

The ultraviolet light induction of tandem double mutations in a reverse mutation system was shown using trpA mutants which are characterized by the codon sequences GAA and AAG in codon position 211. Among 597 Trp + independent revertants of the trpA (AAG211) strain 3 full revertants were detected arising from UV-induced tandem double base exchanges. In the codon unit 211 full revertants due to single base exchanges are at least 20 times as frequent as full revertants due to tandem double base exchanges. (author)

The Bacterial Sequential Markov Coalescent.

Science.gov (United States)

De Maio, Nicola; Wilson, Daniel J

2017-05-01

Bacteria can exchange and acquire new genetic material from other organisms directly and via the environment. This process, known as bacterial recombination, has a strong impact on the evolution of bacteria, for example, leading to the spread of antibiotic resistance across clades and species, and to the avoidance of clonal interference. Recombination hinders phylogenetic and transmission inference because it creates patterns of substitutions (homoplasies) inconsistent with the hypothesis of a single evolutionary tree. Bacterial recombination is typically modeled as statistically akin to gene conversion in eukaryotes, i.e. , using the coalescent with gene conversion (CGC). However, this model can be very computationally demanding as it needs to account for the correlations of evolutionary histories of even distant loci. So, with the increasing popularity of whole genome sequencing, the need has emerged for a faster approach to model and simulate bacterial genome evolution. We present a new model that approximates the coalescent with gene conversion: the bacterial sequential Markov coalescent (BSMC). Our approach is based on a similar idea to the sequential Markov coalescent (SMC)-an approximation of the coalescent with crossover recombination. However, bacterial recombination poses hurdles to a sequential Markov approximation, as it leads to strong correlations and linkage disequilibrium across very distant sites in the genome. Our BSMC overcomes these difficulties, and shows a considerable reduction in computational demand compared to the exact CGC, and very similar patterns in simulated data. We implemented our BSMC model within new simulation software FastSimBac. In addition to the decreased computational demand compared to previous bacterial genome evolution simulators, FastSimBac provides more general options for evolutionary scenarios, allowing population structure with migration, speciation, population size changes, and recombination hotspots. FastSimBac is

An evolutionary reduction principle for mutation rates at multiple Loci.

Science.gov (United States)

Altenberg, Lee

2011-06-01

A model of mutation rate evolution for multiple loci under arbitrary selection is analyzed. Results are obtained using techniques from Karlin (Evolutionary Biology, vol. 14, pp. 61-204, 1982) that overcome the weak selection constraints needed for tractability in prior studies of multilocus event models.A multivariate form of the reduction principle is found: reduction results at individual loci combine topologically to produce a surface of mutation rate alterations that are neutral for a new modifier allele. New mutation rates survive if and only if they fall below this surface-a generalization of the hyperplane found by Zhivotovsky et al. (Proc. Natl. Acad. Sci. USA 91, 1079-1083, 1994) for a multilocus recombination modifier. Increases in mutation rates at some loci may evolve if compensated for by decreases at other loci. The strength of selection on the modifier scales in proportion to the number of germline cell divisions, and increases with the number of loci affected. Loci that do not make a difference to marginal fitnesses at equilibrium are not subject to the reduction principle, and under fine tuning of mutation rates would be expected to have higher mutation rates than loci in mutation-selection balance.Other results include the nonexistence of 'viability analogous, Hardy-Weinberg' modifier polymorphisms under multiplicative mutation, and the sufficiency of average transmission rates to encapsulate the effect of modifier polymorphisms on the transmission of loci under selection. A conjecture is offered regarding situations, like recombination in the presence of mutation, that exhibit departures from the reduction principle. Constraints for tractability are: tight linkage of all loci, initial fixation at the modifier locus, and mutation distributions comprising transition probabilities of reversible Markov chains.

Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3

OpenAIRE

Bøsling, Jacob; Poulsen, Susan M.; Vester, Birte; Long, Katherine S.

2003-01-01

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ri...

Colorectal cancer mutational profiles correlate with defined microbial communities in the tumor microenvironment.

Science.gov (United States)

Burns, Michael B; Montassier, Emmanuel; Abrahante, Juan; Priya, Sambhawa; Niccum, David E; Khoruts, Alexander; Starr, Timothy K; Knights, Dan; Blekhman, Ran

2018-06-20

Variation in the gut microbiome has been linked to colorectal cancer (CRC), as well as to host genetic variation. However, we do not know whether, in addition to baseline host genetics, somatic mutational profiles in CRC tumors interact with the surrounding tumor microbiome, and if so, whether these changes can be used to understand microbe-host interactions with potential functional biological relevance. Here, we characterized the association between CRC microbial communities and tumor mutations using microbiome profiling and whole-exome sequencing in 44 pairs of tumors and matched normal tissues. We found statistically significant associations between loss-of-function mutations in tumor genes and shifts in the abundances of specific sets of bacterial taxa, suggestive of potential functional interaction. This correlation allows us to statistically predict interactions between loss-of-function tumor mutations in cancer-related genes and pathways, including MAPK and Wnt signaling, solely based on the composition of the microbiome. In conclusion, our study shows that CRC microbiomes are correlated with tumor mutational profiles, pointing towards possible mechanisms of molecular interaction.

Mini-review: Strategies for Variation and Evolution of Bacterial Antigens

Science.gov (United States)

Foley, Janet

2015-01-01

Across the eubacteria, antigenic variation has emerged as a strategy to evade host immunity. However, phenotypic variation in some of these antigens also allows the bacteria to exploit variable host niches as well. The specific mechanisms are not shared-derived characters although there is considerable convergent evolution and numerous commonalities reflecting considerations of natural selection and biochemical restraints. Unlike in viruses, mechanisms of antigenic variation in most bacteria involve larger DNA movement such as gene conversion or DNA rearrangement, although some antigens vary due to point mutations or modified transcriptional regulation. The convergent evolution that promotes antigenic variation integrates various evolutionary forces: these include mutations underlying variant production; drift which could remove alleles especially early in infection or during life history phases in arthropod vectors (when the bacterial population size goes through a bottleneck); selection not only for any particular variant but also for the mechanism for the production of variants (i.e., selection for mutability); and overcoming negative selection against variant production. This review highlights the complexities of drivers of antigenic variation, in particular extending evaluation beyond the commonly cited theory of immune evasion. A deeper understanding of the diversity of purpose and mechanisms of antigenic variation in bacteria will contribute to greater insight into bacterial pathogenesis, ecology and coevolution with hosts. PMID:26288700

Reversion in variants from a duplication strain of Aspergillus nidulans

International Nuclear Information System (INIS)

Menezes, E.M.; Azevedo, J.L.

1978-01-01

Strains of Aspergillus nidulans with a chromosome segment in duplicate, one in normal position and one translocated to another chromosome, are unstable at mitosis. In addition to variants which result from deletions in either of the duplicate segments, which usually have improved morphology, they produce variants with deteriorated morphology. Three deteriorated variants reverted frequently to parental type morphology, both spontaneously and after ultra-violet treatment. Of six reversions analysed genetically, five were due to suppressors and one was probably due to back mutation. The suppressors segregated as single genes and were not linked to the mutation which they suppress. The instability of these so-called 'deteriorated' variants is discussed in relation to mitotic instability phenomena in A. nidulans. (orig.) [de

Control of intestinal bacterial proliferation in regulation of lifespan in Caenorhabditis elegans

Directory of Open Access Journals (Sweden)

Portal-Celhay Cynthia

2012-03-01

Full Text Available Abstract Background A powerful approach to understanding complex processes such as aging is to use model organisms amenable to genetic manipulation, and to seek relevant phenotypes to measure. Caenorhabditis elegans is particularly suited to studies of aging, since numerous single-gene mutations have been identified that affect its lifespan; it possesses an innate immune system employing evolutionarily conserved signaling pathways affecting longevity. As worms age, bacteria accumulate in the intestinal tract. However, quantitative relationships between worm genotype, lifespan, and intestinal lumen bacterial load have not been examined. We hypothesized that gut immunity is less efficient in older animals, leading to enhanced bacterial accumulation, reducing longevity. To address this question, we evaluated the ability of worms to control bacterial accumulation as a functional marker of intestinal immunity. Results We show that as adult worms age, several C. elegans genotypes show diminished capacity to control intestinal bacterial accumulation. We provide evidence that intestinal bacterial load, regulated by gut immunity, is an important causative factor of lifespan determination; the effects are specified by bacterial strain, worm genotype, and biologic age, all acting in concert. Conclusions In total, these studies focus attention on the worm intestine as a locus that influences longevity in the presence of an accumulating bacterial population. Further studies defining the interplay between bacterial species and host immunity in C. elegans may provide insights into the general mechanisms of aging and age-related diseases.

Microbial ecology of bacterially mediated PCB biodegradation

International Nuclear Information System (INIS)

Pettigrew, C.A. Jr.

1989-01-01

The roles of plasmid mediated and consortia mediated polychlorinated biphenyl (PCB) biodegradation by bacterial populations isolated from PCB contaminated freshwater sediments were investigated. PCB degrading bacteria were isolated by DNA:DNA colony hybridization, batch enrichments, and chemostat enrichment. Analysis of substrate removal and metabolite production were done using chlorinated biphenyl spray plates, reverse phase high pressure liquid chromatography, Cl - detection, and 14 C-labeled substrate mineralization methods. A bacterial consortium, designated LPS10, involved in a concerted metabolic attack on chlorinated biphenyls, was shown to mineralize 4-chlorobiphenyl (4CB) and 4,4'-dichlorobiphenyl (4,4' CB). The LPS10 consortium was isolated by both batch and chemostat enrichment using 4CB and biphenyl (BP) as sole carbon source and was found to have tree bacterial isolates that predominated; these included: Pseudomonas, testosteroni LPS10A which mediated the breakdown of 4CB and 4,4' CB to the putative meta-cleavage product and subsequently to 4-chlorobenzoic acid (4CBA), an isolate tentatively identified as an Arthrobacter sp. LPS10B which mediated 4CBA degradation, and Pseudomonas putida by A LPS10C whose role in the consortium has not been determined

77 FR 38199 - Propiconazole; Pesticide Tolerances

Science.gov (United States)

2012-06-27

... reported at doses that were not maternally toxic. In the 2-generation reproduction study in rats, offspring... the in vitro BALB/C 3T3 cell transformation assay, bacterial reverse mutation assay, Chinese hamster... fetuses to in utero exposure to propiconazole was observed in this study. In the 2-generation reproduction...

40 CFR 158.230 - Experimental use permit data requirements for toxicology.

Science.gov (United States)

2010-07-01

... NR TGAI TGAI 6 Developmental Toxicity and Reproduction 870.3700 Prenatal Developmental toxicity - rat and rabbit, preferred R NR TGAI TGAI 7, 8 870.3800 Reproduction R NR TGAI TGAI 6 Mutagenicity Testing 870.5100 Bacterial reverse mutation assay R NR TGAI TGAI 9 870.5300870.5375 In vitro mammalian cell...

Sex reversal in zebrafish fancl mutants is caused by Tp53-mediated germ cell apoptosis.

Directory of Open Access Journals (Sweden)

Adriana Rodríguez-Marí

2010-07-01

Full Text Available The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53 mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.

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

Science.gov (United States)

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

2016-11-01

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

Neighborhood properties are important determinants of temperature sensitive mutations.

Directory of Open Access Journals (Sweden)

Svetlana Lockwood

Full Text Available Temperature-sensitive (TS mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method-logistic regression-to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

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

International Nuclear Information System (INIS)

Yoshikawa, Isao; Hoshi, Masaharu; Ikenaga, Mituo

1996-01-01

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

Mechanical interactions in bacterial colonies and the surfing probability of beneficial mutations

Science.gov (United States)

Farrell, Fred D.

2017-01-01

Bacterial conglomerates such as biofilms and microcolonies are ubiquitous in nature and play an important role in industry and medicine. In contrast to well-mixed cultures routinely used in microbial research, bacteria in a microcolony interact mechanically with one another and with the substrate to which they are attached. Here, we use a computer model of a microbial colony of rod-shaped cells to investigate how physical interactions between cells determine their motion in the colony and how this affects biological evolution. We show that the probability that a faster-growing mutant ‘surfs’ at the colony's frontier and creates a macroscopic sector depends on physical properties of cells (shape, elasticity and friction). Although all these factors contribute to the surfing probability in seemingly different ways, their effects can be summarized by two summary statistics that characterize the front roughness and cell alignment. Our predictions are confirmed by experiments in which we measure the surfing probability for colonies of different front roughness. Our results show that physical interactions between bacterial cells play an important role in biological evolution of new traits, and suggest that these interactions may be relevant to processes such as de novo evolution of antibiotic resistance. PMID:28592660

Mechanical interactions in bacterial colonies and the surfing probability of beneficial mutations.

Science.gov (United States)

Farrell, Fred D; Gralka, Matti; Hallatschek, Oskar; Waclaw, Bartlomiej

2017-06-01

Bacterial conglomerates such as biofilms and microcolonies are ubiquitous in nature and play an important role in industry and medicine. In contrast to well-mixed cultures routinely used in microbial research, bacteria in a microcolony interact mechanically with one another and with the substrate to which they are attached. Here, we use a computer model of a microbial colony of rod-shaped cells to investigate how physical interactions between cells determine their motion in the colony and how this affects biological evolution. We show that the probability that a faster-growing mutant 'surfs' at the colony's frontier and creates a macroscopic sector depends on physical properties of cells (shape, elasticity and friction). Although all these factors contribute to the surfing probability in seemingly different ways, their effects can be summarized by two summary statistics that characterize the front roughness and cell alignment. Our predictions are confirmed by experiments in which we measure the surfing probability for colonies of different front roughness. Our results show that physical interactions between bacterial cells play an important role in biological evolution of new traits, and suggest that these interactions may be relevant to processes such as de novo evolution of antibiotic resistance. © 2017 The Author(s).

Molecular characterization of three novel Fanconi anemia mutations in Israeli Arabs.

Science.gov (United States)

Tamary, Hannah; Dgany, Orly; Toledano, Helen; Shalev, Zvi; Krasnov, Tatyana; Shalmon, Lea; Schechter, Tali; Bercovich, Dani; Attias, Dina; Laor, Ruth; Koren, Ariel; Yaniv, Isaac

2004-05-01

In a previous study, we investigated the molecular basis of Fanconi anemia (FA) in 13 unrelated Israeli Jewish FA patients and identified four ethnicity specific mutations. In the present study we extended our study to Israeli Arab patients. We studied three consanguineous families with nine FA patients and an additional unrelated patient. DNA single-strand conformation polymorphism of each exon of the FANCA and FANCG genes was followed by sequence analysis of the aberrantly migrating fragments and by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the splice-site mutations identified. Three unique disease-causing mutations were identified: (i) FANCA gross deletion of exons 6-31; (ii) FANCA splice-site mutation IVS 42-2A>C; (iii) FANCG splice-site mutation IVS4+3A>G. Sequence analysis of the FANCA gross deletion revealed recombination between two highly homologous Alu elements. cDNA analysis of the two splice mutations suggested intron 42 retention in FANCA IVS 42-2A>C and exon 4 skipping in FANCG IVS4+3A>G. The clinical condition of eight patients with FANCA mutations was severe. Two unique FANCA mutations and one FANCG mutation were identified in Israeli Arab FA patients. Deletion of FANCA exon 6-31 as in previously described gross deletions was within introns rich in Alu repeats. To the best of our knowledge, the FANCA IVS 42-2A>C mutation is the first in this gene to result in intron retention. Further analysis of FA mutations will enable prenatal diagnosis and a rational therapeutic approach including frequent monitoring and early bone marrow transplantation. Copyright Blackwell Munksgaard 2004.

Accumulation of neutral mutations in growing cell colonies with competition.

Science.gov (United States)

Sorace, Ron; Komarova, Natalia L

2012-12-07

Neutral mutations play an important role in many biological processes including cancer initiation and progression, the generation of drug resistance in bacterial and viral diseases as well as cancers, and the development of organs in multicellular organisms. In this paper we study how neutral mutants are accumulated in nonlinearly growing colonies of cells subject to growth constraints such as crowding or lack of resources. We investigate different types of growth control which range from "division-controlled" to "death-controlled" growth (and various mixtures of both). In division-controlled growth, the burden of handling overcrowding lies with the process of cell-divisions, the divisions slow down as the carrying capacity is approached. In death-controlled growth, it is death rate that increases to slow down expansion. We show that division-controlled growth minimizes the number of accumulated mutations, and death-controlled growth corresponds to the maximum number of mutants. We check that these results hold in both deterministic and stochastic settings. We further develop a general (deterministic) theory of neutral mutations and achieve an analytical understanding of the mutant accumulation in colonies of a given size in the absence of back-mutations. The long-term dynamics of mutants in the presence of back-mutations is also addressed. In particular, with equal forward- and back-mutation rates, if division-controlled and a death-controlled types are competing for space and nutrients, cells obeying division-controlled growth will dominate the population. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bacteriophage Resistance Mechanisms in the Fish Pathogen Flavobacterium psychrophilum: Linking Genomic Mutations to Changes in Bacterial Virulence Factors

DEFF Research Database (Denmark)

Castillo, Daniel; Christiansen, Rói Hammershaimb; Dalsgaard, Inger

2015-01-01

-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage...

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

Science.gov (United States)

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

2017-08-02

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

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

Science.gov (United States)

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

Science.gov (United States)

Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

2017-09-01

Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. Copyright © 2017 American Society for Microbiology.

40 CFR 158.500 - Toxicology data requirements table.

Science.gov (United States)

2010-07-01

... Toxicity and Reproduction 870.3700 Prenatal Developmental toxicity - rat and rabbit, preferred R R TGAI TGAI 22, 23, 24, 25, 26 870.3800 Reproduction and fertility effects R R TGAI TGAI 26, 27, 29 870.6300 Developmental neurotoxicity CR CR TGAI TGAI 27, 28, 29 Mutagenicity Testing 870.5100 Bacterial reverse mutation...

MutaNET: a tool for automated analysis of genomic mutations in gene regulatory networks.

Science.gov (United States)

Hollander, Markus; Hamed, Mohamed; Helms, Volkhard; Neininger, Kerstin

2018-03-01

Mutations in genomic key elements can influence gene expression and function in various ways, and hence greatly contribute to the phenotype. We developed MutaNET to score the impact of individual mutations on gene regulation and function of a given genome. MutaNET performs statistical analyses of mutations in different genomic regions. The tool also incorporates the mutations in a provided gene regulatory network to estimate their global impact. The integration of a next-generation sequencing pipeline enables calling mutations prior to the analyses. As application example, we used MutaNET to analyze the impact of mutations in antibiotic resistance (AR) genes and their potential effect on AR of bacterial strains. MutaNET is freely available at https://sourceforge.net/projects/mutanet/. It is implemented in Python and supported on Mac OS X, Linux and MS Windows. Step-by-step instructions are available at http://service.bioinformatik.uni-saarland.de/mutanet/. volkhard.helms@bioinformatik.uni-saarland.de. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Energetics of bacterial adhesion

International Nuclear Information System (INIS)

Loosdrecht, M.C.M. van; Zehnder, A.J.B.

1990-01-01

For the description of bacterial adhesion phenomena two different physico-chemical approaches are available. The first one, based on a surface Gibbs energy balance, assumes intimate contact between the interacting surfaces. The second approach, based on colloid chemical theories (DLVO theory), allows for two types of adhesion: 1) secondary minimum adhesion, which is often weak and reversible, and 2) irreversible primary minimum adhesion. In the secondary minimum adhesion a thin water film remains present between the interacting surface. The merits of both approaches are discussed in this paper. In addition, the methods available to measure the physico-chemical surface characteristics of bacteria and the influence of adsorbing (in)organic compounds, extracellular polymers and cell surface appendages on adhesion are summarized. (author) 2 figs., 1 tab., 50 refs

Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli

DEFF Research Database (Denmark)

Sánchez-Céspedes, Javier; Sáez-López, Emma; Frimodt-Møller, N

2015-01-01

the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced......Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase...

Conditional Function of Autoaggregative Protein Cah and Common cah Mutations in Shiga Toxin-Producing Escherichia coli.

Science.gov (United States)

Carter, Michelle Qiu; Brandl, Maria T; Kudva, Indira T; Katani, Robab; Moreau, Matthew R; Kapur, Vivek

2018-01-01

Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro : it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the

40 CFR 158.2050 - Biochemical pesticides human health assessment data requirements table.

Science.gov (United States)

2010-07-01

... Mutagenicity Testing 870.5100 Bacterial reverse mutation test R CR TGAI TGAI 10 870.5300870.5375 In vitro... Testing 880.3800 Immune response CR CR TGAI TGAI 14 870.3800 Reproduction and fertility effects CR CR TGAI... reproduction study in rodents (guideline 870.3800) as a basic protocol for the addition of other endpoints or...

HIV Drug Resistance-Associated Mutations in Antiretroviral Naïve HIV-1-Infected Latin American Children

Directory of Open Access Journals (Sweden)

Luis E. Soto-Ramirez

2010-01-01

Full Text Available Our goal was to describe the presence of HIV drug resistance among HIV-1-infected, antiretroviral (ARV naïve children and adolescents in Latin America and to examine resistance in these children in relation to drug exposure in the mother. Genotyping was performed on plasma samples obtained at baseline from HIV-1-infected participants in a prospective cohort study in Brazil, Argentina, and Mexico (NISDI Pediatric Study. Of 713 HIV-infected children enrolled, 69 were ARV naïve and eligible for the analysis. At enrollment, mean age was 7.3 years; 81.2% were infected with HIV perinatally. Drug resistance mutations (DRMs were detected in 6 (8.7%; 95% confidence interval 3.1–18.2% ARV-naïve subjects; none of the mothers of these 6 received ARVs during their pregnancies and none of the children received ARV prophylaxis. Reverse transcriptase mutations K70R and K70E were detected in 3 and 2 subjects, respectively; protease mutation I50 V was detected in 1 subject. Three of the 6 children with DRMs initiated ARV therapy during followup, with a good response in 2. The overall rate of primary drug resistance in this pediatric HIV-infected population was low, and no subjects had more than 1 DRM. Mutations associated with resistance to nucleoside reverse transcriptase inhibitors were the most prevalent.

Mutations That Alter the Bacterial Cell Envelope Increase Lipid Production

Energy Technology Data Exchange (ETDEWEB)

Lemmer, Kimberly C.; Zhang, Weiping; Langer, Samantha J.; Dohnalkova, Alice; 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 inRhodobacter sphaeroides. By screening anR. sphaeroidesTn5mutant 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.

IMPORTANCEThis 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

Structure of a bacterial toxin-activating acyltransferase.

Science.gov (United States)

Greene, Nicholas P; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2015-06-09

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.

The role of the bacterial mismatch repair system in SOS-induced mutagenesis: a theoretical background

International Nuclear Information System (INIS)

Belov, O.V.; Kapralov, M.I.; Chuluunbaatar, O.; Sweilam, N.H.

2012-01-01

A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, a mathematical model of the bacterial mismatch repair system is developed. Within this model, the key pathways of this type of repair are simulated on the basis of modern experimental data related to its mechanisms. Here we have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Using our calculations, we have tested the hypothesis that the bacterial mismatch repair system is responsible for the removal of the nucleotides misincorporated by DNA polymerase V (the UmuD' 2 C complex) during ultraviolet-induced SOS response. For the theoretical analysis of the mutation frequency, we have combined the proposed mathematical approach with the model of SOS-induced mutagenesis in the E.coli bacterial cell developed earlier. Our calculations support the hypothesis that methyl-directed mismatch repair influences the mutagenic effect of ultraviolet radiation

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation

OpenAIRE

Pailhoux , Eric; Vigier , Bernard; Schibler , Laurent; Edmond P. Cribiu ,; Cotinot , Corinne; Vaiman , Daniel

2005-01-01

Abstract In goats, the PIS (polled intersex syndrome) mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4–5 days is observed in comparison with normal testis differentiation in XY males...

Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli

DEFF Research Database (Denmark)

Adler, Marlen; Anjum, Mehreen; Andersson, Dan I.

2016-01-01

of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance...... levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure...

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

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Miguel Viveiros

2017-04-01

Full Text Available Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality

An anticholinergic reverses motor control and corticostriatal LTD deficits in Dyt1 ΔGAG knock-in mice

OpenAIRE

Dang, Mai T.; Yokoi, Fumiaki; Cheetham, Chad C.; Lu, Jun; Vo, Viet; Lovinger, David M.; Li, Yuqing

2011-01-01

DYT1 early-onset generalized torsion dystonia is an inherited movement disorder associated with mutations in DYT1 that codes for torsinA protein. The most common mutation seen in this gene is a trinucleotide deletion of GAG. We previously reported a motor control deficit on a beam-walking task in our Dyt1 ΔGAG knock-in heterozygous mice. In this report we show the reversal of this motor deficit with the anticholinergic trihexyphenidyl (THP), a drug commonly used to treat movement problems in ...

APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

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Kate N Bishop

2008-12-01

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

TERT promoter mutations and long telomere length predict poor survival and radiotherapy resistance in gliomas.

Science.gov (United States)

Gao, Ke; Li, Gang; Qu, Yiping; Wang, Maode; Cui, Bo; Ji, Meiju; Shi, Bingyin; Hou, Peng

2016-02-23

Increasing evidences have implicated somatic gain-of-function mutations at the telomerase reverse transcriptase (TERT) promoter as one of the major mechanisms that promote transcriptional activation of TERT and subsequently maintain telomere length in human cancers including glioma. To investigate the prognostic value of these mutations and telomere length, individually and their coexistence, in gliomas, we analyzed two somatic mutations C228T and C250T in the TERT promoter, relative telomere length (RTL), IDH1 mutation and MGMT methylation in 389 glioma patients, and explored their associations with patient characteristics and clinical outcomes. Our data showed that C228T and C250T mutations were found in 17.0% (66 of 389) and 11.8% (46 of 389) of gliomas, respectively, and these two mutations were mutually exclusive in this cancer. Moreover, they were significantly associated with WHO grade. We also found that the RTL was significant longer in gliomas than in meningiomas and normal brain tissues (Median, 0.89 vs. 0.44 and 0.50; P radiotherapy. Collectively, TERT promoter mutations and long RTL are not only prognostic factors for poor clinical outcomes, but also the predictors of radiotherapy resistance in gliomas.

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

Energy Technology Data Exchange (ETDEWEB)

Wu, R; Felton, J

2007-10-12

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

Resistance to the peptidyl transferase inhibitor tiamulin caused by mutation of ribosomal protein l3.

Science.gov (United States)

Bøsling, Jacob; Poulsen, Susan M; Vester, Birte; Long, Katherine S

2003-09-01

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

Posterior Reversible Encephalopathy Syndrome in Our Setup

International Nuclear Information System (INIS)

Ahmad, I.; Zubair, U. B.; Mumtaz, H.; Yousaf, M. A.; Muhammad, W. W.

2013-01-01

Objectives: To assess the clinical presentation and neuroimaging abnormalities in a series of patients diagnosed as posterior reversible encephalopathy syndrome at Military Hospital Rawalpindi. Study Design: Case series study. Place and Duration: Study was carried out at Military Hospital Rawalpindi form December 01st, 2011 to May 31st, 2012. Patients and Methods: Study included all the cases of the Posterior reversible encephalopathy syndrome (PRES) admitted in the wards and intensive care unit (ITC). Neuroimaging was done and all the studies were reviewed by independent neuroradiologist. Different clinical and laboratory variables were also studied and correlated with neuroimaging. Follow up ws done to look for the prognosis. Results: Of the seven patients labelled as PRES two were male and five were female. Two patients were over 50 years of age, out of them one was male and one was female. One patient had end stage renal disease (ESRD) secondary to diabetes mellitus (DM) and hypertension (HTN), one had eclampsia, one had pregnancy-induced hypertension (PIH) and one had just uncontrolled HTN. Peak spontaneous bacterial peritonitis (SBP) in 5 cases was 210 mm of Hg, four of which had seizures. Rest two had spontaneous bacterial peritonitis (SBP) of 160 out of which one developed seizures. Total out of 7, 5 experienced seizures and altered conscious state, rest two only had confusion. One patient had papilloedema. Follow up was done after 06 weeks, 02 patients died, 05 remained alive and symptoms of PRES had vanished. Conclusion: PRES is a neurological emergency, presents with a variety of symptoms and has a specific neuroimaging pattern. Early recognition and prompt treatment result in a good neurological outcome. (author)

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

Science.gov (United States)

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

2015-10-01

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

Molecular mechanisms underlying the emergence of bacterial pathogens: an ecological perspective.

Science.gov (United States)

Bartoli, Claudia; Roux, Fabrice; Lamichhane, Jay Ram

2016-02-01

The rapid emergence of new bacterial diseases negatively affects both human health and agricultural productivity. Although the molecular mechanisms underlying these disease emergences are shared between human- and plant-pathogenic bacteria, not much effort has been made to date to understand disease emergences caused by plant-pathogenic bacteria. In particular, there is a paucity of information in the literature on the role of environmental habitats in which plant-pathogenic bacteria evolve and on the stress factors to which these microbes are unceasingly exposed. In this microreview, we focus on three molecular mechanisms underlying pathogenicity in bacteria, namely mutations, genomic rearrangements and the acquisition of new DNA sequences through horizontal gene transfer (HGT). We briefly discuss the role of these mechanisms in bacterial disease emergence and elucidate how the environment can influence the occurrence and regulation of these molecular mechanisms by directly impacting disease emergence. The understanding of such molecular evolutionary mechanisms and their environmental drivers will represent an important step towards predicting bacterial disease emergence and developing sustainable management strategies for crops. © 2015 BSPP AND JOHN WILEY & SONS LTD.

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

Science.gov (United States)

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

2017-08-01

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

Biological significance of TERT promoter mutation in papillary urothelial neoplasm of low malignant potential.

Science.gov (United States)

Wang, Chung-Chieh; Huang, Chao-Yuan; Jhuang, Yu-Lin; Chen, Chih-Chi; Jeng, Yung-Ming

2018-04-01

Mutations in FGFR3 and the promoter region of the telomerase reverse transcriptase (TERT) gene have been found frequently in urothelial carcinoma of the urinary bladder. However, related data for papillary urothelial neoplasm of low malignant potential (PUNLMP) are limited. In this study, we investigated the mutation status of the TERT promoter, FGFR3 and HRAS in low-grade papillary urothelial neoplasms and evaluated their prognostic significance. The cases included in this study comprised 21 inverted papillomas, 30 PUNLMPs and 34 low-grade non-invasive papillary urothelial carcinomas (NIPUCs). TERT promoter mutations were observed in 10 (33%) PUNLMPs and 17 (50%) low-grade NIPUCs, but not in any inverted papilloma. FGFR3 mutations were observed more frequently in PUNLMP and low-grade NIPUC than in inverted papillomas (P = 0.009), whereas the opposite trend was noted for HRAS mutations (P low-grade NIPUC (P = 0.530). Notably, PUNLMP cases with TERT promoter mutations had a similar recurrence rate to that in low-grade NIPUC cases (P = 0.487). Our results suggest that the status of the TERT promoter mutation may serve as a biomarker of prognostic stratification in patients with PUNLMP. © 2017 John Wiley & Sons Ltd.

DNA mutations mediate microevolution between host-adapted forms of the pathogenic fungus Cryptococcus neoformans.

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Denise A Magditch

Full Text Available The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a "switch" from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.

Weakly Deleterious Mutations and Low Rates of Recombination Limit the Impact of Natural Selection on Bacterial Genomes.

Science.gov (United States)

Price, Morgan N; Arkin, Adam P

2015-12-15

Free-living bacteria are usually thought to have large effective population sizes, and so tiny selective differences can drive their evolution. However, because recombination is infrequent, "background selection" against slightly deleterious alleles should reduce the effective population size (Ne) by orders of magnitude. For example, for a well-mixed population with 10(12) individuals and a typical level of homologous recombination (r/m = 3, i.e., nucleotide changes due to recombination [r] occur at 3 times the mutation rate [m]), we predict that Ne is selection should be sufficient to drive evolution if Ne × s is >1, where s is the selection coefficient. We found that this remains approximately correct if background selection is occurring or when population structure is present. Overall, we predict that even for free-living bacteria with enormous populations, natural selection is only a significant force if s is above 10(-7) or so. Because bacteria form huge populations with trillions of individuals, the simplest theoretical prediction is that the better allele at a site would predominate even if its advantage was just 10(-9) per generation. In other words, virtually every nucleotide would be at the local optimum in most individuals. A more sophisticated theory considers that bacterial genomes have millions of sites each and selection events on these many sites could interfere with each other, so that only larger effects would be important. However, bacteria can exchange genetic material, and in principle, this exchange could eliminate the interference between the evolution of the sites. We used simulations to confirm that during multisite evolution with realistic levels of recombination, only larger effects are important. We propose that advantages of less than 10(-7) are effectively neutral. Copyright © 2015 Price and Arkin.

The role of metabolism in bacterial persistence

Directory of Open Access Journals (Sweden)

Stephanie M. Amato

2014-03-01

Full Text Available Bacterial persisters are phenotypic variants with extraordinary tolerances toward antibiotics. Persister survival has been attributed to inhibition of essential cell functions during antibiotic stress, followed by reversal of the process and resumption of growth upon removal of the antibiotic. Metabolism plays a critical role in this process, since it participates in the entry, maintenance, and exit from the persister phenotype. Here, we review the experimental evidence that demonstrates the importance of metabolism to persistence, highlight the successes and potential for targeting metabolism in the search for anti-persister therapies, and discuss the current methods and challenges to understand persister physiology.

Development of a Premature Stop Codon-detection method based on a bacterial two-hybrid system

Directory of Open Access Journals (Sweden)

Mayorga Luis S

2006-09-01

Full Text Available Abstract Background The detection of Premature Stop Codons (PSCs in human genes is very useful for the genetic diagnosis of different hereditary cancers, e.g. Familial Breast Cancer and Hereditary Non-Polyposis Colorectal Cancer (HNPCC. The products of these PSCs are truncated proteins, detectable in vitro by the Protein Truncation Test and in vivo by using the living translation machinery of yeast or bacteria. These living strategies are based on the construction of recombinant plasmids where the human sequence of interest is inserted upstream of a reporter gene. Although simple, these assays have their limitations. The yeast system requires extensive work to enhance its specificity, and the bacterial systems yield many false results due to translation re-initiation events occurring post PSCs. Our aim was to design a recombinant plasmid useful for detecting PSCs in human genes and resistant to bacterial translation re-initiation interferences. Results A functional recombinant plasmid (pREAL was designed based on a bacterial two-hybrid system. In our design, the in vivo translation of fused fragments of the Bordetella pertussis adenylate cyclase triggers the production of cAMP giving rise to a selectable bacterial phenotype. When a gene of interest is inserted between the two fragments, any PSC inhibits the enzymatic activity of the product, and translation re-initiation events post-PSC yield separated inactive fragments. We demonstrated that the system can accurately detect PSCs in human genes by inserting mutated fragments of the brca1 and msh2 gene. Western Blot assays revealed translation re-initiation events in all the tested colonies, implying that a simpler plasmid would not be resistant to this source of false negative results. The application of the system to a HNPCC family with a nonsense mutation in the msh2 gene correctly diagnosed wild type homozygous and heterozygous patients. Conclusion The developed pREAL is applicable to the

Long-term foscarnet therapy remodels thymidine analogue mutations and alters resistance to zidovudine and lamivudine in HIV-1

DEFF Research Database (Denmark)

Mathiesen, Sofie; Dam, Elisabeth; Roge, Birgit

2007-01-01

OBJECTIVE: To study the evolution of multi-drug-resistant HIV-1 in treatment-experienced patients receiving foscarnet (PFA) as part of salvage therapy and to investigate the virological consequences of emerging mutations. METHODS: Genotypic and phenotypic resistance tests were performed on plasma...... viruses from seven patients at baseline and during treatment with PFA. The phenotypic effects of mutations suspected to be associated with PFA resistance were evaluated by site-directed mutagenesis of wild-type or thymidine analogue mutations (TAM)-carrying pNL4-3. Reversion of single mutations...... was performed in a patient-derived recombinant clone. RESULTS: Baseline multi-drug-resistant isolates exhibited hypersusceptibility to PFA. In two patients who received > 12 months of PFA treatment, a novel mutation pattern including K70G, V75T, K219R and L228R emerged. These viruses had 3-6-fold resistance...

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

Science.gov (United States)

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

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

Science.gov (United States)

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

2016-04-22

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

A model capturing novel strand symmetries in bacterial DNA

International Nuclear Information System (INIS)

Sobottka, Marcelo; Hart, Andrew G.

2011-01-01

Highlights: → We propose a simple stochastic model to construct primitive DNA sequences. → The model provide an explanation for Chargaff's second parity rule in primitive DNA sequences. → The model is also used to predict a novel type of strand symmetry in primitive DNA sequences. → We extend the results for bacterial DNA sequences and compare distributional properties intrinsic to the model to statistical estimates from 1049 bacterial genomes. → We find out statistical evidences that the novel type of strand symmetry holds for bacterial DNA sequences. -- Abstract: Chargaff's second parity rule for short oligonucleotides states that the frequency of any short nucleotide sequence on a strand is approximately equal to the frequency of its reverse complement on the same strand. Recent studies have shown that, with the exception of organellar DNA, this parity rule generally holds for double-stranded DNA genomes and fails to hold for single-stranded genomes. While Chargaff's first parity rule is fully explained by the Watson-Crick pairing in the DNA double helix, a definitive explanation for the second parity rule has not yet been determined. In this work, we propose a model based on a hidden Markov process for approximating the distributional structure of primitive DNA sequences. Then, we use the model to provide another possible theoretical explanation for Chargaff's second parity rule, and to predict novel distributional aspects of bacterial DNA sequences.

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

Science.gov (United States)

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

2015-01-01

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

Dynamic of Mutational Events in Variable Number Tandem Repeats of Escherichia coli O157:H7

Directory of Open Access Journals (Sweden)

A. V. Bustamante

2013-01-01

Full Text Available VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10−05 to 1.8 × 10−03 mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10−03 mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.

Reversing Bacterial Resistance to Antibiotics by Phage-Mediated Delivery of Dominant Sensitive Genes

OpenAIRE

Edgar, Rotem; Friedman, Nir; Molshanski-Mor, Shahar; Qimron, Udi

2012-01-01

Pathogen resistance to antibiotics is a rapidly growing problem, leading to an urgent need for novel antimicrobial agents. Unfortunately, development of new antibiotics faces numerous obstacles, and a method that resensitizes pathogens to approved antibiotics therefore holds key advantages. We present a proof of principle for a system that restores antibiotic efficiency by reversing pathogen resistance. This system uses temperate phages to introduce, by lysogenization, the genes rpsL and gyrA...

Towards allele mining of bacterial wilt disease resistance gene in tomato

International Nuclear Information System (INIS)

Galvez, H.F.; Narciso, J.O.; Opina, N.L.; Canama, A.O.; Colle, M.G.; Latiza, M.A.; Caspillo, C.L.; Bituin, J.L.; Frankie, R.B.; Hautea, D.M.

2005-01-01

Tomato (Lycopersicon esculentum Mill.) is the most important vegetable commodity of the Philippines. Bacterial wilt caused by Ralstonia solanacearum is one serious constraint in tomato production particularly during off-season planting. A major locus derived from H7996 that confers resistance to bacterial wilt has been mapped in the tomato genome. To validate the biological function of the resistance locus and generate multiple allele -mimics-, targeted mutation was induced in tomato using gamma ray and ethyl methane sulfonate (EMS) mutagens. Suitable mutagen treatment was established by evaluating a wide range of mutagen doses/concentrations for a) percent seed germination, b) reduction in plant height, and c) loss of resistance. Six hundred Gy and 1.0% EMS were identified to generate large M1 families of H7996. From 10,000 initial seeds treated with either gamma ray or EMS, a total of 3,663 M1 plants were generated. M2 seeds were harvested from all surviving M1 plants. Several DNA markers have been resourced and are being developed specific to the bacterial wilt resistant gene. In the large M2 population, of H7996, both the phenotypic manifestation of bacterial wilt susceptibility and nucleotide changes in the resistance locus will be evaluated. Large M3 families for the different allele series of the bacterial wilt resistance gene will be established for future high throughput TILLING (Targeting Induced Local Lesions in Genomes) analysis in the gene region

Familial knockin mutation of LRRK2 causes lysosomal dysfunction and accumulation of endogenous insoluble α-synuclein in neurons.

Science.gov (United States)

Schapansky, Jason; Khasnavis, Saurabh; DeAndrade, Mark P; Nardozzi, Jonathan D; Falkson, Samuel R; Boyd, Justin D; Sanderson, John B; Bartels, Tim; Melrose, Heather L; LaVoie, Matthew J

2018-03-01

Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD. Copyright © 2017 Elsevier Inc. All rights reserved.

Protease inhibitor associated mutations compromise the efficacy of therapy in human immunodeficiency virus – 1 (HIV-1 infected pediatric patients: a cross-sectional study

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Petrova Anna

2007-07-01

Full Text Available Abstract Background Although the introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in considerable decrease in HIV related mortality; it has also induced the development of multiple drug-resistant HIV-1 variants. The few studies on HIV-1 mutagenesis in HIV infected children have not evaluated the impact of HIV-1 mutations on the clinical, virological and immunological presentation of HIV disease that is fundamental to optimizing the treatment regimens for these patients. Results A cross sectional study was conducted to evaluate the impact of treatment regimens and resistance mutation patterns on the clinical, virological, and immunological presentation of HIV disease in 41 children (25 male and 16 female at the Robert Wood Johnson Pediatric AIDS Program in New Brunswick, New Jersey. The study participants were symptomatic and had preceding treatment history with combined ARV regimens including protease inhibitors (PIs, nucleoside reverse transcriptase inhibitors (NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs. Fifteen (36.6% children were treated with NRTI+NNRTI+ PI, 6 (14.6% with NRTI+NNRTIs, 13 (31.7% with NRTI+PIs, and the remaining 7 (17.1% received NRTIs only. Combined ARV regimens did not significantly influence the incidence of NRTI and NNRTI associated mutations. The duration of ARV therapy and the child's age had no significant impact on the ARV related mutations. The clinico-immunological presentation of the HIV disease was not associated with ARV treatment regimens or number of resistance mutations. However, primary mutations in the protease (PR gene increased the likelihood of plasma viral load (PVL ≥ 10,000 copies/mL irrespective of the child's age, duration of ARV therapy, presence of NRTI and NNRTI mutation. Viremia ≥ 10,000 copies/mL was recorded in almost all the children with primary mutations in the PR region (n = 12/13, 92.3% as compared with only 50.0% (n

Coinheritance of hereditary spherocytosis and reversibility of cirrhosis in a young female patient with hereditary hemochromatosis.

Science.gov (United States)

Höblinger, A; Erdmann, C; Strassburg, C P; Sauerbruch, T; Lammert, F

2009-04-16

Here we report a 33-years-old woman with hereditary spherocytosis and hemochromatosis due to homozygosity for the C282Y mutation of the HFE gene. The coinheritance of both conditions led to severe iron overload and liver cirrhosis at young age. The patient was treated by repeated phlebotomy, and reversibility of cirrhosis was documented by transient elastography. This report discusses the pathophysiology of iron accumulation in patients with hemolytic anemia combined with HFE C282Y homozygosity. The case indicates that patients with hematological disorders characterized by increased erythropoetic activity should be screened for HFE mutations.

A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated.

Science.gov (United States)

Hornemann, Thorsten; Penno, Anke; Richard, Stephane; Nicholson, Garth; van Dijk, Fleur S; Rotthier, Annelies; Timmerman, Vincent; von Eckardstein, Arnold

2009-04-01

Hereditary sensory neuropathy type 1 (HSAN I) is an autosomal dominant inherited neurodegenerative disorder of the peripheral nervous system associated with mutations in the SPTLC1 subunit of the serine palmitoyltransferase (SPT). Four missense mutations (C133W, C133Y, V144D and G387A) in SPTLC1 were reported to cause HSAN I. SPT catalyses the condensation of Serine and Palmitoyl-CoA, which is the first and rate-limiting step in the de novo synthesis of ceramides. Earlier studies showed that C133W and C133Y mutants have a reduced activity, whereas the impact of the V144D and G387A mutations on the human enzyme was not tested yet. In this paper, we show that none of the HSAN I mutations interferes with SPT complex formation. We demonstrate that also V144D has a reduced SPT activity, however to a lower extent than C133W and C133Y. In contrast, the G387A mutation showed no influence on SPT activity. Furthermore, the growth phenotype of LY-B cells--a SPTLC1 deficient CHO cell line--could be reversed by expressing either the wild-type SPTLC1 or the G387A mutant, but not the C133W mutant. This indicates that the G387A mutation is most likely not directly associated with HSAN I. These findings were genetically confirmed by the identification of a nuclear HSAN family which showed segregation of the G387A variant as a non-synonymous SNP.

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

Science.gov (United States)

Paquet, Dominik; Kwart, Dylan; Chen, Antonia; Sproul, Andrew; Jacob, Samson; Teo, Shaun; Olsen, Kimberly Moore; Gregg, Andrew; Noggle, Scott; Tessier-Lavigne, Marc

2016-05-05

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in

Reverse transcriptase real-time PCR for detection and quantification of viable Campylobacter jejuni directly from poultry faecal samples

DEFF Research Database (Denmark)

Bui, Thanh Xuan; Wolff, Anders; Madsen, Mogens

2012-01-01

Campylobacter spp. is the most common cause of bacterial diarrhoea in humans worldwide. Therefore, rapid and reliable methods fordetection and quantification of this pathogen are required. In this study, we have developed a reverse transcription quantitative real-time PCR(RT-qPCR) for detection a...

Selection of protective antigens in Lawsonia intracellularis by reverse vaccinology

DEFF Research Database (Denmark)

Vadekær, Dorte Fink; Lundegaard, Claus; Riber, Ulla

protection against L. intracellularis. To this end, a reverse vaccinology approach was applied: the entire L. intracellularis genome encoding 1340 proteins was screened in silico using bioinformatics tools to identify potential protein antigens. Advanced software algorithms predicted 150 secreted and outer...... present in top 30 of both lists, and a combined rank was calculated. The two highest ranking proteins were initially selected for production. Synthetic genes have been designed, and the proteins are currently being produced in recombinant forms in bacterial expression systems. They will be analyzed...

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

OpenAIRE

Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.; Rock, Charles O.

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...

Clinical and molecular characterization of a Brazilian cohort of campomelic dysplasia patients, and identification of seven new SOX9 mutations

Directory of Open Access Journals (Sweden)

Eduardo P. Mattos

2015-03-01

Full Text Available Campomelic dysplasia (CD is an autosomal, dominantly inherited, skeletal abnormality belonging to the subgroup of bent bone dysplasias. In addition to bowed lower limbs, CD typically includes the following: disproportionate short stature, flat face, micrognathia, cleft palate, bell-shaped thorax, and club feet. Up to three quarters of 46, XY individuals may be sex-reversed. Radiological signs include scapular and pubic hypoplasia, narrow iliac wings, spaced ischia, and bowed femora and tibiae. Lethal CD is usually due to heterozygous mutations in SOX9, a major regulator of chondrocytic development. We present a detailed clinical and molecular characterization of nine Brazilian CD patients. Infants were either stillborn (n = 2 or died shortly after birth and presented similar phenotypes. Sex-reversal was observed in one of three chromosomally male patients. Sequencing of SOX9 revealed new heterozygous mutations in seven individuals. Six patients had mutations that resulted in premature transcriptional termination, while one infant had a single-nucleotide substitution at the conserved splice-site acceptor of intron 1. No clear genotype-phenotype correlations were observed. This study highlights the diversity of SOX9 mutations leading to lethal CD, and expands the group of known genetic alterations associated with this skeletal dysplasia.

The waaL gene mutation compromised the inhabitation of Enterobacter sp. Ag1 in the mosquito gut environment.

Science.gov (United States)

Pei, Dong; Jiang, Jinjin; Yu, Wanqin; Kukutla, Phanidhar; Uentillie, Alejandro; Xu, Jiannong

2015-08-27

The mosquito gut harbors a variety of bacteria that are dynamically associated with mosquitoes in various contexts. However, little is known about bacterial factors that affect bacterial inhabitation in the gut microbial community. Enterobacter sp. Ag1 is a predominant Gram negative bacterium in the mosquito midgut. In a mutant library that was generated using transposon Tn5-mediated mutagenesis, a mutant was identified, in which the gene waaL was disrupted by the Tn5 insertion. The waaL encodes O antigen ligase, which is required for the attachment of O antigen to the outer core oligosaccharide of the lipopolysaccharide (LPS). The waaL(-) mutation caused the O antigen repeat missing in the LPS. The normal LPS structure was restored when the mutant was complemented with a plasmid containing waaL gene. The waaL(-) mutation did not affect bacterial proliferation in LB culture, the mutant cells grew at a rate the same as the wildtype (wt) cells. However, when waaL(-) strain were co-cultured with the wt strain or complemented strain, the mutant cells proliferated with a slower rate, indicating that the mutants were less competitive than wt cells in a community setting. Similarly, in a co-feeding assay, when fluorescently tagged wt strain and waaL(-) strain were orally co-introduced into the gut of Anopheles stephensi mosquitoes, the mutant cells were less prevalent in both sugar-fed and blood-fed guts. The data suggest that the mutation compromised the bacterial inhabitation in the gut community. Besides, the mutant was more sensitive to oxidative stress, demonstrated by lower survival rate upon exposure to 20 mM H₂O₂. Lack of the O antigen structure in LPS of Enterobacter compromised the effective growth in co-culture and co-feeding assays. In addition, O-antigen was involved in protection against oxidative stress. The findings suggest that intact LPS is crucial for the bacteria to steadily stay in the gut microbial community.

Factors affecting mutational specificity in mammalian cells: Informal [technical] progress report, February 1, 1987-January 31, 1988

International Nuclear Information System (INIS)

Strauss, B.S.

1986-01-01

We analyzed the different c-H-ras mutations produced in cells after treatment with chemical carcinogens. The overall goal of this work is an understanding of the changes produced by environmental mutagens and carcinogens to learn how closely the results obtained with bacterial and cellular assay systems apply to the in vivo situation

Absence of Mutagenicity in Three Nigerian Medicinal Plants ...

African Journals Online (AJOL)

Erah

of the medicinal plant trade in the region [2]. One of the basic criteria set by World Health. Organization (WHO) for the use of herbs as medicines is that they should be shown to be non-toxic [3,4]. Bacterial reverse mutation assay (Ames test) was used in this work to evaluate the mutagenic potential of the methanolic extracts ...

ITGB6 loss-of-function mutations cause autosomal recessive amelogenesis imperfecta.

Science.gov (United States)

Wang, Shih-Kai; Choi, Murim; Richardson, Amelia S; Reid, Bryan M; Lin, Brent P; Wang, Susan J; Kim, Jung-Wook; Simmer, James P; Hu, Jan C-C

2014-04-15

Integrins are cell-surface adhesion receptors that bind to extracellular matrices (ECM) and mediate cell-ECM interactions. Some integrins are known to play critical roles in dental enamel formation. We recruited two Hispanic families with generalized hypoplastic amelogenesis imperfecta (AI). Analysis of whole-exome sequences identified three integrin beta 6 (ITGB6) mutations responsible for their enamel malformations. The female proband of Family 1 was a compound heterozygote with an ITGB6 transition mutation in Exon 4 (g.4545G > A c.427G > A p.Ala143Thr) and an ITGB6 transversion mutation in Exon 6 (g.27415T > A c.825T > A p.His275Gln). The male proband of Family 2 was homozygous for an ITGB6 transition mutation in Exon 11 (g.73664C > T c.1846C > T p.Arg616*) and hemizygous for a transition mutation in Exon 6 of Nance-Horan Syndrome (NHS Xp22.13; g.355444T > C c.1697T > C p.Met566Thr). These are the first disease-causing ITGB6 mutations to be reported. Immunohistochemistry of mouse mandibular incisors localized ITGB6 to the distal membrane of differentiating ameloblasts and pre-ameloblasts, and then ITGB6 appeared to be internalized by secretory stage ameloblasts. ITGB6 expression was strongest in the maturation stage and its localization was associated with ameloblast modulation. Our findings demonstrate that early and late amelogenesis depend upon cell-matrix interactions. Our approach (from knockout mouse phenotype to human disease) demonstrates the power of mouse reverse genetics in mutational analysis of human genetic disorders and attests to the need for a careful dental phenotyping in large-scale knockout mouse projects.

Reversible flowchart languages and the structured reversible program theorem

DEFF Research Database (Denmark)

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

2008-01-01

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

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

DEFF Research Database (Denmark)

Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.

2015-01-01

organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10ºC. Multivariate statistical analysis of the bacterial diversity data (DNA......The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78º......N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable...

Lateral Gene Transfer Dynamics in the Ancient Bacterial Genus Streptomyces.

Science.gov (United States)

McDonald, Bradon R; Currie, Cameron R

2017-06-06

Lateral gene transfer (LGT) profoundly shapes the evolution of bacterial lineages. LGT across disparate phylogenetic groups and genome content diversity between related organisms suggest a model of bacterial evolution that views LGT as rampant and promiscuous. It has even driven the argument that species concepts and tree-based phylogenetics cannot be applied to bacteria. Here, we show that acquisition and retention of genes through LGT are surprisingly rare in the ubiquitous and biomedically important bacterial genus Streptomyces Using a molecular clock, we estimate that the Streptomyces bacteria are ~380 million years old, indicating that this bacterial genus is as ancient as land vertebrates. Calibrating LGT rate to this geologic time span, we find that on average only 10 genes per million years were acquired and subsequently maintained. Over that same time span, Streptomyces accumulated thousands of point mutations. By explicitly incorporating evolutionary timescale into our analyses, we provide a dramatically different view on the dynamics of LGT and its impact on bacterial evolution. IMPORTANCE Tree-based phylogenetics and the use of species as units of diversity lie at the foundation of modern biology. In bacteria, these pillars of evolutionary theory have been called into question due to the observation of thousands of lateral gene transfer (LGT) events within and between lineages. Here, we show that acquisition and retention of genes through LGT are exceedingly rare in the bacterial genus Streptomyces , with merely one gene acquired in Streptomyces lineages every 100,000 years. These findings stand in contrast to the current assumption of rampant genetic exchange, which has become the dominant hypothesis used to explain bacterial diversity. Our results support a more nuanced understanding of genetic exchange, with LGT impacting evolution over short timescales but playing a significant role over long timescales. Deeper understanding of LGT provides new

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

Directory of Open Access Journals (Sweden)

Miriam E. Szurman-Zubrzycka

2018-02-01

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

A study of gamma-ray mutagenesis in Saccharomyces Cerevisiae. Analysis of reversion production kinetics in a wild-type haploid strain

International Nuclear Information System (INIS)

Levkovich, N.V.; Chepurnoj, A.I.

1992-01-01

After γ-irradiation of a synchronized yeast culture in the G 1 -phase of the cell cycle, radiation-induced leu2 reversions are found to be formed in the first three postradiation phases of DNA replication. Without replication and in periods between two phases of DNA replication, formation of induced reversions was not observed. The analysis of postradiation formation kinetics of induced mutants is proposed to be used together with other know approaches for studying mutation processes. Hypothetical schemes are proposed to explain the ascending descending trend in formation of γ-induced reversions during postradiation growth of irradiated cells. 7 refs.; 5 figs.; 1 tab

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

Science.gov (United States)

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

2014-07-01

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

Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

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Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

2016-01-29

Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

Persistence of transmitted HIV-1 drug resistance mutations associated with fitness costs and viral genetic backgrounds.

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Wan-Lin Yang

2015-03-01

Full Text Available Transmission of drug-resistant pathogens presents an almost-universal challenge for fighting infectious diseases. Transmitted drug resistance mutations (TDRM can persist in the absence of drugs for considerable time. It is generally believed that differential TDRM-persistence is caused, at least partially, by variations in TDRM-fitness-costs. However, in vivo epidemiological evidence for the impact of fitness costs on TDRM-persistence is rare. Here, we studied the persistence of TDRM in HIV-1 using longitudinally-sampled nucleotide sequences from the Swiss-HIV-Cohort-Study (SHCS. All treatment-naïve individuals with TDRM at baseline were included. Persistence of TDRM was quantified via reversion rates (RR determined with interval-censored survival models. Fitness costs of TDRM were estimated in the genetic background in which they occurred using a previously published and validated machine-learning algorithm (based on in vitro replicative capacities and were included in the survival models as explanatory variables. In 857 sequential samples from 168 treatment-naïve patients, 17 TDRM were analyzed. RR varied substantially and ranged from 174.0/100-person-years;CI=[51.4, 588.8] (for 184V to 2.7/100-person-years;[0.7, 10.9] (for 215D. RR increased significantly with fitness cost (increase by 1.6[1.3,2.0] per standard deviation of fitness costs. When subdividing fitness costs into the average fitness cost of a given mutation and the deviation from the average fitness cost of a mutation in a given genetic background, we found that both components were significantly associated with reversion-rates. Our results show that the substantial variations of TDRM persistence in the absence of drugs are associated with fitness-cost differences both among mutations and among different genetic backgrounds for the same mutation.

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

Energy Technology Data Exchange (ETDEWEB)

Matney, T.S.

1978-01-01

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

KIAA1549-BRAF fusions and IDH mutations can coexist in diffuse gliomas of adults.

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Badiali, Manuela; Gleize, Vincent; Paris, Sophie; Moi, Loredana; Elhouadani, Selma; Arcella, Antonietta; Morace, Roberta; Antonelli, Manila; Buttarelli, Francesca Romana; Figarella-Branger, Dominique; Kim, Young-Ho; Ohgaki, Hiroko; Mokhtari, Karima; Sanson, Marc; Giangaspero, Felice

2012-11-01

KIAA1549-BRAF fusion gene and isocitrate dehydrogenase (IDH) mutations are considered two mutually exclusive genetic events in pilocytic astrocytomas and diffuse gliomas, respectively. We investigated the presence of the KIAA1549-BRAF fusion gene in conjunction with IDH mutations and 1p/19q loss in 185 adult diffuse gliomas. Moreover BRAF(v600E) mutation was also screened. The KIAA1549-BRAF fusion gene was evaluated by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We found IDH mutations in 125 out 175 cases (71.4%). There were KIAA1549-BRAF fusion gene in 17 out of 180 (9.4%) cases and BRAF(v600E) in 2 out of 133 (1.5%) cases. In 11 of these 17 cases, both IDH mutations and the KIAA1549-BRAF fusion were present, as independent molecular events. Moreover, 6 of 17 cases showed co-presence of 1p/19q loss, IDH mutations and KIAA1549-BRAF fusion. Among the 17 cases with KIAA1549-BRAF fusion gene 15 (88.2%) were oligodendroglial neoplasms. Similarly, the two cases with BRAF(v600E) mutation were both oligodendroglioma and one had IDH mutations and 1p/19q co-deletion. Our results suggest that in a small fraction of diffuse gliomas, KIAA1549-BRAF fusion gene and BRAF(v600E) mutation may be responsible for deregulation of the Ras-RAF-ERK signaling pathway. Such alterations are more frequent in oligodendroglial neoplasm and may be co-present with IDH mutations and 1p/19q loss. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

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Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Mutational spectrum analysis of umuC-independent and umuC-dependent γ-radiation mutagenesis in Escherichia coli

International Nuclear Information System (INIS)

Sargentini, N.J.; Smith, K.C.

1989-01-01

γ-radiation mutagenesis Escherichia coli K-12. Mutagenesis (argE3(OC) A rg + ) was blocked in a δ(recA-srlR)306 strain at the same doses that induced mutations in umuC122::Tn5 and wild-type strains, indicating that both umuC-independent and umuC-dependent mechanisms function within recA-dependent misrepair. Analyses of various suppressor and back mutations that result in argE3 and hisG4 ochre reversion and an analysis of trpE9777 reversion were performed on umuC and wild-type cells irradiated in the presence and absence of oxygen. While the umuC strain showed the γ-radiation induction of base substitution and frameshifts when irradiated in the absence of oxygen, the umuC mutation blocked all oxygen-dependent base-substitution mutagenesis, but non all oxygen-dependent frameshift mutagenesis. For anoxically irradiated cells, the yields of GC T and AT GC transitions were essentially umuC independent, while the yields of (AT or GC) TA transversions were heavily umuC dependent. These data suggest new concepts about the nature of the DNA lesions and the mutagenic mechanisms that lead to γ-radiation mutagenesis. (author). 48 refs.; 1 tab.; 6 refs

Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene

International Nuclear Information System (INIS)

Blanco, M.; Rebollo, J.E.

1981-01-01

Bacterial survival after UV irradiation was increased in E. coli K12 lex B30 and tif zab-53 mutants harboring plasmid pKM101. Mutagenesis in response to UV was observed in these bacteria which, in absence of pKM101, are not UV-mutable. The mutator effect observed in unirradiated wild-type cells containing pKM101 was higher after incubation at 30 0 C with adenine than at 37 0 C. This effect was still enhanced by tif mutation, even in the tif zab-53 strain, but it was abolished by lexB30 mutation. In the tif zab-53 (pKM101) strain, repair and mutagenesis of UV-irradiated phage lambda was observed, but not in the lexB30 mutant carrying pKM101. The pKM101 mutant, pGW1, was unable to protect tif zab-53 bacteria against killing by UV, whereas the protection of lexB30 was intermediate; moreover, it did not promote the mutator effect at 30 0 C or enhance phage repair and mutagenesis in tif zab-53 cells. All UV-induced bacterial mutations in lexB30 (pKM101) strain were suppressors; in contrast, true revertants were found after UV irradiation of the tif zab-53 (pKM101) cells. (orig./AJ)

Limitations to estimating bacterial cross-speciestransmission using genetic and genomic markers: inferencesfrom simulation modeling

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Julio Andre, Benavides; Cross, Paul C.; Luikart, Gordon; Scott, Creel

2014-01-01

Cross-species transmission (CST) of bacterial pathogens has major implications for human health, livestock, and wildlife management because it determines whether control actions in one species may have subsequent effects on other potential host species. The study of bacterial transmission has benefitted from methods measuring two types of genetic variation: variable number of tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). However, it is unclear whether these data can distinguish between different epidemiological scenarios. We used a simulation model with two host species and known transmission rates (within and between species) to evaluate the utility of these markers for inferring CST. We found that CST estimates are biased for a wide range of parameters when based on VNTRs and a most parsimonious reconstructed phylogeny. However, estimations of CST rates lower than 5% can be achieved with relatively low bias using as low as 250 SNPs. CST estimates are sensitive to several parameters, including the number of mutations accumulated since introduction, stochasticity, the genetic difference of strains introduced, and the sampling effort. Our results suggest that, even with whole-genome sequences, unbiased estimates of CST will be difficult when sampling is limited, mutation rates are low, or for pathogens that were recently introduced.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

Science.gov (United States)

Tabassum, Najia; Saboor, Mohammed; Ghani, Rubina; Moinuddin, Moinuddin

2014-01-01

Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

Reverse Algols

Science.gov (United States)

Leung, K. C.

1989-01-01

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

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination

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Laura Novellasdemunt

2017-10-01

Full Text Available The tumor suppressor gene adenomatous polyposis coli (APC is mutated in most colorectal cancers (CRCs, resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs.

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

Energy Technology Data Exchange (ETDEWEB)

Brown, R; Stretch, A; Thacker, J

1986-04-01

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

Towards rationally redesigning bacterial signaling systems using information encoded in abundant sequence data

Science.gov (United States)

Cheng, Ryan; Morcos, Faruck; Levine, Herbert; Onuchic, Jose

2014-03-01

An important challenge in biology is to distinguish the subset of residues that allow bacterial two-component signaling (TCS) proteins to preferentially interact with their correct TCS partner such that they can bind and transfer signal. Detailed knowledge of this information would allow one to search sequence-space for mutations that can systematically tune the signal transmission between TCS partners as well as re-encode a TCS protein to preferentially transfer signals to a non-partner. Motivated by the notion that this detailed information is found in sequence data, we explore the mutual sequence co-evolution between signaling partners to infer how mutations can positively or negatively alter their interaction. Using Direct Coupling Analysis (DCA) for determining evolutionarily conserved interprotein interactions, we apply a DCA-based metric to quantify mutational changes in the interaction between TCS proteins and demonstrate that it accurately correlates with experimental mutagenesis studies probing the mutational change in the in vitro phosphotransfer. Our methodology serves as a potential framework for the rational design of TCS systems as well as a framework for the system-level study of protein-protein interactions in sequence-rich systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1308264).

The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope.

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Shuai Chang

Full Text Available This study was designed to identify common HIV-1 mutation complexes affecting the slope of inhibition curve, and to propose a new parameter incorporating both the IC50 and the slope to evaluate phenotypic resistance.Utilizing site-directed mutagenesis, we constructed 22 HIV-1 common mutation complexes. IC50 and slope of 10 representative approved drugs and a novel agent against these mutations were measured to determine the resistance phenotypes. The values of new parameter incorporating both the IC50 and the slope of the inhibition curve were calculated, and the correlations between parameters were assessed.Depending on the class of drug, there were intrinsic differences in how the resistance mutations affected the drug parameters. All of the mutations resulted in large increases in the IC50s of nucleoside reverse transcriptase inhibitors. The effects of the mutations on the slope were the most apparent when examining their effects on the inhibition of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. For example, some mutations, such as V82A, had no effect on IC50, but reduced the slope. We proposed a new concept, termed IIPatoxic, on the basis of IC50, slope and the maximum limiting concentrations of the drug. The IIPatoxic values of 10 approved drugs and 1 novel agent were calculated, and were closely related to the IIPmax values (r > 0.95, p < 0.001.This study confirms that resistance mutations cannot be accurately assessed by IC50 alone, because it tends to underestimate the degree of resistance. The slope parameter is of very importance in the measurement of drug resistance and the effect can be applied to more complex patterns of resistance. This is the most apparent when testing the effects of the mutations on protease inhibitors activity. We also propose a new index, IIPatoxic, which incorporates both the IC50 and the slope. This new index could complement current IIP indices, thereby enabling predict the

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions

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Andy Hesketh

2017-07-01

Full Text Available We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP, cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription.

Science.gov (United States)

Nair, Smita; Zlotnick, Adam

2018-05-15

Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. IMPORTANCE Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription

Recombination-Driven Genome Evolution and Stability of Bacterial Species.

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Dixit, Purushottam D; Pang, Tin Yau; Maslov, Sergei

2017-09-01

While bacteria divide clonally, horizontal gene transfer followed by homologous recombination is now recognized as an important contributor to their evolution. However, the details of how the competition between clonality and recombination shapes genome diversity remains poorly understood. Using a computational model, we find two principal regimes in bacterial evolution and identify two composite parameters that dictate the evolutionary fate of bacterial species. In the divergent regime, characterized by either a low recombination frequency or strict barriers to recombination, cohesion due to recombination is not sufficient to overcome the mutational drift. As a consequence, the divergence between pairs of genomes in the population steadily increases in the course of their evolution. The species lacks genetic coherence with sexually isolated clonal subpopulations continuously formed and dissolved. In contrast, in the metastable regime, characterized by a high recombination frequency combined with low barriers to recombination, genomes continuously recombine with the rest of the population. The population remains genetically cohesive and temporally stable. Notably, the transition between these two regimes can be affected by relatively small changes in evolutionary parameters. Using the Multi Locus Sequence Typing (MLST) data, we classify a number of bacterial species to be either the divergent or the metastable type. Generalizations of our framework to include selection, ecologically structured populations, and horizontal gene transfer of nonhomologous regions are discussed as well. Copyright © 2017 by the Genetics Society of America.

Surface contact stimulates the just-in-time deployment of bacterial adhesins.

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Li, Guanglai; Brown, Pamela J B; Tang, Jay X; Xu, Jing; Quardokus, Ellen M; Fuqua, Clay; Brun, Yves V

2012-01-01

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive. © 2011 Blackwell Publishing Ltd.

Ultraviolet light protection, enhancement of ultraviolet light mutagenesis, and mutator effect of plasmid R46 in Salmonella typhimurium

International Nuclear Information System (INIS)

Mortelmans, K.E.; Stocker, B.A.D.

1976-01-01

Plasmid R46 partially protected Salmonella typhimurium, wild type or uvrB or polA, against the lethal effect of ultraviolet (uv) irradiation, but did not protect recA mutants. The plasmid also increased frequency of uv-induced reversion to His + in all tested his point mutants (wild type for uv sensitivity), including amber, ochre, UGA, missense, and frame-shift mutants. Plasmid R46 also increased uv-induced reversion to His + in uvrB and polA strains, but no uv mutagenic effect was detected in R - or R46-carrying recA derivatives of a his(amber) mutant. The spontaneous reversion frequency of his nonsense mutants of all classes, and of some his missense mutants, was increased about 10-fold when the strains carried R46, but the plasmid had no effect on the spontaneous reversion frequency of some other his missense mutations or of reversion rate of his frame-shift mutants (except for two uvrB derivatives of one single-base insertion mutant). The plasmid increased the ability of wild type, polA, and uvrB hosts to support plaque production by uv-irradiated phage, and made strain LT2 his G46 less sensitive to methyl methane sulfonate and to x rays and more responsive to the mutagenic effect of visible-light irradiation. R46 increased spontaneous reversion frequency of a his(amber) rec + strain, but had no such effect in its recA sublines. Since the plasmid in the absence of host recA function fails to produce its mutator effect, or to confer uv protection or to enhance uv mutagenesis, these three effects may be produced via some mechanism involved in recA-dependent deoxyribonucleic acid repair, perhaps by an increase in activity of the ''error-prone'' component of the inducible repair pathway

Managing Reverse Logistics or Reversing Logistics Management?

OpenAIRE

Brito, Marisa

2004-01-01

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

Genome-Wide Mutation Rate Response to pH Change in the Coral Reef Pathogen Vibrio shilonii AK1.

Science.gov (United States)

Strauss, Chloe; Long, Hongan; Patterson, Caitlyn E; Te, Ronald; Lynch, Michael

2017-08-22

Recent application of mutation accumulation techniques combined with whole-genome sequencing (MA/WGS) has greatly promoted studies of spontaneous mutation. However, such explorations have rarely been conducted on marine organisms, and it is unclear how marine habitats have influenced genome stability. This report resolves the mutation rate and spectrum of the coral reef pathogen Vibrio shilonii , which causes coral bleaching and endangers the biodiversity maintained by coral reefs. We found that its mutation rate and spectrum are highly similar to those of other studied bacteria from various habitats, despite the saline environment. The mutational properties of this marine bacterium are thus controlled by other general evolutionary forces such as natural selection and genetic drift. We also found that as pH drops, the mutation rate decreases and the mutation spectrum is biased in the direction of generating G/C nucleotides. This implies that evolutionary features of this organism and perhaps other marine microbes might be altered by the increasingly acidic ocean water caused by excess CO 2 emission. Nonetheless, further exploration is needed as the pH range tested in this study was rather narrow and many other possible mutation determinants, such as carbonate increase, are associated with ocean acidification. IMPORTANCE This study explored the pH dependence of a bacterial genome-wide mutation rate. We discovered that the genome-wide rates of appearance of most mutation types decrease linearly and that the mutation spectrum is biased in generating more G/C nucleotides with pH drop in the coral reef pathogen V. shilonii . Copyright © 2017 Strauss et al.

Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

Science.gov (United States)

Joensuu, T; Hämäläinen, R; Yuan, B; Johnson, C; Tegelberg, S; Gasparini, P; Zelante, L; Pirvola, U; Pakarinen, L; Lehesjoki, A E; de la Chapelle, A; Sankila, E M

2001-10-01

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

Recurrent Reverse Evolution Maintains Polymorphism after Strong Bottlenecks in Commensal Gut Bacteria.

Science.gov (United States)

Sousa, Ana; Ramiro, Ricardo S; Barroso-Batista, João; Güleresi, Daniela; Lourenço, Marta; Gordo, Isabel

2017-11-01

The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (∼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Active PI3K pathway causes an invasive phenotype which can be reversed or promoted by blocking the pathway at divergent nodes.

Directory of Open Access Journals (Sweden)

Jeffrey J Wallin

Full Text Available The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP(3 production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.

Cell culture-adaptive mutations of NS5A affect replication of hepatitis C virus differentially depending on the viral genotypes.

Science.gov (United States)

Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek

2017-01-01

Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication

International Nuclear Information System (INIS)

Lee, Changhee; Hodgins, Douglas; Calvert, Jay G.; Welch, Siao-Kun W.; Jolie, Rika; Yoo, Dongwan

2006-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a

A376S in the connection subdomain of HIV-1 reverse transcriptase confers increased risk of virological failure to nevirapine therapy

NARCIS (Netherlands)

Paredes, Roger; Puertas, Maria Carmen; Bannister, Wendy; Kisic, Mónica; Cozzi-Lepri, Alessandro; Pou, Christian; Bellido, Rocío; Betancor, Gilberto; Bogner, Johannes; Gargalianos, Panagiotis; Bánhegyi, Dénes; Clotet, Bonaventura; Lundgren, Jens; Menéndez-Arias, Luis; Martinez-Picado, Javier; Losso, M.; Elias, C.; Vetter, N.; Zangerle, R.; Karpov, I.; Vassilenko, A.; Mitsura, V. M.; Suetnov, O.; Clumeck, N.; de Wit, S.; Poll, B.; Colebunders, R.; Vandekerckhove, L.; Hadziosmanovic, V.; Kostov, K.; Begovac, J.; Machala, L.; Rozsypal, H.; Sedlacek, D.; Nielsen, J.; Kronborg, G.; Benfield, T.; Larsen, M.; Gerstoft, J.; Katzenstein, T.; Hansen, A.-B. E.; Skinhøj, P.; Pedersen, C.; Oestergaard, L.; Zilmer, K.; Ristola, M.; Katlama, C.; Viard, J.-P.; Girard, P.-M.; Livrozet, J. M.; Vanhems, P.; Pradier, C.; Dabis, F.; Neau, D.; Rockstroh, J.; Schmidt, R.; van Lunzen, J.; Degen, O.; Stellbrink, H. J.; Staszewski, S.; Fätkenheuer, G.; Kosmidis, J.; Gargalianos, P.; Xylomenos, G.; Perdios, J.; Panos, G.; Filandras, A.; Karabatsaki, E.; Sambatakou, H.; Banhegyi, D.; Mulcahy, F.; Yust, I.; Turner, D.; Burke, M.; Pollack, S.; Hassoun, G.; Maayan, S.; Vella, S.; Esposito, R.; Mazeu, I.; Mussini, C.; Arici, C.; Pristera, R.; Mazzotta, F.; Gabbuti, A.; Vullo, V.; Lichtner, M.; Chirianni, A.; Montesarchio, E.; Gargiulo, M.; Antonucci, G.; Iacomi, F.; Narciso, P.; Vlassi, C.; Zaccarelli, M.; Lazzarin, A.; Finazzi, R.; Galli, M.; Ridolfo, A.; d'Arminio, A.; Rozentale, B.; Aldins, P.; Chaplinskas, S.; Hemmer, R.; Staub, T.; Reiss, P.; Ormaasen, V.; Maeland, A.; Brunn, J.; Knysz, B.; Gasiorowski, J.; Horban, A.; Bakowska, E.; Prokopowicz, D.; Flisiak, R.; Boron-Kaczmarska, A.; Pynka, M.; Beniowski, M.; Mularska, E.; Trocha, H.; Jablonowska, E.; Malolepsza, E.; Wojcik, K.; Antunes, F.; Valadas, E.; Mansinho, K.; Maltez, F.; Duiculescu, D.; Rakhmanova, A.; Vinogradova, E.; Buzunova, S.; Jevtovic, D.; Mokrás, M.; Staneková, D.; Tomazic, J.; González-Lahoz, J.; Soriano, V.; Martin-Carbonero, L.; Labarga, P.; Moreno, S.; Clotet, B.; Jou, A.; Paredes, R.; Tural, C.; Puig, J.; Bravo, I.; Gatell, J. M.; Miró, J. M.; Domingo, P.; Gutierrez, M.; Mateo, G.; Sambeat, M. A.; Karlsson, A.; Persson, P. O.; Ledergerber, B.; Weber, R.; Francioli, P.; Cavassini, M.; Hirschel, B.; Boffi, E.; Furrer, H.; Battegay, M.; Elzi, L.; Kravchenko, E.; Chentsova, N.; Kutsyna, G.; Servitskiy, S.; Krasnov, M.; Barton, S.; Johnson, A. M.; Mercey, D.; Phillips, A.; Johnson, M. A.; Murphy, M.; Weber, J.; Scullard, G.; Fisher, M.; Leen, C.; Gatell, J.; Gazzard, B.; Lundgren, J.; d'Arminio Monforte, A.; Kirk, O.; Mocroft, A.; Cozzi-Lepri, A.; Grint, D.; Ellefson, M.; Podlekareva, D.; Kjaer, J.; Peters, L.; Reekie, J.; Kowalska, J.; Tverland, J.; Fischer, A. H.

2011-01-01

The clinical relevance of mutations in the connection subdomain and the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) is uncertain. The risk of virological failure to nonnucleoside RT inhibitor (NNRTI)-based antiretroviral therapy (ART) was evaluated in NNRTI-naive patients who

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins

DEFF Research Database (Denmark)

Al-Saaidi, Rasha Abdelkadhem; Rasmussen, Torsten Bloch; Palmfeldt, Johan

2013-01-01

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems...... however are still not clearly established. In this study, we used a representative LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR...

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

Science.gov (United States)

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The

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

Science.gov (United States)

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

2010-12-01

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

Results after laparoscopic partial splenectomy for children with hereditary spherocytosis: Are outcomes influenced by genetic mutation?

Science.gov (United States)

Pugi, Jakob; Carcao, Manuel; Drury, Luke J; Langer, Jacob C

2018-05-01

Laparoscopic partial splenectomy (LPS) theoretically maintains long-term splenic immune function for children with hereditary spherocytosis (HS). Our goal was to review our results after LPS and to determine if specific genetic mutations influence outcome. All children with HS undergoing LPS between 2005 and 2016 were reviewed. Thirty-one children underwent LPS (16 male) at a median age of 9 (range 2-18) years. All experienced an increase in hemoglobin and decrease in reticulocyte count early after LPS and at last follow-up. Twenty-two were sent for genetic analysis. Mutations in α-spectrin, β-spectrin, and Ankyrin were identified in 6, 5, and 11 patients, respectively. Gene mutation was not correlated with complications, perioperative transfusion, length of hospital stay, or median hemoglobin, platelet, or reticulocyte counts. Three children required completion splenectomy at 10.9, 6.9, and 3.2years post-LPS, each with a different gene mutation. LPS is effective in reversing anemia and reducing reticulocytosis. So far less than 10% have required completion splenectomy, and those children did benefit from delaying the risks of asplenia. In this preliminary analysis, genetic mutation did not influence outcome after LPS. A larger multicenter study is necessary to further investigate potential correlations with specific genetic mutations. Prognosis Study. IV. Copyright © 2018. Published by Elsevier Inc.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice.

Science.gov (United States)

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, Hongbin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-04-17

The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

Directory of Open Access Journals (Sweden)

Roe Bruce A

2007-04-01

Full Text Available Abstract Background The long bone abnormality (lbab mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. Results A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. Conclusion A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

Predicting the dynamics of bacterial growth inhibition by ribosome-targeting antibiotics

Science.gov (United States)

Greulich, Philip; Doležal, Jakub; Scott, Matthew; Evans, Martin R.; Allen, Rosalind J.

2017-12-01

Understanding how antibiotics inhibit bacteria can help to reduce antibiotic use and hence avoid antimicrobial resistance—yet few theoretical models exist for bacterial growth inhibition by a clinically relevant antibiotic treatment regimen. In particular, in the clinic, antibiotic treatment is time-dependent. Here, we use a theoretical model, previously applied to steady-state bacterial growth, to predict the dynamical response of a bacterial cell to a time-dependent dose of ribosome-targeting antibiotic. Our results depend strongly on whether the antibiotic shows reversible transport and/or low-affinity ribosome binding (‘low-affinity antibiotic’) or, in contrast, irreversible transport and/or high affinity ribosome binding (‘high-affinity antibiotic’). For low-affinity antibiotics, our model predicts that growth inhibition depends on the duration of the antibiotic pulse, and can show a transient period of very fast growth following removal of the antibiotic. For high-affinity antibiotics, growth inhibition depends on peak dosage rather than dose duration, and the model predicts a pronounced post-antibiotic effect, due to hysteresis, in which growth can be suppressed for long times after the antibiotic dose has ended. These predictions are experimentally testable and may be of clinical significance.

Kinetics of bacterial potentiometric titrations: the effect of equilibration time on buffering capacity of Pantoea agglomerans suspensions.

Science.gov (United States)

Kapetas, Leon; Ngwenya, Bryne T; Macdonald, Alan M; Elphick, Stephen C

2011-07-15

Several recent studies have made use of continuous acid-base titration data to describe the surface chemistry of bacterial cells as a basis for accurately modelling metal adsorption to bacteria and other biomaterials of potential industrial importance. These studies do not share a common protocol; rather they titrate in different pH ranges and they use different stability criteria to define equilibration time during titration. In the present study we investigate the kinetics of bacterial titrations and test the effect they have on the derivation of functional group concentrations and acidity constants. We titrated suspensions of Pantoea agglomerans by varying the equilibration time between successive titrant additions until stability of 0.1 or 0.001 mV s(-1) was attained. We show that under longer equilibration times, titration results are less reproducible and suspensions exhibit marginally higher buffering. Fluorescence images suggest that cell lysis is not responsible for these effects. Rather, high DOC values and titration reversibility hysterisis after long equilibration times suggest that variability in buffering is due to the presence of bacterial exudates, as demonstrated by titrating supernatants separated from suspensions of different equilibration times. It is recommended that an optimal equilibration time is always determined with variable stability control and preliminary reversibility titration experiments. Copyright © 2011 Elsevier Inc. All rights reserved.

X-linked primary immunodeficiency associated with hemizygous mutations in the moesin (MSN) gene.

Science.gov (United States)

Lagresle-Peyrou, Chantal; Luce, Sonia; Ouchani, Farid; Soheili, Tayebeh Shabi; Sadek, Hanem; Chouteau, Myriam; Durand, Amandine; Pic, Isabelle; Majewski, Jacek; Brouzes, Chantal; Lambert, Nathalie; Bohineust, Armelle; Verhoeyen, Els; Cosset, François-Loïc; Magerus-Chatinet, Aude; Rieux-Laucat, Frédéric; Gandemer, Virginie; Monnier, Delphine; Heijmans, Catherine; van Gijn, Marielle; Dalm, Virgil A; Mahlaoui, Nizar; Stephan, Jean-Louis; Picard, Capucine; Durandy, Anne; Kracker, Sven; Hivroz, Claire; Jabado, Nada; de Saint Basile, Geneviève; Fischer, Alain; Cavazzana, Marina; André-Schmutz, Isabelle

2016-12-01

We investigated 7 male patients (from 5 different families) presenting with profound lymphopenia, hypogammaglobulinemia, fluctuating monocytopenia and neutropenia, a poor immune response to vaccine antigens, and increased susceptibility to bacterial and varicella zoster virus infections. We sought to characterize the genetic defect involved in a new form of X-linked immunodeficiency. We performed genetic analyses and an exhaustive phenotypic and functional characterization of the lymphocyte compartment. We observed hemizygous mutations in the moesin (MSN) gene (located on the X chromosome and coding for MSN) in all 7 patients. Six of the latter had the same missense mutation, which led to an amino acid substitution (R171W) in the MSN four-point-one, ezrin, radixin, moesin domain. The seventh patient had a nonsense mutation leading to a premature stop codon mutation (R533X). The naive T-cell counts were particularly low for age, and most CD8 + T cells expressed the senescence marker CD57. This phenotype was associated with impaired T-cell proliferation, which was rescued by expression of wild-type MSN. MSN-deficient T cells also displayed poor chemokine receptor expression, increased adhesion molecule expression, and altered migration and adhesion capacities. Our observations establish a causal link between an ezrin-radixin-moesin protein mutation and a primary immunodeficiency that could be referred to as X-linked moesin-associated immunodeficiency. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells

International Nuclear Information System (INIS)

Bredberg, A.; Kraemer, K.H.; Seidman, M.M.

1986-01-01

A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans

Robust biological nitrogen fixation in a model grass-bacterial association.

Science.gov (United States)

Pankievicz, Vânia C S; do Amaral, Fernanda P; Santos, Karina F D N; Agtuca, Beverly; Xu, Youwen; Schueller, Michael J; Arisi, Ana Carolina M; Steffens, Maria B R; de Souza, Emanuel M; Pedrosa, Fábio O; Stacey, Gary; Ferrieri, Richard A

2015-03-01

Nitrogen-fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C4 grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen-13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen-limiting conditions when inoculated with an ammonium-excreting strain of Azospirillum brasilense. (11)C-labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen-starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen-sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Life history trade-offs and relaxed selection can decrease bacterial virulence in environmental reservoirs.

Directory of Open Access Journals (Sweden)

Lauri Mikonranta

Full Text Available Pathogen virulence is usually thought to evolve in reciprocal selection with the host. While this might be true for obligate pathogens, the life histories of opportunistic pathogens typically alternate between within-host and outside-host environments during the infection-transmission cycle. As a result, opportunistic pathogens are likely to experience conflicting selection pressures across different environments, and this could affect their virulence through life-history trait correlations. We studied these correlations experimentally by exposing an opportunistic bacterial pathogen Serratia marcescens to its natural protist predator Tetrahymena thermophila for 13 weeks, after which we measured changes in bacterial traits related to both anti-predator defence and virulence. We found that anti-predator adaptation (producing predator-resistant biofilm caused a correlative attenuation in virulence. Even though the direct mechanism was not found, reduction in virulence was most clearly connected to a predator-driven loss of a red bacterial pigment, prodigiosin. Moreover, life-history trait evolution was more divergent among replicate populations in the absence of predation, leading also to lowered virulence in some of the 'predator absent' selection lines. Together these findings suggest that the virulence of non-obligatory, opportunistic bacterial pathogens can decrease in environmental reservoirs through life history trade-offs, or random accumulation of mutations that impair virulence traits under relaxed selection.

Marine bacterial transparent exopolymer particles (TEP) and TEP precursors: Characterization and RO fouling potential

KAUST Repository

Li, Sheng

2015-10-31

This paper investigated the characteristics and membrane fouling potential of bacterial transparent exopolymer particles (TEP)/TEP precursors released from two marine bacteria, Pseudidiomarina homiensis (P. homiensis) and Pseudoalteromonas atlantica (P. atlantica), isolated from the Red Sea. Results showed that both bacteria grew at the similar rate, but the production of TEP/TEP precursors from P. atlantica was higher than that from P. homiensis. During the 168. h of incubation time, production rates of TEP/TEP precursors from P. atlantica and P. homiensis were 0.30 and 0.08 xanthan gum eq. mg/L-h, respectively. Isolated bacterial TEP precursors were mainly biopolymer, and P. atlantica produced a significantly higher concentration of biopolymer than that produced by P. homiensis. TEP/TEP precursors from both marine bacteria possessed protein-like material and were very similar in composition to previously reported foulants isolated from a fouled reverse osmosis (RO) membrane. Bacterial TEP/TEP precursors mostly consisted of aliphatic hydrocarbon from amino acids and amide group carbon of proteins (around 55%). Bacterial TEP precursors caused obvious fouling on RO membranes, which may create an ideal environment for bacteria attachment and promote to biofouling.

Linezolid Decreases Susceptibility to Secondary Bacterial Pneumonia Post-Influenza Infection in Mice Through its Effects on Interferon-γ

Science.gov (United States)

Breslow-Deckman, Jessica M.; Mattingly, Cynthia M.; Birket, Susan E.; Hoskins, Samantha N.; Ho, Tam N.; Garvy, Beth A.; Feola, David J.

2013-01-01

Influenza infection predisposes patients to secondary bacterial pneumonia that contributes significantly to morbidity and mortality. While this association is well documented, the mechanisms that govern this synergism are poorly understood. A window of hyporesponsiveness following influenza infection has been associated with a substantial increase in local and systemic IFNγ concentrations. Recent data suggests that the oxazolidinone antibiotic linezolid decreases IFNγ and TNFα production in vitro from stimulated peripheral blood mononuclear cells. We therefore sought to determine whether linezolid would reverse immune hyporesponsiveness after influenza infection in mice through its effects on IFNγ. In vivo dose response studies demonstrated that oral linezolid administration sufficiently decreased bronchoalveolar lavage fluid levels of IFNγ at day 7 post-influenza infection in a dose-dependent manner. The drug also decreased morbidity as measured by weight loss compared to vehicle-treated controls. When mice were challenged intranasally with S. pneumoniae 7 days after infection with influenza, linezolid pre-treatment led to decreased IFNγ and TNFα production, decreased weight loss, and lower bacterial burdens at 24 hours post bacterial infection in comparison to vehicle-treated controls. To determine whether these effects were due to suppression of IFNγ, linezolid-treated animals were given intranasal instillations of recombinant IFNγ before challenge with S. pneumoniae. This partially reversed the protective effects observed in the linezolid-treated mice, suggesting that the modulatory effects of linezolid are mediated partially by its ability to blunt IFNγ production. These results suggest that IFNγ, and potentially TNFα, may be useful drug targets for prophylaxis against secondary bacterial pneumonia following influenza infection. PMID:23833238

Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies

Directory of Open Access Journals (Sweden)

Aniello Russo

2010-02-01

Full Text Available RNA interference (RNAi is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2 protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA. The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site.

Reverse Logistics

OpenAIRE

Kulikova, Olga

2016-01-01

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

Structure-Activity Relationship in TLR4 Mutations: Atomistic Molecular Dynamics Simulations and Residue Interaction Network Analysis

Science.gov (United States)

Anwar, Muhammad Ayaz; Choi, Sangdun

2017-03-01

Toll-like receptor 4 (TLR4), a vital innate immune receptor present on cell surfaces, initiates a signaling cascade during danger and bacterial intrusion. TLR4 needs to form a stable hexamer complex, which is necessary to dimerize the cytoplasmic domain. However, D299G and T399I polymorphism may abrogate the stability of the complex, leading to compromised TLR4 signaling. Crystallography provides valuable insights into the structural aspects of the TLR4 ectodomain; however, the dynamic behavior of polymorphic TLR4 is still unclear. Here, we employed molecular dynamics simulations (MDS), as well as principal component and residue network analyses, to decipher the structural aspects and signaling propagation associated with mutations in TLR4. The mutated complexes were less cohesive, displayed local and global variation in the secondary structure, and anomalous decay in rotational correlation function. Principal component analysis indicated that the mutated complexes also exhibited distinct low-frequency motions, which may be correlated to the differential behaviors of these TLR4 variants. Moreover, residue interaction networks (RIN) revealed that the mutated TLR4/myeloid differentiation factor (MD) 2 complex may perpetuate abnormal signaling pathways. Cumulatively, the MDS and RIN analyses elucidated the mutant-specific conformational alterations, which may help in deciphering the mechanism of loss-of-function mutations.

Bacterial prostatitis.

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Gill, Bradley C; Shoskes, Daniel A

2016-02-01

The review provides the infectious disease community with a urologic perspective on bacterial prostatitis. Specifically, the article briefly reviews the categorization of prostatitis by type and provides a distillation of new findings published on bacterial prostatitis over the past year. It also highlights key points from the established literature. Cross-sectional prostate imaging is becoming more common and may lead to more incidental diagnoses of acute bacterial prostatitis. As drug resistance remains problematic in this condition, the reemergence of older antibiotics such as fosfomycin, has proven beneficial. With regard to chronic bacterial prostatitis, no clear clinical risk factors emerged in a large epidemiological study. However, bacterial biofilm formation has been associated with more severe cases. Surgery has a limited role in bacterial prostatitis and should be reserved for draining of a prostatic abscess or the removal of infected prostatic stones. Prostatitis remains a common and bothersome clinical condition. Antibiotic therapy remains the basis of treatment for both acute and chronic bacterial prostatitis. Further research into improving prostatitis treatment is indicated.

Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains.

Science.gov (United States)

Lavania, Mallika; Singh, Itu; Turankar, Ravindra P; Gupta, Anuj Kumar; Ahuja, Madhvi; Pathak, Vinay; Sengupta, Utpal

2018-01-01

Despite more than three decades of multidrug therapy (MDT), leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae) is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains - comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain - was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB , that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s) in RNA polymerase gene(s), resulting in rifampicin resistance in relapsed leprosy patients.

Profiling of oligosaccharides and p53 gene mutation in Filipino breast tumors

International Nuclear Information System (INIS)

Deocaris, Custer C.; De Vera, Azucena C.; Magno, Jose Donato A.; Cruz, Michael Joseph B.; Prodigalidad, Abelardo-Alan T.; Jacinto, Sonia D.

2010-01-01

Majority of patients are diagnosed with benign tumors, however, such benign tumors can progress to an invasive disease. Since carbohydrate-mediated cell-cell adhesion and proliferative potential play crucial roles in tumorigenesis and tumor aggressive behavior, we analyzed the qualitative changes in oligosaccharide expression and analyzed for presence of mutation in the tumor suppressor p53 gene, the most mutated gene in all human cancers. Forty-three (43) breast tumors were screened for p53 mutation in exons 2-11 using polymerase chain reaction (PCR)-amplification coupled to temporal temperature gradient electrophoresis (TTGE). Paraffin-embedded tissues were stained with biotinylated-glycoproteins containing the following sugar groups: mannose (Man), lactose (Lac), fucoidan (Fuc), N-acetyl-glucosamine (GlcNac), N-acetyl-b-galactosamine (GalNAc) and hyaluronic acid (Hya). Expression of carbohydrate receptors was significantly elevated (p=0.003) in malignant compared with benign tumors, particularly at receptors for GalNAc, lac and Fuc. No change in overall glycan signatures using our panel of neoglycoconjugates was noted when grouped according to p53 mutation status in both benign and malignant cases. Although the prognostic value of carbohydrate-receptors in breast cancer has not been validated to date, our results indicate that benign and malignant tumors can be defined by their affinities to our battery of neoglyconjugates. However, result from our reverse lectin histochemistry failed to correlated glycan signature with presence of p53 mutations. (author)

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

International Nuclear Information System (INIS)

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

1999-01-01

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

ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia.

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Zhang, Kang; Liu, Qing; Liu, Keqiang; Shen, Dongchao; Tai, Hongfei; Shu, Shi; Ding, Qingyun; Fu, Hanhui; Liu, Shuangwu; Wang, Zhili; Li, Xiaoguang; Liu, Mingsheng; Zhang, Xue; Cui, Liying

2018-06-01

To investigate the genetic contribution of ANXA11 , a gene associated with amyotrophic lateral sclerosis (ALS), in Chinese ALS patients with and without cognitive dementia. Sequencing all the coding exons of ANXA11 and intron-exon boundaries in 18 familial amyotrophic lateral sclerosis (FALS), 353 unrelated sporadic amyotrophic lateral sclerosis (SALS), and 12 Chinese patients with ALS-frontotemporal lobar dementia (ALS-FTD). The transcripts in peripheral blood generated from a splicing mutation were examined by reverse transcriptase PCR. We identified 6 nonsynonymous heterozygous mutations (5 novel and 1 recurrent), 1 splice site mutation, and 1 deletion of 10 amino acids (not accounted in the mutant frequency) in 11 unrelated patients, accounting for a mutant frequency of 5.6% (1/18) in FALS, 2.3% (8/353) in SALS, and 8.3% (1/12) in ALS-FTD. The deletion of 10 amino acids was detected in 1 clinically undetermined male with an ALS family history who had atrophy in hand muscles and myotonic discharges revealed by EMG. The novel p. P36R mutation was identified in 1 FALS index, 1 patient with SALS, and 1 ALS-FTD. The splicing mutation (c.174-2A>G) caused in-frame skipping of the entire exon 6. The rest missense mutations including p.D40G, p.V128M, p.S229R, p.R302C and p.G491R were found in 6 unrelated patients with SALS. The ANXA11 gene is one of the most frequently mutated genes in Chinese patients with SALS. A canonical splice site mutation leading to skipping of the entire exon 6 further supports the loss-of-function mechanism. In addition, the study findings further expand the ANXA11 phenotype, first highlighting its pathogenic role in ALS-FTD.

Evolution of Bacterial Suicide

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Tchernookov, Martin; Nemenman, Ilya

2013-03-01

While active, controlled cellular suicide (autolysis) in bacteria is commonly observed, it has been hard to argue that autolysis can be beneficial to an individual who commits it. We propose a theoretical model that predicts that bacterial autolysis is evolutionarily advantageous to an individualand would fixate in physically structured environments for stationary phase colonies. We perform spatially resolved agent-based simulations of the model, which predict that lower mixing in the environment results in fixation of a higher autolysis rate from a single mutated cell, regardless of the colony's genetic diversity. We argue that quorum sensing will fixate as well, even if initially rare, if it is coupled to controlling the autolysis rate. The model does not predict a strong additional competitive advantage for cells where autolysis is controlled by quorum sensing systems that distinguish self from nonself. These predictions are broadly supported by recent experimental results in B. subtilisand S. pneumoniae. Research partially supported by the James S McDonnell Foundation grant No. 220020321 and by HFSP grant No. RGY0084/2011.

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

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Hane James K

2010-11-01

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

Induction of bacterial blight resistance in elite Indian rice cultivars using gamma-rays and ethyl methanesulfonate

International Nuclear Information System (INIS)

Agrawal, P.K.; Gosal, S.S.; Sidhu, G.S.

2001-01-01

Rice is the most important cereal crop in the world feeding more than 50 percent of the human population. During the last 30 years, induced mutation breeding has played a significant role in rice breeding programmes. Rice mutants with higher yield, greater tolerance to diseases and pests and other agronomic qualities have been released for commercial cultivation in many countries. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is the second important disease in Southeast Asia. In the Basmati field sometime the yield loss is up to 100%. Moreover, there is no resistance source available. In Basmati rice, which is known for its quality and aroma. Induction of bacterial blight resistance in Basmati will help in developing high yielding Basmati type cultivars without compromising the quality

Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

Science.gov (United States)

Matamoros, Tania; Barrioluengo, Verónica; Abia, David; Menéndez-Arias, Luis

2013-12-23

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

Novel CFI mutation in a patient with leukocytoclastic vasculitis may redefine the clinical spectrum of Complement Factor I deficiency

DEFF Research Database (Denmark)

Bay, Jakob Thaning; Katzenstein, Terese Lea; Kofoed, Kristian

2015-01-01

presentation of Factor I deficiency varies and includes severe recurrent bacterial infections, glomerulonephritis and autoimmune diseases. The patient, a 28-years old woman with consanguineous parents, presented with recurrent leukocytoclastic vasculitis in the lower extremities with no associated systemic...... mutations vary among patients sole association with leukocytoclastic vasculitis redefines the clinical spectrum of complete Factor I deficiency....

A genome-wide association study points out the causal implication of SOX9 in the sex-reversal phenotype in XX pigs.

Science.gov (United States)

Rousseau, Sarah; Iannuccelli, Nathalie; Mercat, Marie-José; Naylies, Claire; Thouly, Jean-Claude; Servin, Bertrand; Milan, Denis; Pailhoux, Eric; Riquet, Juliette

2013-01-01

Among farm animals, pigs are known to show XX sex-reversal. In such cases the individuals are genetically female but exhibit a hermaphroditism, or a male phenotype. While the frequency of this congenital disease is quite low (less than 1%), the economic losses are significant for pig breeders. These losses result from sterility, urogenital infections and the carcasses being downgraded because of the risk of boar taint. It has been clearly demonstrated that the SRY gene is not involved in most cases of sex-reversal in pigs, and that autosomal recessive mutations remain to be discovered. A whole-genome scan analysis was performed in the French Large-White population to identify candidate genes: 38 families comprising the two non-affected parents and 1 to 11 sex-reversed full-sib piglets were genotyped with the PorcineSNP60 BeadChip. A Transmission Disequilibrium Test revealed a highly significant candidate region on SSC12 (most significant p-valueTesco. However, no causal mutations could be identified in either of the two sequenced regions. Further haplotype analyses did not identify a shared homozygous segment between the affected pigs, suggesting either a lack of power due to the SNP properties of the chip, or a second causative locus. Together with information from humans and mice, this study in pigs adds to the field of knowledge, which will lead to characterization of novel molecular mechanisms regulating sexual differentiation and dysregulation in cases of sex reversal.

Genotoxicological Evaluation of NUTRALYS Pea Protein Isolate

OpenAIRE

Aouatif, Chentouf; Looten, Ph.; Parvathi, M. V. S.; Raja Ganesh, S.; Paranthaman, V.

2013-01-01

NUTRALYS Pea Protein Isolate, a protein supplement, is a high-quality source of protein which is primarily emulsifying functional protein. We evaluated the genotoxic potential of NUTRALYS isolated from dry yellow pea, using three established genotoxicity tests (AMES test in vitro chromosomal aberration test, and in vivo micronucleus test) employing OECD guidelines under GLP conditions. In the bacterial reverse mutation test, NUTRALYS did not show positive responses in strains detecting point ...

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

Science.gov (United States)

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

Science.gov (United States)

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-07-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.

Science.gov (United States)

Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-06-26

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination.

Science.gov (United States)

Novellasdemunt, Laura; Foglizzo, Valentina; Cuadrado, Laura; Antas, Pedro; Kucharska, Anna; Encheva, Vesela; Snijders, Ambrosius P; Li, Vivian S W

2017-10-17

The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs. Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

Science.gov (United States)

Atik, Tahir; Aykut, Ayca; Hazan, Filiz; Onay, Huseyin; Goksen, Damla; Darcan, Sukran; Tukun, Ajlan; Ozkinay, Ferda

2016-06-01

To evaluate the spectrum of PTPN11 gene mutations in Noonan syndrome patients and to study the genotype-phenotype associations. In this study, twenty Noonan syndrome patients with PTPN11 mutations were included. The patients underwent a detailed clinical and physical evaluation. To identify inherited cases, parents of all mutation positive patients were analyzed. Thirteen different PTPN11 mutations, two of them being novel, were detected in the study group. These mutations included eleven missense mutations: p.G60A, p.D61N, p.Y62D, p.Y63C, p.E69Q, p.Q79R, p.Y279C,p.N308D, p.N308S, p.M504V, p.Q510R and two novel missense mutations: p.I56V and p.I282M. The frequency of cardiac abnormalities and short stature were found to be 80 % and 80 %, respectively. Mental retardation was not observed in patients having exon 8 mutations. No significant correlations were detected between other phenotypic features and genotypes. By identifying genotype-phenotype correlations, this study provides information on phenotypes observed in NS patients with different PTPN11 mutations.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

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Wu Xuhong

2006-08-01

Full Text Available Abstract Background INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN. It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. Results We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Conclusion Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

Science.gov (United States)

Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

2006-08-31

INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene.

Science.gov (United States)

Poussier, Stéphane; Thoquet, Philippe; Trigalet-Demery, Danièle; Barthet, Séverine; Meyer, Damien; Arlat, Matthieu; Trigalet, André

2003-08-01

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

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

NARCIS (Netherlands)

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

2013-01-01

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

Bacterial Population Genetics in a Forensic Context

Energy Technology Data Exchange (ETDEWEB)

Velsko, S P

2009-11-02

This report addresses the recent Department of Homeland Security (DHS) call for a Phase I study to (1) assess gaps in the forensically relevant knowledge about the population genetics of eight bacterial agents of concern, (2) formulate a technical roadmap to address those gaps, and (3) identify new bioinformatics tools that would be necessary to analyze and interpret population genetic data in a forensic context. The eight organisms that were studied are B. anthracis, Y. pestis, F. tularensis, Brucella spp., E. coli O157/H7, Burkholderia mallei, Burkholderia pseudomallei, and C. botulinum. Our study focused on the use of bacterial population genetics by forensic investigators to test hypotheses about the possible provenance of an agent that was used in a crime or act of terrorism. Just as human population genetics underpins the calculations of match probabilities for human DNA evidence, bacterial population genetics determines the level of support that microbial DNA evidence provides for or against certain well-defined hypotheses about the origins of an infecting strain. Our key findings are: (1) Bacterial population genetics is critical for answering certain types of questions in a probabilistic manner, akin (but not identical) to 'match probabilities' in DNA forensics. (2) A basic theoretical framework for calculating likelihood ratios or posterior probabilities for forensic hypotheses based on microbial genetic comparisons has been formulated. This 'inference-on-networks' framework has deep but simple connections to the population genetics of mtDNA and Y-STRs in human DNA forensics. (3) The 'phylogeographic' approach to identifying microbial sources is not an adequate basis for understanding bacterial population genetics in a forensic context, and has limited utility, even for generating 'leads' with respect to strain origin. (4) A collection of genotyped isolates obtained opportunistically from international locations

Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers cisplatin resistance and a migratory phenotype in cervical cancer cells

Science.gov (United States)

Arjumand, Wani; Merry, Cole D.; Wang, Chen; Saba, Elias; McIntyre, John B.; Fang, Shujuan; Kornaga, Elizabeth; Ghatage, Prafull; Doll, Corinne M.; Lees, Susan P.

2016-01-01

The phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR signaling pathway is activated in many human cancers. Previously, we reported that patients with early stage cervical cancer whose tumours harbour PIK3CA exon 9 or 20 mutations have worse overall survival in response to treatment with radiation and cisplatin than patients with wild-type PIK3CA. The purpose of this study was to determine whether PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin and/or radiation, and whether PI3K inhibition reverses the phenotype. We found that CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. Our results suggests that cervical cancer patients whose tumours are positive for the PIK3CA-E545K mutation may benefit from PI3K inhibitor therapy in concert with standard cisplatin and radiation therapy. PMID:27489350

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

Directory of Open Access Journals (Sweden)

Irene Bianconi

2011-02-01

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

Prevalence of drug-resistant mutation among drug-treated HIV/AIDS inpatient in Airlangga University teaching hospital, Surabaya, Indonesia

Science.gov (United States)

Rachman, B. E.; Khairunisa, S. Q.; Witaningrum, A. M.; Yunifiar, M. Q.; Widiyanti, P.; Nasronudin

2018-03-01

Increased use of antiretroviral therapy did not completely reduce the incidence of HIV/AIDShospitalization. Various factors can be involved. The aim of this study is to examine HIV-1 drug resistance mutations profile in drug-treated HIV/AIDS patients who underwent hospitalization. HIV/AIDS patients who are admitted to hospital who had received ART are included in the study and then examined for the presence of drug resistance-associated mutations. A total of 17 samples were included in the study, but only 11 samples that could be sequence analyzed. On the mutation examination of drug resistance in reverse transcriptase gene, it werefound a major mutation in K103N (9%) and G190A (9%). Most minor mutations were found in A98S (18.1%), followed by M41L, M184V, L210W, T215Y, V108l, Y181C and H221Y at 9% each. Whereas, on examination of drug resistance mutations in protease genes, there is a major mutation in I84V of 9%. Most minor mutations on M36I (45.4%), followed by L10I (36.3%), H69K (36.3%), I93L (27.2%), G16E, L89M, K20R 18.1%, L64V and V771I 9% respectively.A large number of mutated samples pose a challenge in long-term antiretroviral treatment, so a breakthrough policy is needed to minimize the impact.

Mechanics of torque generation in the bacterial flagellar motor.

Science.gov (United States)

Mandadapu, Kranthi K; Nirody, Jasmine A; Berry, Richard M; Oster, George

2015-08-11

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual "power stroke." Specifically, we propose that ion-induced conformational changes about a proline "hinge" residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque-speed and speed-ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak.

Science.gov (United States)

Dietzel, Erik; Schudt, Gordian; Krähling, Verena; Matrosovich, Mikhail; Becker, Stephan

2017-01-15

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

Science.gov (United States)

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Bacterial Adhesion & Blocking Bacterial Adhesion

DEFF Research Database (Denmark)

Vejborg, Rebecca Munk

2008-01-01

, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...... tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters...

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

DEFF Research Database (Denmark)

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

1992-01-01

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

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

Directory of Open Access Journals (Sweden)

David Warrilow

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

Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

Energy Technology Data Exchange (ETDEWEB)

Argaw, Takele; Wilson, Carolyn A., E-mail: carolyn.wilson@fda.hhs.gov

2015-01-15

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

Science.gov (United States)

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.

Directory of Open Access Journals (Sweden)

Raul A Cernadas

2014-02-01

Full Text Available Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo, which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

An integrative genomic and proteomic analysis of PIK3CA, PTEN and AKT mutations in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Stemke-Hale, Katherine; Gonzalez-Angulo, Ana Maria; Lluch, Ana; Neve, Richard M.; Kuo, Wen-Lin; Davies, Michael; Carey, Mark; Hu, Zhi; Guan, Yinghui; Sahin, Aysegul; Symmans, W. Fraser; Pusztai, Lajos; Nolden, Laura K.; Horlings, Hugo; Berns, Katrien; Hung, Mien-Chie; van de Vijver, Marc J.; Valero, Vicente; Gray, Joe W.; Bernards, Rene; Mills, Gordon B.; Hennessy, Bryan T.

2008-05-06

Phosphatidylinositol-3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT and PTEN mutations, and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in-vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor positive (33.8%) and HER2-positive (24.6%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers with PTEN protein levels also being significantly lower in hormone receptor-positive cancers. Unlike AKT1 mutations, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant impact on outcome in 166 hormone receptor-positive breast cancer patients after adjuvant tamoxifen. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and indeed inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines, and PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss but not PIK3CA mutations rendered cells sensitive to growth inhibition by the PI3K inhibitor LY294002. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

Bacterial community structure and variation in a full-scale seawater desalination plant for drinking water production

KAUST Repository

Belila, Abdelaziz

2016-02-18

Microbial processes inevitably play a role in membrane-based desalination plants, mainly recognized as membrane biofouling. We assessed the bacterial community structure and diversity during different treatment steps in a full-scale seawater desalination plant producing 40,000 m3/d of drinking water. Water samples were taken over the full treatment train consisting of chlorination, spruce media and cartridge filters, de-chlorination, first and second pass reverse osmosis (RO) membranes and final chlorine dosage for drinking water distribution. The water samples were analyzed for water quality parameters (total bacterial cell number, total organic carbon, conductivity, pH, etc.) and microbial community composition by 16S rRNA gene pyrosequencing. The planktonic microbial community was dominated by Proteobacteria (48.6%) followed by Bacteroidetes (15%), Firmicutes (9.3%) and Cyanobacteria (4.9%). During the pretreatment step, the spruce media filter did not impact the bacterial community composition dominated by Proteobacteria. In contrast, the RO and final chlorination treatment steps reduced the Proteobacterial relative abundance in the produced water where Firmicutes constituted the most dominant bacterial group. Shannon and Chao1 diversity indices showed that bacterial species richness and diversity decreased during the seawater desalination process. The two-stage RO filtration strongly reduced the water conductivity (>99%), TOC concentration (98.5%) and total bacterial cell number (>99%), albeit some bacterial DNA was found in the water after RO filtration. About 0.25% of the total bacterial operational taxonomic units (OTUs) were present in all stages of the desalination plant: the seawater, the RO permeates and the chlorinated drinking water, suggesting that these bacterial strains can survive in different environments such as high/low salt concentration and with/without residual disinfectant. These bacterial strains were not caused by contamination during

In Vitro and In Vivo Genotoxicity Assessment of Aristolochia manshuriensis Kom.

Directory of Open Access Journals (Sweden)

Youn-Hwan Hwang

2012-01-01

Full Text Available Arisolochiae species plants containing aristolochic acids I and II (AA I and AA II are well known to cause aristolochic acid nephropathy (AAN. Recently, there are various approaches to use AAs-containing herbs after the removal of their toxic factors. However, there is little information about genotoxicity of Arisolochiae manshuriensis Kom. (AMK per se. To obtain safety information for AMK, its genotoxicity was evaluated in accordance with OECD guideline. To evaluate genotoxicity of AMK, we tested bacterial reverse mutation assay, chromosomal aberration test, and micronucleus test. Here, we also determined the amounts of AA I and II in AMK (2.85 ± 0.08 and 0.50 ± 0.02 mg/g extract, resp.. In bacterial reverse mutation assay, AMK dose-dependently increased revertant colony numbers in TA98, TA100 and TA1537 regardless of metabolic activation. AMK increased the incidence of chromosomal aberration in Chinese hamster ovary-K1 cells, but there was no statistically significant difference. The incidences of micronucleus in bone marrow erythrocyte were significantly increased in mice after oral administration of AMK (5000 mg/kg, comparing with those of vehicle group (P<0.05. The results of three standard tests suggest that the genotoxicity of AMK is directly related to the AAs contents in AMK.

XX male sex reversal with genital abnormalities associated with a de novo SOX3 gene duplication.

Science.gov (United States)

Moalem, Sharon; Babul-Hirji, Riyana; Stavropolous, Dmitri J; Wherrett, Diane; Bägli, Darius J; Thomas, Paul; Chitayat, David

2012-07-01

Differentiation of the bipotential gonad into testis is initiated by the Y chromosome-linked gene SRY (Sex-determining Region Y) through upregulation of its autosomal direct target gene SOX9 (Sry-related HMG box-containing gene 9). Sequence and chromosome homology studies have shown that SRY most probably evolved from SOX3, which in humans is located at Xq27.1. Mutations causing SOX3 loss-of-function do not affect the sex determination in mice or humans. However, transgenic mouse studies have shown that ectopic expression of Sox3 in the bipotential gonad results in upregulation of Sox9, resulting in testicular induction and XX male sex reversal. However, the mechanism by which these rearrangements cause sex reversal and the frequency with which they are associated with disorders of sex development remains unclear. Rearrangements of the SOX3 locus were identified recently in three cases of human XX male sex reversal. We report on a case of XX male sex reversal associated with a novel de novo duplication of the SOX3 gene. These data provide additional evidence that SOX3 gain-of-function in the XX bipotential gonad causes XX male sex reversal and further support the hypothesis that SOX3 is the evolutionary antecedent of SRY. Copyright © 2012 Wiley Periodicals, Inc.

Nanotechnology to rescue bacterial bidirectional extracellular electron transfer in bioelectrochemical systems

KAUST Repository

Kalathil, Shafeer

2016-03-17

An electrically active bacterium transports its metabolically generated electrons to insoluble substrates such as electrodes via a process known as extracellular electron transport (EET). Bacterial EET is a crucial process in the geochemical cycling of metals, bioremediation and bioenergy devices such as microbial fuel cells (MFCs). Recently, it has been found that electroactive bacteria can reverse their respiratory pathways by accepting electrons from a negatively poised electrode to produce high-value chemicals such as ethanol in a process termed as microbial electrosynthesis (MES). A poor electrical connection between bacteria and the electrode hinders the EET and MES processes significantly. Also, the bidirectional EET process is sluggish and needs to be improved drastically to extend its practical applications. Several attempts have been undertaken to improve the bidirectional EET by employing various advanced nanostructured materials such as carbon nanotubes and graphene. This review covers the recent progress in the bacterial bidirectional EET processes using advanced nanostructures in the light of current understandings of bacteria–nanomaterial interactions.

Mutations to Less-Preferred Synonymous Codons in a Highly Expressed Gene of Escherichia coli: Fitness and Epistatic Interactions.

Directory of Open Access Journals (Sweden)

David J Hauber

Full Text Available Codon-tRNA coevolution to maximize protein production has been, until recently, the dominant hypothesis to explain codon-usage bias in highly expressed bacterial genes. Two predictions of this hypothesis are 1 selection is weak; and 2 similar silent replacements at different codons should have similar fitness consequence. We used an allele-replacement strategy to change five specific 3rd-codon-position (silent sites in the highly expressed Escherichia coli ribosomal protein gene rplQ from the wild type to a less-preferred alternative. We introduced the five mutations within a 10-codon region. Four of the silent sites were chosen to test the second prediction, with a CTG to CTA mutation being introduced at two closely linked leucine codons and an AAA to AAG mutation being introduced at two closely linked lysine codons. We also introduced a fifth silent mutation, a GTG to GTA mutation at a valine codon in the same genic region. We measured the fitness effect of the individual mutations by competing each single-mutant strain against the parental wild-type strain, using a disrupted form of the araA gene as a selectively neutral phenotypic marker to distinguish between strains in direct competition experiments. Three of the silent mutations had a fitness effect of |s| > 0.02, which is contradictory to the prediction that selection will be weak. The two leucine mutations had significantly different fitness effects, as did the two lysine mutations, contradictory to the prediction that similar mutations at different codons should have similar fitness effects. We also constructed a strain carrying all five silent mutations in combination. Its fitness effect was greater than that predicted from the individual fitness values, suggesting that negative synergistic epistasis acts on the combination allele.

Mutation Induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis

International Nuclear Information System (INIS)

Tanooka, H.; Munakata, N.; Kitahara, S.

1978-01-01

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr - , ssp - , hcr - ssp - ) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotropic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity

TERT promoter hot spot mutations are frequent in Indian cervical and oral squamous cell carcinomas.

Science.gov (United States)

Vinothkumar, Vilvanathan; Arunkumar, Ganesan; Revathidevi, Sundaramoorthy; Arun, Kanagaraj; Manikandan, Mayakannan; Rao, Arunagiri Kuha Deva Magendhra; Rajkumar, Kottayasamy Seenivasagam; Ajay, Chandrasekar; Rajaraman, Ramamurthy; Ramani, Rajendren; Murugan, Avaniyapuram Kannan; Munirajan, Arasambattu Kannan

2016-06-01

Squamous cell carcinoma (SCC) of the uterine cervix and oral cavity are most common cancers in India. Telomerase reverse transcriptase (TERT) overexpression is one of the hallmarks for cancer, and activation through promoter mutation C228T and C250T has been reported in variety of tumors and often shown to be associated with aggressive tumors. In the present study, we analyzed these two hot spot mutations in 181 primary tumors of the uterine cervix and oral cavity by direct DNA sequencing and correlated with patient's clinicopathological characteristics. We found relatively high frequency of TERT hot spot mutations in both cervical [21.4 % (30/140)] and oral [31.7 % (13/41)] squamous cell carcinomas. In cervical cancer, TERT promoter mutations were more prevalent (25 %) in human papilloma virus (HPV)-negative cases compared to HPV-positive cases (20.6 %), and both TERT promoter mutation and HPV infection were more commonly observed in advanced stage tumors (77 %). Similarly, the poor and moderately differentiated tumors of the uterine cervix had both the TERT hot spot mutations and HPV (16 and 18) at higher frequency (95.7 %). Interestingly, we observed eight homozygous mutations (six 228TT and two 250TT) only in cervical tumors, and all of them were found to be positive for high-risk HPV. To the best of our knowledge, this is the first study from India reporting high prevalence of TERT promoter mutations in primary tumors of the uterine cervix and oral cavity. Our results suggest that TERT reactivation through promoter mutation either alone or in association with the HPV oncogenes (E6 and E7) could play an important role in the carcinogenesis of cervical and oral cancers.

Disease-associated mutations that alter the RNA structural ensemble.

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Matthew Halvorsen

2010-08-01

Full Text Available Genome-wide association studies (GWAS often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs from the Human Gene Mutation Database (HGMD that map to the untranslated regions (UTRs of a gene. Rather than using minimum free energy approaches (e.g. mFold, we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, beta-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD, and Hypertension, we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5' UTRs of FTL and RB1 SNP-induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a "RiboSNitch," that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

Genotoxicity of triiodothyronine: Effects on Salmonella typhimurium TA100 and human lymphocytes in vitro

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Bošnjak-Neumüller Jasna

2017-01-01

Full Text Available There is increasing evidence that substances which are normally present in human or animal bodies may, under the certain circumstances, exhibit deleterious effects on genetic material, therefore acting as endogenous mutagenic agents. Since hormones represent one of the best studied endogenous mutagens, some research focused on the possible role of thyroid hormone in mutagenesis and carcinogenesis. Indeed, thyroid hormones accelerate aerobic metabolism and production of reactive oxygen species (ROS and, therefore, may exhibit mutagenic effects in various test systems on mammalian cells. However, possible mutagenic effects on prokaryotic DNA has not been investigated so far. Hence, the aim of this research was to compare the sensitivity of TA 100 Salmonella typhimurium with and without metabolic activation with S9 fraction, and human lymphocytes to possible genotoxic effects of triiodothyronine (T3. Therefore, we used the reverse mutation assay on S. typhimurium (Ames test and in vitro Comet assay in isolated peripheral blood human lymphocytes. In both tests-systems a broad spectrum of T3 concentrations was applied. The obtained results showed absence of genotoxic effects of T3 in bacterial reverse mutation assay and very profound genotoxic effects in human lymphocytes at concentrations higher than 15 μM. We only observed cytotoxic effects in bacterial system at very high T3 concentrations (300 and 500 μM. In conclusion, T3 was unable to increase the level of reverse mutations in Ames test both with and without S9 mix. Therefore, it seems that ROS production in mitochondria may be the primary cause of DNA damage caused by T3 in mammalian cells. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III46002

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation

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Pailhoux Eric

2005-12-01

Full Text Available Abstract In goats, the PIS (polled intersex syndrome mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4–5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation.

Dual Inhibition of EGFR with Afatinib and Cetuximab in Kinase Inhibitor-Resistant EGFR-Mutant Lung Cancer with and without T790M Mutations

NARCIS (Netherlands)

Janjigian, Yelena Y.; Smit, Egbert F.; Groen, Harry J. M.; Horn, Leora; Gettinger, Scott; Camidge, D. Ross; Riely, Gregory J.; Wang, Bushi; Fu, Yali; Chand, Vikram K.; Miller, Vincent A.; Pao, William

EGFR-mutant lung cancers responsive to reversible EGFR inhibitors (gefitinib/erlotinib) develop acquired resistance, mediated by second-site EGFR T790M mutation in >50% of cases. Preclinically, afatinib (irreversible ErbB family blocker) plus cetuximab (anti-EGFR monoclonal antibody) overcomes

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

OpenAIRE

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-01-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was d...

Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action

DEFF Research Database (Denmark)

Xiong, L; Kloss, P; Douthwaite, S

2000-01-01

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction......, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis...

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

Science.gov (United States)

Orndorff, Paul E

2016-11-01

Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

The CDC Hemophilia B mutation project mutation list: a new online resource.

Science.gov (United States)

Li, Tengguo; Miller, Connie H; Payne, Amanda B; Craig Hooper, W

2013-11-01

Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

Chlorpheniramine Analogues Reverse Chloroquine Resistance in Plasmodium falciparum by Inhibiting PfCRT.

Science.gov (United States)

Deane, Karen J; Summers, Robert L; Lehane, Adele M; Martin, Rowena E; Barrow, Russell A

2014-05-08

The emergence and spread of malaria parasites that are resistant to chloroquine (CQ) has been a disaster for world health. The antihistamine chlorpheniramine (CP) partially resensitizes CQ-resistant (CQR) parasites to CQ but possesses little intrinsic antiplasmodial activity. Mutations in the parasite's CQ resistance transporter (PfCRT) confer resistance to CQ by enabling the protein to transport the drug away from its site of action, and it is thought that resistance-reversers such as CP exert their effect by blocking this CQ transport activity. Here, a series of new structural analogues and homologues of CP have been synthesized. We show that these compounds (along with other in vitro CQ resistance-reversers) inhibit the transport of CQ via a resistance-conferring form of PfCRT expressed in Xenopus laevis oocytes. Furthermore, the level of PfCRT-inhibition was found to correlate well with both the restoration of CQ accumulation and the level of CQ resensitization in CQR parasites.

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

Science.gov (United States)

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-02-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

Science.gov (United States)

Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

2014-01-01

Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

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Wei Cheng

Full Text Available Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1. Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART.

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Jose Manuel Vazquez-Guillen

Full Text Available Although Structured Treatment Interruptions (STI are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM's at levels under limit of detection of conventional genotyping (<20% of quasispecies could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM's in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM's during STI.

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

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Muriaux Delphine

2007-08-01

Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

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

Science.gov (United States)

Maisnier-Patin, Sophie; Roth, John R.

2015-01-01

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

Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

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Zhong-bin LI

2012-06-01

Full Text Available Objective 　To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods 　A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results 　HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions 　During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

Reversible Thermoset Adhesives

Science.gov (United States)

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

2016-01-01

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

Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy.

Science.gov (United States)

De Francesco, Vincenzo; Zullo, Angelo; Giorgio, Floriana; Saracino, Ilaria; Zaccaro, Cristina; Hassan, Cesare; Ierardi, Enzo; Di Leo, Alfredo; Fiorini, Giulia; Castelli, Valentina; Lo Re, Giovanna; Vaira, Dino

2014-03-01

Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8%), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88% (Presistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

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Inna Biela

Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

[Analysis of USH2A gene mutation in a Chinese family affected with Usher syndrome].

Science.gov (United States)

Li, Pengcheng; Liu, Fei; Zhang, Mingchang; Wang, Qiufen; Liu, Mugen

2015-08-01

To investigate the disease-causing mutation in a Chinese family affected with Usher syndrome type II. All of the 11 members from the family underwent comprehensive ophthalmologic examination and hearing test, and their genomic DNA were isolated from venous leukocytes. PCR and direct sequencing of USH2A gene were performed for the proband. Wild type and mutant type minigene vectors containing exon 42, intron 42 and exon 43 of the USH2A gene were constructed and transfected into Hela cells by lipofectamine reagent. Reverse transcription (RT)-PCR was carried out to verify the splicing of the minigenes. Pedigree analysis and clinical diagnosis indicated that the patients have suffered from autosomal recessive Usher syndrome type II. DNA sequencing has detected a homozygous c.8559-2A>G mutation of the USH2A gene in the proband, which has co-segregated with the disease in the family. The mutation has affected a conserved splice site in intron 42, which has led to inactivation of the splice site. Minigene experiment has confirmed the retaining of intron 42 in mature mRNA. The c.8559-2A>G mutation in the USH2A gene probably underlies the Usher syndrome type II in this family. The splice site mutation has resulted in abnormal splicing of USH2A pre-mRNA.

Identification and characterization of a novel XK splice site mutation in a patient with McLeod syndrome.

Science.gov (United States)

Arnaud, Lionel; Salachas, François; Lucien, Nicole; Maisonobe, Thierry; Le Pennec, Pierre-Yves; Babinet, Jérôme; Cartron, Jean-Pierre

2009-03-01

McLeod syndrome is a rare X-linked neuroacanthocytosis syndrome with hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused by mutations in the XK gene whose product is expressed at the red blood cell (RBC) surface but whose function is currently unknown. A variety of XK mutations has been reported but no clear phenotype-genotype correlation has been found, especially for the point mutations affecting splicing sites. A man suspected of neuroacanthocytosis was evaluated by neurologic examination, electromyography, muscle biopsy, muscle computed tomography, and cerebral magnetic resonance imaging. The McLeod RBC phenotype was disclosed by blood smear and immunohematology analyses and then confirmed at the biochemical level by Western blot analysis. The responsible XK mutation was characterized at the mRNA level by reverse transcription-polymerase chain reaction (PCR), identified by genomic DNA sequencing, and verified by allele-specific PCR. A novel XK splice site mutation (IVS1-1G>A) has been identified in a McLeod patient who has developed hematologic, neuromuscular, and neurologic symptoms. This is the first reported example of a XK point mutation affecting the 3' acceptor splice site of Intron 1, and it was demonstrated that this mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at the RBC membrane. The detailed characterization at the molecular biology level of this novel XK splice site mutation associated with the clinical description of the patient contributes to a better understanding of the phenotype-genotype correlation in the McLeod syndrome.

Molecular basis of hereditary C1q deficiency-revisited: identification of several novel disease-causing mutations

DEFF Research Database (Denmark)

Schejbel, L; Skattum, L; Hagelberg, S

2011-01-01

C1q is the central pattern-recognition molecule in the classical pathway of the complement system and is known to have a key role in the crossroads between adaptive and innate immunity. Hereditary C1q deficiency is a rare genetic condition strongly associated with systemic lupus erythematosus...... and increased susceptibility to bacterial infections. However, the clinical symptoms may vary. For long, the molecular basis of C1q deficiency was ascribed to only six different mutations. In the present report, we describe five new patients with C1q deficiency, present the 12 causative mutations described till...... now and review the clinical spectrum of symptoms found in patients with C1q deficiency. With the results presented here, confirmed C1q deficiency is reported in 64 patients from at least 38 families....

Zero field reversal probability in thermally assisted magnetization reversal

Science.gov (United States)

Prasetya, E. B.; Utari; Purnama, B.

2017-11-01

This paper discussed about zero field reversal probability in thermally assisted magnetization reversal (TAMR). Appearance of reversal probability in zero field investigated through micromagnetic simulation by solving stochastic Landau-Lifshitz-Gibert (LLG). The perpendicularly anisotropy magnetic dot of 50×50×20 nm3 is considered as single cell magnetic storage of magnetic random acces memory (MRAM). Thermally assisted magnetization reversal was performed by cooling writing process from near/almost Curie point to room temperature on 20 times runs for different randomly magnetized state. The results show that the probability reversal under zero magnetic field decreased with the increase of the energy barrier. The zero-field probability switching of 55% attained for energy barrier of 60 k B T and the reversal probability become zero noted at energy barrier of 2348 k B T. The higest zero-field switching probability of 55% attained for energy barrier of 60 k B T which corespond to magnetif field of 150 Oe for switching.

The role and regulation of catalase in respiratory tract opportunistic bacterial pathogens.

Science.gov (United States)

Eason, Mia M; Fan, Xin

2014-09-01

Respiratory tract bacterial pathogens are the etiologic agents of a variety of illnesses. The ability of these bacteria to cause disease is imparted through survival within the host and avoidance of pathogen clearance by the immune system. Respiratory tract pathogens are continually bombarded by reactive oxygen species (ROS), which may be produced by competing bacteria, normal metabolic function, or host immunological responses. In order to survive and proliferate, bacteria have adapted defense mechanisms to circumvent the effects of ROS. Bacteria employ the use of anti-oxidant enzymes, catalases and catalase-peroxidases, to relieve the effects of the oxidative stressors to which they are continually exposed. The decomposition of ROS has been shown to provide favorable conditions in which respiratory tract opportunistic bacterial pathogens such as Haemophilus influenzae, Mycobacterium tuberculosis, Legionella pneumophila, and Neisseria meningitidis are able to withstand exposure to highly reactive molecules and yet survive. Bacteria possessing mutations in the catalase gene have a decreased survival rate, yet may be able to compensate for the lack of catalatic activity if peroxidatic activity is present. An incomplete knowledge of the mechanisms by which catalase and catalase-peroxidases are regulated still persists, however, in some bacterial species, a regulatory factor known as OxyR has been shown to either up-regulate or down-regulate catalase gene expression. Yet, more research is still needed to increase the knowledge base in relation to this enzyme class. As with this review, we focus on major respiratory tract opportunistic bacterial pathogens in order to elucidate the function and regulation of catalases. The importance of the research could lead to the development of novel treatments against respiratory bacterial infections. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sanitization of an Automatic Reverse-Osmosis Watering System: Removal of a Clinically Significant Biofilm

Science.gov (United States)

Molk, Denise M; Karr-May, Charlene L; Trang, Elaine D; Sanders, George E

2013-01-01

During environmental monitoring of our institution's rodent watering systems, one vivarium was found to have high bacterial loads in the reverse-osmosis (RO) automatic water system. These findings prompted evaluation of the entire RO water production and distribution system. Investigation revealed insufficient rack and RO system sanitization, leading to heavy biofilm accumulation within the system. Approximately 2 wk after discovery in the water system, one of the bacterial organisms isolated in the water supply, Sphingomonas paucimobilis, was isolated from a peritoneal abscess of a severely immunodeficient B6.Cg-Slc11a1r Rag1tm1Mom/Cwi mouse housed in the same vivarium, suggesting that rodents drinking from this system were being exposed randomly to fragments of biofilm. Plans were developed to sanitize the entire system. Hypercholorination was used first, followed by treatment with a combination of peracetic acid and hydrogen peroxide. Between system sanitizations, a low-level chlorine infusion was added to the system as a biocide. Heterotrophic plate counts and bacterial isolation were performed on water samples obtained before and after sanitization procedures. We here discuss the process of identifying and correcting this important water-quality issue. PMID:23562105

Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands.

Science.gov (United States)

Vercoe, Reuben B; Chang, James T; Dy, Ron L; Taylor, Corinda; Gristwood, Tamzin; Clulow, James S; Richter, Corinna; Przybilski, Rita; Pitman, Andrew R; Fineran, Peter C

2013-04-01

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas-mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA-targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.

Synthetic Decapeptide Enhances Bacterial Clearance and Accelerates Healing in the Wounds of Restraint-Stressed Mice

Science.gov (United States)

2012-02-06

reverse phase HPLC (series 1100; Hewlett Packard) on a Vydac C18 column. Peptide purity was con firmed by MALDI TOF (matrix assisted laser desorption...tissue surrounding the wounds, with the help of Micro Fine IV syringes 28G1/2 (Becton Dickinson). Careful wound manipulation ensured no direct ...Sheep blood agar plates. All bacterial assays were performed in triplicate. 2.12. Release kinetics of KSLW peptide in Pluronic F68 Measurements of

Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

International Nuclear Information System (INIS)

Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

1984-01-01

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate

[Bacterial prostatitis and prostatic fibrosis: modern view on the treatment and prophylaxis].

Science.gov (United States)

Zaitsev, A V; Pushkar, D Yu; Khodyreva, L A; Dudareva, A A

2016-08-01

Treatments of chronic bacterial prostatitis (CP) remain difficult problem. Bacterial prostatitis is a disease entity diagnosed clinically and by evidence of inflammation and infection localized to the prostate. Risk factors for UTI in men include urological interventions, such as transrectal prostate biopsy. Ensuing infections after prostate biopsy, such as UTI and bacterial prostatitis, are increasing due to increasing rates of fluoroquinolone resistance. The increasing global antibiotic resistance also significantly affects management of UTI in men, and therefore calls for alternative strategies. Prostatic inflammation has been suggested to contribute to the etiology of lower urinary tract symptoms (LUTS) by inducing fibrosis. Several studies have shown that prostatic fibrosis is strongly associated with impaired urethral function and LUTS severity. Fibrosis resulting from excessive deposition of collagen is traditionally recognized as a progressive irreversible condition and an end stage of inflammatory diseases; however, there is compelling evidence in both animal and human studies to support that the development of fibrosis could potentially be a reversible process. Prostate inflammation may induce fibrotic changes in periurethral prostatic tissues, promote urethral stiffness and LUTS. Patients experiencing CP and prostate-related LUTS could benefit from anti-inflammatory therapies, especially used in combination with the currently prescribed enzyme treatment with Longidase. Treatment results showed that longidase is highly effective in bacterial and abacterial CP. Longidase addition to standard therapeutic methods significantly reduced the disease symptoms and regression of inflammatory-proliferative alterations in the prostate.

Reverse Osmosis

Indian Academy of Sciences (India)

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

Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

Directory of Open Access Journals (Sweden)

Lei Jia

Full Text Available Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG and melting temperature change (dTm were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models.

Mutation Detection with Next-Generation Resequencing through a Mediator Genome

Energy Technology Data Exchange (ETDEWEB)

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

2010-12-31

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

Correlation of oxidative stress in patients with HBV-induced liver disease with HBV genotypes and drug resistance mutations.

Science.gov (United States)

Xianyu, Jianbo; Feng, Jiafu; Yang, Yuwei; Tang, Jie; Xie, Gang; Fan, Lingying

2018-05-01

This study aims to explore the correlation of oxidative stress (OxS) in patients with chronic hepatitis B (CHB) and the disease severity with HBV genotypes and drug resistance mutations. A total of 296 patients with CHB were enrolled into the study. PCR-reverse dot-blot hybridization was used to detect the HBV genotypes (B, C, and D) and the drug resistance-causing HBV mutant genes. In addition, the total oxidative stress (TOS) and total antioxidant status (TAS) were determined, and oxidative stress index (OSI) was calculated and compared. Serum levels of TOS and OSI, the B/C ratio, and drug resistance mutation rate were increased along with the elevated disease severity degree (CHBHBV mutation had higher serum TOS and OSI levels, while lower serum TAS levels (P HBV-induced liver disease, and the damage degree is correlated with the HBV genotype and drug resistance mutation. Oxidative stress might be a useful indicator of the progression of HBV-induced liver disease in patients. Copyright © 2018. Published by Elsevier Inc.

A second chance for telomerase reverse transcriptase in anticancer immunotherapy.

Science.gov (United States)

Zanetti, Maurizio

2017-02-01

Telomerase reverse transcriptase (TERT) is a self-antigen that is expressed constitutively in many tumours, and is, therefore, an important target for anticancer immunotherapy. In the past 10 years, trials of immunotherapy with TERT-based vaccines have demonstrated only modest benefits. In this Perspectives, I discuss the possible immunological reasons for this limited antitumour efficacy, and propose that advances in our understanding of the genetics and biology of the involvement of TERT in cancer provides the basis for renewed interest in TERT- based immunotherapy. Telomerase and TERT are expressed in cancer cells at every stage of tumour evolution, from the cancer stem cell to circulating tumour cells and tumour metastases. Many cancer types also harbour cells with mutations in the TERT promoter region, which increase transcriptional activation of this gene. These new findings should spur new interest in the development of TERT-based immunotherapies that are redesigned in line with established immunological considerations and working principles, and are tailored to patients stratified on the basis of TERT-promoter mutations and other underlying tumour characteristics. Thus, despite the disappointment of previous clinical trials, TERT offers the potential for personalized immunotherapy, perhaps in combination with immune-checkpoint inhibition.

The Bacterial Actin MamK

Science.gov (United States)

Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

2013-01-01

It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

Methane production in an anaerobic osmotic membrane bioreactor using forward osmosis: Effect of reverse salt flux

KAUST Repository

Li, Sheng

2017-05-10

This study investigated the impact of reverse salt flux (RSF) on microbe community and bio-methane production in a simulated fertilizer driven FO-AnMBR system using KCl, KNO3 and KH2PO4 as draw solutes. Results showed that KH2PO4 exhibited the lowest RSF in terms of molar concentration 19.1 mM/(m2.h), while for KCl and KNO3 it was 32.2 and 120.8 mM/(m2.h), respectively. Interestingly, bio-methane production displayed an opposite order with KH2PO4, followed by KCl and KNO3. Pyrosequencing results revealed the presence of different bacterial communities among the tested fertilizers. Bacterial community of sludge exposed to KH2PO4 was very similar to that of DI-water and KCl. However, results with KNO3 were different since the denitrifying bacteria were found to have a higher percentage than the sludge with other fertilizers. This study demonstrated that RSF has a negative effect on bio-methane production, probably by influencing the sludge bacterial community via environment modification.

TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma.

Directory of Open Access Journals (Sweden)

Karine Pallier

Full Text Available Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT. The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33 and showed that TWIST1 expression was linked to EGFR mutations (P<0.001, to low CDH1 expression (P<0.05 and low disease free survival (P = 0.044. To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.

Viral resuppression and detection of drug resistance following interruption of a suppressive non-nucleoside reverse transcriptase inhibitor-based regimen

DEFF Research Database (Denmark)

Fox, Zoe; Phillips, Andrew; Cohen, Cal

2008-01-01

the NRTIs, or by replacing the NNRTI with another drug before interruption. Simultaneous interruption of all antiretrovirals was discouraged. Resuppression rates 4-8 months after reinitiating NNRTI-therapy were assessed, as was the detection of drug-resistance mutations within 2 months of the treatment...... regimen. NNRTI drug-resistance mutations were observed in a relatively high proportion of patients. These data provide additional support for a staggered or switched interruption strategy for NNRTI drugs.......BACKGROUND: Interruption of a non-nucleoside reverse transcriptase inhibitor (NNRTI)-regimen is often necessary, but must be performed with caution because NNRTIs have a low genetic barrier to resistance. Limited data exist to guide clinical practice on the best interruption strategy to use...

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

Directory of Open Access Journals (Sweden)

Pedersen Niels C

2007-04-01

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

Report of Chinese family with severe dermatitis, multiple allergies and metabolic wasting syndrome caused by novel homozygous desmoglein-1 gene mutation.

Science.gov (United States)

Cheng, Ruhong; Yan, Ming; Ni, Cheng; Zhang, Jia; Li, Ming; Yao, Zhirong

2016-10-01

Recently, homozygous mutations in the desmoglein-1 (DSG1) gene and heterozygous mutation in the desmoplakin (DSP) gene have been demonstrated to be associated with severe dermatitis, multiple allergies and metabolic wasting (SAM) syndrome (Mendelian Inheritance in Man no. 615508). We aim to identify the molecular basis for a Chinese pedigree of SAM syndrome. A Chinese pedigree of SAM syndrome was subjected to mutation detection in the DSG1 gene. Sequence analysis of the DSG1 gene and quantitative reverse transcriptase polymerase chain reaction analysis for gene expression of DSG1 using cDNA derived from the epidermis of patients and controls were both performed. Skin biopsies were also taken from patients for pathological study and transmission electron microscopy observation. Novel homozygous splicing mutation c.1892-1delG in the exon-intron border of the DSG1 gene has been demonstrated to be associated with SAM syndrome. We report a new family of SAM syndrome of Asian decent and expand the spectrum of mutations in the DSG1 gene. © 2016 Japanese Dermatological Association.

Hypertrophic Cardiomyopathy: A Vicious Cycle Triggered by Sarcomere Mutations and Secondary Disease Hits.

Science.gov (United States)

Wijnker, Paul J M; Sequeira, Vasco; Kuster, Diederik W D; Velden, Jolanda van der

2018-04-11

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction, and myocardial disarray. Disease onset occurs between 20 and 50 years of age, thus affecting patients in the prime of their life. HCM is caused by mutations in sarcomere proteins, the contractile building blocks of the heart. Despite increased knowledge of causal mutations, the exact path from genetic defect leading to cardiomyopathy is complex and involves additional disease hits. Recent Advances: Laboratory-based studies indicate that HCM development not only depends on the primary sarcomere impairment caused by the mutation but also on secondary disease-related alterations in the heart. Here we propose a vicious mutation-induced disease cycle, in which a mutation-induced energy depletion alters cellular metabolism with increased mitochondrial work, which triggers secondary disease modifiers that will worsen disease and ultimately lead to end-stage HCM. Evidence shows excessive cellular reactive oxygen species (ROS) in HCM patients and HCM animal models. Oxidative stress markers are increased in the heart (oxidized proteins, DNA, and lipids) and serum of HCM patients. In addition, increased mitochondrial ROS production and changes in endogenous antioxidants are reported in HCM. Mutant sarcomeric protein may drive excessive levels of cardiac ROS via changes in cardiac efficiency and metabolism, mitochondrial activation and/or dysfunction, impaired protein quality control, and microvascular dysfunction. Interventions restoring metabolism, mitochondrial function, and improved ROS balance may be promising therapeutic approaches. We discuss the effects of current HCM pharmacological therapies and potential future therapies to prevent and reverse HCM. Antioxid. Redox Signal. 00, 000-000.

DamX Controls Reversible Cell Morphology Switching in Uropathogenic Escherichia coli

DEFF Research Database (Denmark)

Khandige, Surabhi; Antoinette Asferg, Cecilie; Rasmussen, Karina Juhl

2016-01-01

undertaking targeted investigations that are challenging to perform in animal infection models. IMPORTANCE: Urinary tract infections (UTIs) are most often caused by uropathogenic Escherichia coli (UPEC) and account for a considerable health care burden. UPEC exhibits a dynamic lifestyle in the course....... In aiming to uncover genes underlying the phenomenon of UPEC morphotype switching, this study identifies damX, a cell division gene, as a mediator of reversible filamentation during UTI. DamX-mediated filamentation represents an additional pathway for bacterial cell shape control, an alternative to Sul......A-mediated FtsZ sequestration during E. coli uropathogenesis, and hence represents a potential target for combating UTI....

Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries.

Science.gov (United States)

Toussay, Xavier; Morel, Jean-Luc; Biendon, Nathalie; Rotureau, Lolita; Legeron, François-Pierre; Boutonnet, Marie-Charlotte; Cho, Yoon H; Macrez, Nathalie

2017-10-01

Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca 2+ ) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca 2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca 2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca 2+ -release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca 2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca 2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca 2+ signals in PS1dE9 mutant mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile, and accurate RNA structure analysis

Science.gov (United States)

Smola, Matthew J.; Rice, Greggory M.; Busan, Steven; Siegfried, Nathan A.; Weeks, Kevin M.

2016-01-01

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures. PMID:26426499

Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin.

Science.gov (United States)

Lin, Dachuan; Chen, Kaichao; Li, Ruichao; Liu, Lizhang; Guo, Jiubiao; Yao, Wen; Chen, Sheng

2014-01-01

It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry.

PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens

DEFF Research Database (Denmark)

Zankari, Ea; Allesøe, Rosa Lundbye; Joensen, Katrine Grimstrup

2017-01-01

enterica, Escherichia coli and Campylobacter jejuni. The web-server ResFinder-2.1 was used to identify acquired antimicrobial resistance genes and two methods, the novel PointFinder (using BLAST) and an in-house method (mapping of raw WGS reads), were used to identify chromosomal point mutations. Results...... or when mapping the reads. Conclusions PointFinder proved, with high concordance between phenotypic and predicted antimicrobial susceptibility, to be a user-friendly web tool for detection of chromosomal point mutations associated with antimicrobial resistance....

Genetic and rat toxicity studies of cyclodextrin glucanotransferase

Directory of Open Access Journals (Sweden)

Robert R. Maronpot

Full Text Available Introduction: Microbiologically derived cyclodextrin glucanotransferase (CGTase is used commercially as a processing agent in manufacture of food, pharmaceuticals, and cosmetics. Its toxic potential was evaluated in anticipation of use in the production of alpha-glycosyl isoquercitrin, a water-soluble form of quercetin. Methods: Following OECD guidelines, CGTase, produced by Bacillus pseudalcaliphilus DK-1139, was evaluated in a genotoxicity battery consisting of a bacterial reverse mutation assay, an in vitro micronucleus (MN assay and MN and comet assays using B6C3F1 male and female mice. These same genotoxicity assays were also conducted for sodium sulfate, a contaminant of CGTase preparation. In a 90-day Sprague Dawley rat toxicity study, CGTase was administered by gavage in water at daily doses of 0, 250, 500, and 1000 mg/kg/day. Results: CGTase did not induce mutations with or without metabolic activation in the bacterial reverse mutation assay. Formation of micronuclei was not induced in either in vitro or in vivo MN assays with or without metabolic activation. No induction of DNA damage was detected in male or female mouse liver, stomach, or duodenum in the comet assay. Sodium sulfate also tested negative in these same genotoxicity assays. In the 90-day repeated dose rat study there were no treatment-related adverse clinical or pathological findings. Conclusion: The genotoxicity assays and repeated dose toxicity study support the safe use of CGTase in production of alpha-glycosyl isoquercitrin. Keywords: Micronucleus assay, Comet assay, Enzymatically modified isoquercitrin (EMIQ, Food additive, Flavonol, Sodium sulfate

Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes.

Science.gov (United States)

Santini, Alessandro; Ronchi, Dario; Garbellini, Manuela; Piga, Daniela; Protti, Alessandro

2017-07-01

Linezolid inhibits bacterial growth by targeting bacterial ribosomes and by interfering with bacterial protein synthesis. Lactic acidosis is a rare, but potentially lethal, side effect of linezolid. Areas covered: The pathogenesis of linezolid-induced lactic acidosis is reviewed with special emphasis on aspects relevant to the recognition, prevention and treatment of the syndrome. Expert opinion: Linezolid-induced lactic acidosis reflects the untoward interaction between the drug and mitochondrial ribosomes. The inhibition of mitochondrial protein synthesis diminishes the respiratory chain enzyme content and thus limits aerobic energy production. As a result, anaerobic glycolysis and lactate generation accelerate independently from tissue hypoxia. In the absence of any confirmatory test, linezolid-induced lactic acidosis should be suspected only after exclusion of other, more common, causes of lactic acidosis such as hypoxemia, anemia or low cardiac output. Normal-to-high whole-body oxygen delivery, high venous oxygen saturation and lack of response to interventions that effectively increase tissue oxygen provision all suggest a primary defect in oxygen use at the mitochondrial level. During prolonged therapy with linezolid, blood drug and lactate levels should be regularly monitored. The current standard-of-care treatment of linezolid-induced lactic acidosis consists of drug withdrawal to reverse mitochondrial intoxication and intercurrent life support.

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

DEFF Research Database (Denmark)

Urbanski, Dorian Fabian

2011-01-01

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

Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

International Nuclear Information System (INIS)

Dumaz, N.; Drougard, C.; Sarasin, A.; Daya-Grosjean, L.

1993-01-01

The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a good target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC → TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues

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

DEFF Research Database (Denmark)

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

2001-01-01

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

Conserved water molecules in bacterial serine hydroxymethyltransferases.

Science.gov (United States)

Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

2015-10-01

Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

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Ana Luiza Chaves Valadão

2015-06-01

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

The Arabidopsis thaliana rlp mutations revert the ectopic leaf blade formation conferred by activation tagging of the LEP gene

DEFF Research Database (Denmark)

van der Graaff, Eric; Nussbaumer, C; Keller, Bente

2003-01-01