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Sample records for affect dna integrity

  1. Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

    PubMed

    Habermeyer, Michael; Fritz, Jessica; Barthelmes, Hans U; Christensen, Morten O; Larsen, Morten K; Boege, Fritz; Marko, Doris

    2005-09-01

    In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM). PMID:16167831

  2. Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response

    PubMed Central

    Moreli, Jusciele B.; Santos, Janine H.; Lorenzon-Ojea, Aline Rodrigues; Corrêa-Silva, Simone; Fortunato, Rodrigo S.; Rocha, Clarissa Ribeiro; Rudge, Marilza V.; Damasceno, Débora C.; Bevilacqua, Estela; Calderon, Iracema M.

    2016-01-01

    Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ and FEN1). A diabetes-like environment was unable to induce apoptosis in the umbilical cord blood cells. Conclusions: Our data show relevant asymmetry between maternal and fetal blood cell susceptibility to DNA damage and apoptosis induction. Maternal cells seem to be more predisposed to changes in an adverse glucose environment. This may be due to differential ability in upregulating multiple genes involved in the activation of DNA repair response, especially the BER mechanism. However if this study shows a more effective adaptive response by the fetal organism, it also calls for

  3. Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro

    PubMed Central

    Salian, Sujith Raj; Kumar, Dayanidhi; Singh, Vikram Jeet; D’Souza, Fiona; Kalthur, Guruprasad; Kamath, Asha; Adiga, Satish Kumar

    2016-01-01

    Background The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome. Objective To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. Methods This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA. Results Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, in vitro incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001). Conclusion Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes. PMID:27043437

  4. DNA Storage under High Temperature Conditions Does Not Affect Performance in Human Leukocyte Antigen Genotyping via Next-Generation Sequencing (DNA Integrity Maintained in Extreme Conditions)

    PubMed Central

    McDevitt, Shana L; Hogan, Michael E; Pappas, Derek J; Wong, Lily Y

    2014-01-01

    Background: Stable dry-state storage of DNA is desirable to minimize required storage space and to reduce electrical and shipping costs. DNA purified from various commercially available dry-state stabilization matrices has been used successfully in downstream molecular applications (e.g., quantitative polymerase chain reaction [qPCR], microarray, and sequence-based genotyping). However, standard DNA storage conditions still include freezing of DNA eluted in aqueous buffers or nuclease-free water. Broad implementation of dry-state, long-term DNA storage requires enhancement of such dry-state DNA stabilization products to control for temperature fluctuations at specimen collection, transit, and storage. This study tested the integrity of genomic DNA subjected to long-term storage on GenTegra™ DNA stabilization matrices (GenTegra LLC, Pleasanton, CA) at extreme conditions, as defined by a 4-year storage period at ambient temperature with an initial incubation for 7 months at 37°C, 56°C, or ambient temperature. Subsequently, purified DNA performance and integrity were measured by qPCR and next-generation sequencing (NGS)-based human leokocyte antigen (HLA) genotyping. Results: High molecular weight genomic DNA samples were recovered from the GenTegra product matrix and exhibited integrity comparable to a highly characterized commercial standard under assessment by qPCR. Samples were genotyped for classical HLA loci using next generation sequencing-based methodolgy on the Roche 454 GS Junior instrument. Amplification efficiency, sequence coverage, and sequence quality were all comparable with those produced from a cell line DNA sequenced as a control. No significant differences were observed in the mean, median, or mode quality scores between samples and controls (p≥0.4). Conclusions: Next generation HLA genotyping was chosen to test the integrity of GenTegra-treated genomic DNA due to the requirment for long sequence reads to genotype the highly polymorphic

  5. How Do Structure and Charge Affect Metal-Complex Binding to DNA? An Upper-Division Integrated Laboratory Project Using Cyclic Voltammetry

    ERIC Educational Resources Information Center

    Kulczynska, Agnieszka; Johnson, Reed; Frost, Tony; Margerum, Lawrence D.

    2011-01-01

    An advanced undergraduate laboratory project is described that integrates inorganic, analytical, physical, and biochemical techniques to reveal differences in binding between cationic metal complexes and anionic DNA (herring testes). Students were guided to formulate testable hypotheses based on the title question and a list of different metal…

  6. Sequence Affects the Cyclization of DNA Minicircles.

    PubMed

    Wang, Qian; Pettitt, B Montgomery

    2016-03-17

    Understanding how the sequence of a DNA molecule affects its dynamic properties is a central problem affecting biochemistry and biotechnology. The process of cyclizing short DNA, as a critical step in molecular cloning, lacks a comprehensive picture of the kinetic process containing sequence information. We have elucidated this process by using coarse-grained simulations, enhanced sampling methods, and recent theoretical advances. We are able to identify the types and positions of structural defects during the looping process at a base-pair level. Correlations along a DNA molecule dictate critical sequence positions that can affect the looping rate. Structural defects change the bending elasticity of the DNA molecule from a harmonic to subharmonic potential with respect to bending angles. We explore the subelastic chain as a possible model in loop formation kinetics. A sequence-dependent model is developed to qualitatively predict the relative loop formation time as a function of DNA sequence. PMID:26938490

  7. Microfluidic-integrated DNA nanobiosensors.

    PubMed

    Ansari, M I Haque; Hassan, Shabir; Qurashi, Ahsanulhaq; Khanday, Firdous Ahmad

    2016-11-15

    Over the last few decades, an increased demand has emerged for integrating biosensors with microfluidic- and nanofluidic-based lab-on-chip (LOC) devices for point-of-care (POC) diagnostics, in the medical industry and environmental monitoring of pathogenic threat agents. Such a merger of microfluidics with biosensing technologies allows for the precise control of volumes, as low as one nanolitre and the integration of various types of bioassays on a single miniaturized platform. This integration offers several favorable advantages, such as low reagent consumption, automation of sample preparation, reduction in processing time, low cost analysis, minimal handling of hazardous materials, high detection accuracy, portability and disposability. This review provides a synopsis of the most recent developments in the microfluidic-integrated biosensing field by delineating the fundamental theory of microfluidics, fabrication techniques and a detailed account of the various transduction methods that are employed. Lastly, the review discusses state-of-the-art DNA biosensors with a focus on optical DNA biosensors. PMID:27179566

  8. Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

    PubMed

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2016-08-22

    Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs. PMID:27430161

  9. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  10. Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription

    PubMed Central

    Kraeva, Rayna I.; Krastev, Dragomir B.; Roguev, Assen; Ivanova, Anna; Nedelcheva-Veleva, Marina N.; Stoynov, Stoyno S.

    2007-01-01

    Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3′-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3′-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription. PMID:17356699

  11. Does Attention Affect Visual Feature Integration?

    ERIC Educational Resources Information Center

    Prinzmetal, William; And Others

    This work investigates, first, whether the integration of color and shape information is affected by attending to the stimulus location, and second, whether attending to a stimulus location enhances the perceptual representation of the stimulus or merely affects decision processes. In three experiments, subjects were briefly presented with colored…

  12. Solar ultraviolet-B radiation affects seedling emergence, DNA integrity, plant morphology, growth rate, and attractiveness to herbivore insects in Datura ferox

    SciTech Connect

    Ballare, C.L.; Scopel, A.L.; Stapleton, A.E.

    1996-09-01

    To study functional relationships between the effects of solar ultraviolet-B radiation (UV0B) on different aspects of the physiology of a wild plant, we carried out exclusion experiments in the field with the summer annual Datura ferrox L. Solar UV-B incident over Buenos Aires reduced daytime seedling emergence, inhibited stem elongation and leaf expansion, and tended to reduce biomass accumulation during early growth. However, UV-B had no effect on calculated net assimilation rate. Using a monoclonal antibody specific to the cyclobutane-pyrimidine dimer (CPD), we found that plants receiving full sunlight had more CPDs per unit of DNA than plants shielded from solar UV-B, but the positive correlation between UV-B and CPD burden tended to level off at high (near solar) UV-B levels. At our field site, Datura plants were consumed by leaf beetles (Coleoptera), and the proportion of plants attacked by insects declined with the amount of UV-B received during growth. Field experiments showed that plant exposure to solar UV-B reduced the likelihood of leaf beetle attack by one-half. Our results highlight the complexities associated with scaling plant responses to solar UV-B, because they show: (a) a lack of correspondence between UV-B effects on net assimilation rate and whole-plant growth rate, (b) nonlinear UV-B dose-response curves, and (c) UV-B effects of plant attractiveness to natural herbivores. 56 refs., 7 figs.

  13. Solar Ultraviolet-B Radiation Affects Seedling Emergence, DNA Integrity, Plant Morphology, Growth Rate, and Attractiveness to Herbivore Insects in Datura ferox.

    PubMed Central

    Ballare, C. L.; Scopel, A. L.; Stapleton, A. E.; Yanovsky, M. J.

    1996-01-01

    To study functional relationships between the effects of solar ultraviolet-B radiation (UV-B) on different aspects of the physiology of a wild plant, we carried out exclusion experiments in the field with the summer annual Datura ferox L. Solar UV-B incident over Buenos Aires reduced daytime seedling emergence, inhibited stem elongation and leaf expansion, and tended to reduce biomass accumulation during early growth. However, UV-B had no effect on calculated net assimilation rate. Using a monoclonal antibody specific to the cyclobutane-pyrimidine dimer (CPD), we found that plants receiving full sunlight had more CPDs per unit of DNA than plants shielded from solar UV-B, but the positive correlation between UV-B and CPD burden tended to level off at high (near solar) UV-B levels. At our field site, Datura plants were consumed by leaf beetles (Coleoptera), and the proportion of plants attacked by insects declined with the amount of UV-B received during growth. Field experiments showed that plant exposure to solar UV-B reduced the likelihood of leaf beetle attack by one-half. Our results highlight the complexities associated with scaling plant responses to solar UV-B, because they show: (a) a lack of correspondence between UV-B effects on net assimilation rate and whole-plant growth rate, (b) nonlinear UV-B dose-response curves, and (c) UV-B effects of plant attractiveness to natural herbivores. PMID:12226382

  14. Affect integration in dreams and dreaming.

    PubMed

    Grenell, Gary

    2008-03-01

    The processes by which dreaming aids in the ongoing integration of affects into the mind are approached here from complementary psychoanalytic and nonpsychoanalytic perspectives. One relevant notion is that the dream provides a psychological space wherein overwhelming, contradictory, or highly complex affects that under waking conditions are subject to dissociation, splitting, or disavowal may be brought together for observation by the dreaming ego. This process serves the need for psychological balance and equilibrium. A brief discussion of how the mind processes information during dreaming is followed by a consideration of four component aspects of the integrative process: the nature and use of the dream-space, the oscillating "me / not me" quality of the dream, the apparent reality of the dream, and the use of nonpathological projective identification in dreaming. Three clinical illustrations are offered and discussed. PMID:18430709

  15. Pentoxifylline affects idarubicin binding to DNA.

    PubMed

    Gołuński, Grzegorz; Borowik, Agnieszka; Lipińska, Andrea; Romanik, Monika; Derewońko, Natalia; Woziwodzka, Anna; Piosik, Jacek

    2016-04-01

    Anticancer drug idarubicin - derivative of doxorubicin - is commonly used in treatment of numerous cancer types. However, in contrast to doxorubicin, its biophysical properties are not well established yet. Additionally, potential direct interactions of idarubicin with other biologically active aromatic compounds, such as pentoxifylline - representative of methylxanthines - were not studied at all. Potential formation of such hetero-aggregates may result in sequestration of the anticancer drug and, in consequence, reduction of its biological activity. This work provide description of the idarubicin biophysical properties as well as assess influence of pentoxifylline on idarubicin interactions with DNA. To achieve these goals we employed spectrophotometric methods coupled with analysis with the appropriate mathematical models as well as flow cytometry and Ames test. Obtained results show influence of pentoxifylline on idarubicin binding to DNA and are well in agreement with the data previously published for other aromatic ligands. Additionally it may be hypothesized that direct interactions between idarubicin and pentoxifylline may influence the anticancer drug biological activity. PMID:26921593

  16. Integrated microfluidic systems for DNA analysis.

    PubMed

    Njoroge, Samuel K; Chen, Hui-Wen; Witek, Małgorzata A; Soper, Steven A

    2011-01-01

    The potential utility of genome-related research in terms of evolving basic discoveries in biology has generated widespread use of DNA diagnostics and DNA forensics and driven the accelerated development of fully integrated microfluidic systems for genome processing. To produce a microsystem with favorable performance characteristics for genetic-based analyses, several key operational elements must be strategically chosen, including device substrate material, temperature control, fluidic control, and reaction product readout. As a matter of definition, a microdevice is a chip that performs a single processing step, for example microchip electrophoresis. Several microdevices can be integrated to a single wafer, or combined on a control board as separate devices to form a microsystem. A microsystem is defined as a chip composed of at least two microdevices. Among the many documented analytical microdevices, those focused on the ability to perform the polymerase chain reaction (PCR) have been reported extensively due to the importance of this processing step in most genetic-based assays. Other microdevices that have been detailed in the literature include those for solid-phase extractions, microchip electrophoresis, and devices composed of DNA microarrays used for interrogating DNA primary structure. Great progress has also been made in the areas of chip fabrication, bonding and sealing to enclose fluidic networks, evaluation of different chip substrate materials, surface chemistries, and the architecture of reaction conduits for basic processing steps such as mixing. Other important elements that have been developed to realize functional systems include miniaturized readout formats comprising optical or electrochemical transduction and interconnect technologies. These discoveries have led to the development of fully autonomous and functional integrated systems for genome processing that can supply "sample in/answer out" capabilities. In this chapter, we focus on

  17. Integrating data from heterogeneous DNA microarray platforms.

    PubMed

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus. PMID:26673932

  18. Microfabricated structures for integrated DNA analysis.

    PubMed Central

    Burns, M A; Mastrangelo, C H; Sammarco, T S; Man, F P; Webster, J R; Johnsons, B N; Foerster, B; Jones, D; Fields, Y; Kaiser, A R; Burke, D T

    1996-01-01

    Photolithographic micromachining of silicon is a candidate technology for the construction of high-throughput DNA analysis devices. However, the development of complex silicon microfabricated systems has been hindered in part by the lack of a simple, versatile pumping method for integrating individual components. Here we describe a surface-tension-based pump able to move discrete nanoliter drops through enclosed channels using only local heating. This thermocapillary pump can accurately mix, measure, and divide drops by simple electronic control. In addition, we have constructed thermal-cycling chambers, gel electrophoresis channels, and radiolabeled DNA detectors that are compatible with the fabrication of thermocapillary pump channels. Since all of the components are made by conventional photolithographic techniques, they can be assembled into more complex integrated systems. The combination of pump and components into self-contained miniaturized devices may provide significant improvements in DNA analysis speed, portability, and cost. The potential of microfabricated systems lies in the low unit cost of silicon-based construction and in the efficient sample handling afforded by component integration. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643614

  19. One-step cloning and chromosomal integration of DNA.

    PubMed

    St-Pierre, François; Cui, Lun; Priest, David G; Endy, Drew; Dodd, Ian B; Shearwin, Keith E

    2013-09-20

    We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids. PMID:24050148

  20. Path integral method for DNA denaturation

    NASA Astrophysics Data System (ADS)

    Zoli, Marco

    2009-04-01

    The statistical physics of homogeneous DNA is investigated by the imaginary time path integral formalism. The base pair stretchings are described by an ensemble of paths selected through a macroscopic constraint, the fulfillment of the second law of thermodynamics. The number of paths contributing to the partition function strongly increases around and above a specific temperature Tc∗ , whereas the fraction of unbound base pairs grows continuously around and above Tc∗ . The latter is identified with the denaturation temperature. Thus, the separation of the two complementary strands appears as a highly cooperative phenomenon displaying a smooth crossover versus T . The thermodynamical properties have been computed in a large temperature range by varying the size of the path ensemble at the lower bound of the range. No significant physical dependence on the system size has been envisaged. The entropy grows continuously versus T while the specific heat displays a remarkable peak at Tc∗ . The location of the peak versus T varies with the stiffness of the anharmonic stacking interaction along the strand. The presented results suggest that denaturation in homogeneous DNA has the features of a second-order phase transition. The method accounts for the cooperative behavior of a very large number of degrees of freedom while the computation time is kept within a reasonable limit.

  1. Integrating the Affective Domain into the Instructional Design Process.

    ERIC Educational Resources Information Center

    Main, Robert G.

    This study begins with a definition of the affective domain and its importance to learning, outlining its impact both in achieving affective behaviors and in facilitating cognitive and psychomotor objectives. The study then develops a model of instructional design that incorporates the affective domain as an integral component. The model combines…

  2. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

    PubMed Central

    Petersen, J; Dandri, M; Bürkle, A; Zhang, L; Rogler, C E

    1997-01-01

    Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency. PMID

  3. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa

    PubMed Central

    Mahmoud, K. Gh. M.; El-Sokary, A. A. E.; Abdel-Ghaffar, A. E.; Abou El-Roos, M. E. A.; Ahmed, Y. F.

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  4. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa.

    PubMed

    Mahmoud, K Gh M; El-Sokary, A A E; Abdel-Ghaffar, A E; Abou El-Roos, M E A; Ahmed, Y F

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  5. The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

    PubMed Central

    Roy, Upasana; Mukherjee, Shivam; Sharma, Anjali; Frank, Ekaterina G.; Schärer, Orlando D.

    2016-01-01

    Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases. PMID:27257072

  6. An integrated workflow for DNA methylation analysis.

    PubMed

    Li, Pingchuan; Demirci, Feray; Mahalingam, Gayathri; Demirci, Caghan; Nakano, Mayumi; Meyers, Blake C

    2013-05-20

    The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes. DNA methylation has a demonstrated role in the genome stability and protection, regulation of gene expression and many other aspects of genome function and maintenance. BS-seq is a relatively unbiased method for profiling the DNA methylation, with a resolution capable of measuring methylation at individual cytosines. Here we describe, as an example, a workflow to handle DNA methylation analysis, from BS-seq library preparation to the data visualization. We describe some applications for the analysis and interpretation of these data. Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu), along with small RNA, RNA-seq and other data types. PMID:23706300

  7. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

  8. The Arithmetic of Emotion: Integration of Incidental and Integral Affect in Judgments and Decisions

    PubMed Central

    Västfjäll, Daniel; Slovic, Paul; Burns, William J.; Erlandsson, Arvid; Koppel, Lina; Asutay, Erkin; Tinghög, Gustav

    2016-01-01

    Research has demonstrated that two types of affect have an influence on judgment and decision making: incidental affect (affect unrelated to a judgment or decision such as a mood) and integral affect (affect that is part of the perceiver’s internal representation of the option or target under consideration). So far, these two lines of research have seldom crossed so that knowledge concerning their combined effects is largely missing. To fill this gap, the present review highlights differences and similarities between integral and incidental affect. Further, common and unique mechanisms that enable these two types of affect to influence judgment and choices are identified. Finally, some basic principles for affect integration when the two sources co-occur are outlined. These mechanisms are discussed in relation to existing work that has focused on incidental or integral affect but not both. PMID:27014136

  9. The Arithmetic of Emotion: Integration of Incidental and Integral Affect in Judgments and Decisions.

    PubMed

    Västfjäll, Daniel; Slovic, Paul; Burns, William J; Erlandsson, Arvid; Koppel, Lina; Asutay, Erkin; Tinghög, Gustav

    2016-01-01

    Research has demonstrated that two types of affect have an influence on judgment and decision making: incidental affect (affect unrelated to a judgment or decision such as a mood) and integral affect (affect that is part of the perceiver's internal representation of the option or target under consideration). So far, these two lines of research have seldom crossed so that knowledge concerning their combined effects is largely missing. To fill this gap, the present review highlights differences and similarities between integral and incidental affect. Further, common and unique mechanisms that enable these two types of affect to influence judgment and choices are identified. Finally, some basic principles for affect integration when the two sources co-occur are outlined. These mechanisms are discussed in relation to existing work that has focused on incidental or integral affect but not both. PMID:27014136

  10. Roles of DNA helicases in the maintenance of genome integrity

    PubMed Central

    Bochman, Matthew L

    2014-01-01

    Genome integrity is achieved and maintained by the sum of all of the processes in the cell that ensure the faithful duplication and repair of DNA, as well as its genetic transmission from one cell division to the next. As central players in virtually all of the DNA transactions that occur in vivo, DNA helicases (molecular motors that unwind double-stranded DNA to produce single-stranded substrates) represent a crucial enzyme family that is necessary for genomic stability. Indeed, mutations in many human helicase genes are linked to a variety of diseases with symptoms that can be generally described as genomic instability, such as predispositions to cancers. This review focuses on the roles of both DNA replication helicases and recombination/repair helicases in maintaining genome integrity and provides a brief overview of the diseases related to defects in these enzymes. PMID:27308340

  11. Integrating DNA strand-displacement circuitry with DNA tile self-assembly

    PubMed Central

    Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

    2013-01-01

    DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

  12. DNA integrity determination in marine invertebrates by Fast Micromethod.

    PubMed

    Jaksić, Zeljko; Batel, Renato

    2003-12-10

    This study was focused toward the adaptation of the previously developed Fast Micromethod for DNA damage determination to marine invertebrates for the establishment of biomonitoring assessment. The Fast Micromethod detects DNA damage (strand breaks, alkali-labile sites and incomplete excision repair) and determines DNA integrity in cell suspensions or tissue homogenates in single microplates. The procedure is based on the ability of the specific fluorochrome dye PicoGreen to preferentially interact with high integrity DNA molecules, dsDNA, in the presence of ssDNA and proteins in high alkaline medium, thereby allowing direct fluorometric measurements of dsDNA denaturation without sample handling and stepwise DNA separations. The results presented herein describe the influence of the DNA amount and the pH of the denaturation media on slopes of the kinetic denaturation curves and calculated strand scission factors (SSFs). The optimal amount of DNA in Mytilus galloprovincialis gills homogenate was found to be 100 ng ml(-1) and the greatest differences in DNA unwinding kinetics (slopes and SSF values) were reached at pH 11.5. The induction of DNA damage and loss of DNA integrity was measured in native DNA isolated from cotton-spinner Holothuria tubulosa, marine sponge Suberites domuncula cells and mussel M. galloprovincialis gills homogenate. DNA damage and loss of DNA integrity were detected after induction by different doses of (gamma-rays, generated by 137Cs 1800 Ci; 0-500 rad in marine sponge S. domuncula cells up to SSFx(-1) values 0.082 +/- 0.012 for the highest radiation dose). Analysis by chemical xenobiotics based on the in vitro action of bleomycin (bleomycin-Fe(II) complex 0-50 or 0-83 microg ml(-1) (microM)) with native DNA from cotton-spinner H. tubulosa and mussel M. galloprovincialis gills homogenate yielded values of 0.537 +/- 0.072 and 0.130 +/- 0.018, respectively. In vivo experiments with mussel M. galloprovincialis gills homogenate by 4

  13. Viral Carcinogenesis: Factors Inducing DNA Damage and Virus Integration

    PubMed Central

    Chen, Yan; Williams, Vonetta; Filippova, Maria; Filippov, Valery; Duerksen-Hughes, Penelope

    2014-01-01

    Viruses are the causative agents of 10%–15% of human cancers worldwide. The most common outcome for virus-induced reprogramming is genomic instability, including accumulation of mutations, aberrations and DNA damage. Although each virus has its own specific mechanism for promoting carcinogenesis, the majority of DNA oncogenic viruses encode oncogenes that transform infected cells, frequently by targeting p53 and pRB. In addition, integration of viral DNA into the human genome can also play an important role in promoting tumor development for several viruses, including HBV and HPV. Because viral integration requires the breakage of both the viral and the host DNA, the integration rate is believed to be linked to the levels of DNA damage. DNA damage can be caused by both endogenous and exogenous factors, including inflammation induced by either the virus itself or by co-infections with other agents, environmental agents and other factors. Typically, cancer develops years to decades following the initial infection. A better understanding of virus-mediated carcinogenesis, the networking of pathways involved in transformation and the relevant risk factors, particularly in those cases where tumorigenesis proceeds by way of virus integration, will help to suggest prophylactic and therapeutic strategies to reduce the risk of virus-mediated cancer. PMID:25340830

  14. FOB1 affects DNA topoisomerase I in vivo cleavages in the enhancer region of the Saccharomyces cerevisiae ribosomal DNA locus

    PubMed Central

    Di Felice, Francesca; Cioci, Francesco; Camilloni, Giorgio

    2005-01-01

    In Saccharomyces cerevisiae the FOB1 gene affects replication fork blocking activity at the replication fork block (RFB) sequences and promotes recombination events within the rDNA cluster. Using in vivo footprinting assays we mapped two in vivo Fob1p-binding sites, RFB1 and RFB3, located in the rDNA enhancer region and coincident with those previously reported to be in vitro binding sites. We previously provided evidences that DNA topoisomerase I is able to cleave two sites within this region. The results reported in this paper, indicate that the DNA topoisomerase I cleavage specific activity at the enhancer region is affected by the presence of Fob1p and independent of replication and transcription activities. We thus hypothesize that the binding to DNA of Fob1p itself may be the cause of the DNA topoisomerase I activity in the rDNA enhancer. PMID:16269824

  15. Mutants affecting nucleotide recognition by T7 DNA polymerase.

    PubMed

    Donlin, M J; Johnson, K A

    1994-12-13

    Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in

  16. Quadruplex Integrated DNA (QuID) Nanosensors for Monitoring Dopamine

    PubMed Central

    Morales, Jennifer M.; Skipwith, Christopher G.; Clark, Heather A.

    2015-01-01

    Dopamine is widely innervated throughout the brain and critical for many cognitive and motor functions. Imbalances or loss in dopamine transmission underlie various psychiatric disorders and degenerative diseases. Research involving cellular studies and disease states would benefit from a tool for measuring dopamine transmission. Here we show a Quadruplex Integrated DNA (QuID) nanosensor platform for selective and dynamic detection of dopamine. This nanosensor exploits DNA technology and enzyme recognition systems to optically image dopamine levels. The DNA quadruplex architecture is designed to be compatible in physically constrained environments (110 nm) with high flexibility, homogeneity, and a lower detection limit of 110 µM. PMID:26287196

  17. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    PubMed Central

    Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual’s DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  18. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    PubMed

    Radenbaugh, Amie J; Ma, Singer; Ewing, Adam; Stuart, Joshua M; Collisson, Eric A; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  19. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  20. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. PMID:26091838

  1. Integrating prokaryotes and eukaryotes: DNA transposases in light of structure.

    PubMed

    Hickman, Alison Burgess; Chandler, Michael; Dyda, Fred

    2010-02-01

    DNA rearrangements are important in genome function and evolution. Genetic material can be rearranged inadvertently during processes such as DNA repair, or can be moved in a controlled manner by enzymes specifically dedicated to the task. DNA transposases comprise one class of such enzymes. These move DNA segments known as transposons to new locations, without the need for sequence homology between transposon and target site. Several biochemically distinct pathways have evolved for DNA transposition, and genetic and biochemical studies have provided valuable insights into many of these. However, structural information on transposases - particularly with DNA substrates - has proven elusive in most cases. On the other hand, large-scale genome sequencing projects have led to an explosion in the number of annotated prokaryotic and eukaryotic mobile elements. Here, we briefly review biochemical and mechanistic aspects of DNA transposition, and propose that integrating sequence information with structural information using bioinformatics tools such as secondary structure prediction and protein threading can lead not only to an additional level of understanding but possibly also to testable hypotheses regarding transposition mechanisms. Detailed understanding of transposition pathways is a prerequisite for the long-term goal of exploiting DNA transposons as genetic tools and as a basis for genetic medical applications. PMID:20067338

  2. How Do Volcanoes Affect Human Life? Integrated Unit.

    ERIC Educational Resources Information Center

    Dayton, Rebecca; Edwards, Carrie; Sisler, Michelle

    This packet contains a unit on teaching about volcanoes. The following question is addressed: How do volcanoes affect human life? The unit covers approximately three weeks of instruction and strives to present volcanoes in an holistic form. The five subject areas of art, language arts, mathematics, science, and social studies are integrated into…

  3. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    PubMed

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. PMID:24861204

  4. Microblogging for Class: An Analysis of Affective, Cognitive, Personal Integrative, and Social Integrative Gratifications

    ERIC Educational Resources Information Center

    Gant, Camilla; Hadley, Patrick D.

    2014-01-01

    This study shows that undergraduate students can gratify cognitive, affective, social integrative, and personal integrative needs microblogging via a learning management system discussion tool. Moreover, the researchers find that microblogging about news regarding mass media events and issues via Blackboard heightened engagement, expanded…

  5. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

    PubMed Central

    Folger, K R; Wong, E A; Wahl, G; Capecchi, M R

    1982-01-01

    We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by

  6. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development.

    PubMed

    Kumar, Rachana A; Oldenburg, Delene J; Bendich, Arnold J

    2014-12-01

    The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR. PMID:25261192

  7. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development

    PubMed Central

    Kumar, Rachana A.; Oldenburg, Delene J.; Bendich, Arnold J.

    2014-01-01

    The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR. PMID:25261192

  8. Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma

    SciTech Connect

    Matsukura, T.; Kanda, T.; Furuno, A.; Yoshikawa, H.; Kawana, T.; Yoshiike, K.

    1986-06-01

    The authors have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1284 and 4471 (numbering system from K. Seedorf, G. Kraemmer, M. Duerst, S. Suhai, and W.G. Roewkamp), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1137 and 1138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.

  9. Affective picture processing: An integrative review of ERP findings

    PubMed Central

    Olofsson, Jonas K.; Nordin, Steven; Sequeira, Henrique; Polich, John

    2008-01-01

    The review summarizes and integrates findings from 40 years of event-related potential (ERP) studies using pictures that differ in valence (unpleasant-to-pleasant) and arousal (low-to-high) and that are used to elicit emotional processing. Affective stimulus factors primarily modulate ERP component amplitude, with little change in peak latency observed. Arousal effects are consistently obtained, and generally occur at longer latencies. Valence effects are inconsistently reported at several latency ranges, including very early components. Some affective ERP modulations vary with recording methodology, stimulus factors, as well as task-relevance and emotional state. Affective ERPs have been linked theoretically to attention orientation for unpleasant pictures at earlier components (< 300 ms). Enhanced stimulus processing has been associated with memory encoding for arousing pictures of assumed intrinsic motivational relevance, with task-induced differences contributing to emotional reactivity at later components (> 300 ms). Theoretical issues, stimulus factors, task demands, and individual differences are discussed. PMID:18164800

  10. An impedance-based integrated biosensor for suspended DNA characterization

    PubMed Central

    Ma, Hanbin; Wallbank, Richard W. R.; Chaji, Reza; Li, Jiahao; Suzuki, Yuji; Jiggins, Chris; Nathan, Arokia

    2013-01-01

    Herein, we describe a novel integrated biosensor for performing dielectric spectroscopy to analyze biological samples. We analyzed biomolecule samples with different concentrations and demonstrated that the solution's impedance is highly correlated with the concentration, indicating that it may be possible to use this sensor as a concentration sensor. In contrast with standard spectrophotometers, this sensor offers a low-cost and purely electrical solution for the quantitative analysis of biomolecule solutions. In addition to determining concentrations, we found that the sample solution impedance is highly correlated with the length of the DNA fragments, indicating that the sizes of PCR products could be validated with an integrated chip-based, sample-friendly system within a few minutes. The system could be the basis of a rapid, low-cost platform for DNA characterization with broad applications in cancer and genetic disease research. PMID:24060937

  11. Does varicocelectomy affect DNA fragmentation in infertile patients?

    PubMed Central

    Telli, Onur; Sarici, Hasmet; Kabar, Mucahit; Ozgur, Berat Cem; Resorlu, Berkan; Bozkurt, Selen

    2015-01-01

    Introduction: The aims of this study were to investigate the effect of varicocelectomy on DNA fragmentation index and semen parameters in infertile patients before and after surgical repair of varicocele. Materials and Methods: In this prospective study, 72 men with at least 1-year history of infertility, varicocele and oligospermia were examined. Varicocele sperm samples were classified as normal or pathological according to the 2010 World Health Organization guidelines. The acridine orange test was used to assess the DNA fragmentation index (DFI) preoperatively and postoperatively. Results: DFI decreased significantly after varicocelectomy from 34.5% to 28.2% (P = 0.024). In addition all sperm parameters such as mean sperm count, sperm concentration, progressive motility and sperm morphology significantly increased from 19.5 × 106 to 30.7 × 106, 5.4 × 106/ml to 14.3 × 106/ml, and 19.9% to 31.2% (P < 0.001) and 2.6% to 3.1% (P = 0.017). The study was limited by the loss to follow-up of some patients and unrecorded pregnancy outcome due to short follow-up. Conclusion: Varicocele causes DNA-damage in spermatozoa. We suggest that varicocelectomy improves sperm parameters and decreases DFI. PMID:25878412

  12. Mechanism of integrating foreign DNA during transformation of Bacillus subtilis.

    PubMed Central

    Duncan, C H; Wilson, G A; Young, F E

    1978-01-01

    Genes encoding thymidylate synthetase from Bacillus subtilis bacteriophages were cloned in Escherichia coli. Chimeric plasmids pCD1 and pCD3 were constructed from site-specific endonuclease digests of bacteriophage phi3T DNA cloned in pMB9 in E. coli. Similar cloning techniques with bacteriophage beta22 DNA yielded chimeric plasmids pCD4, pCD5, and pCD6. Endonuclease digests of DNA from pCD1 and pCD3 propagated in E. coli or from DNA isolated from bacteriophage phi3T propagated in B. subtilis transformed B. subtilis from Thy- to Thy+. Intact DNA from bacteriophage beta22, endonuclease digests of beta22 DNA, and a chimeric plasmid (pCD5) composed only of the thybeta22 gene and pMB9 did not transform B. subtilis from Thy- to Thy+ even though pCD5 could transform Thy- E. coli to Thy+. However, if the thybeta22 fragment from pCD5 was introduced into another chimeric plasmid, pCD2, that contains a region of homology to the chromosome of B. subtilis in addition to pMB9, transformation of Thy- clones of B. subtilis was possible. Furthermore, Southern hybridization analyses of the digests of chromosomal DNA from the Thy+ transformants established that the entire chimeric plasmid was incorporated into the chromosome of B. subtilis. Treatment of these plasmids with site-specific endonucleases abolished transformation. These results indicated that the entire chimeric plasmid can be incorporated into the chromosome of B. subtilis by a Campbell-like model. Therefore, an additional mechanism for transformation exists whereby plasmids can be integrated if sufficient chromosomal homology is maintained. Images PMID:99740

  13. Persistence of DNA in Carcasses, Slime and Avian Feces May Affect Interpretation of Environmental DNA Data

    PubMed Central

    Merkes, Christopher M.; McCalla, S. Grace; Jensen, Nathan R.; Gaikowski, Mark P.; Amberg, Jon J.

    2014-01-01

    The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps. PMID:25402206

  14. Affect Consciousness in children with internalizing problems: Assessment of affect integration.

    PubMed

    Taarvig, Eva; Solbakken, Ole André; Grova, Bjørg; Monsen, Jon T

    2015-10-01

    Affect integration was operationalized through the Affect Consciousness (AC) construct as degrees of awareness, tolerance, nonverbal expression and conceptual expression of 11 affects. These aspects are assessed through a semi-structured Affect Consciousness Interview (ACI) and separate rating scales (Affect Consciousness Scales (ACSs)) developed for use in research and clinical work with adults with psychopathological disorders. Age-adjusted changes were made in the interview and rating system. This study explored the applicability of the adjusted ACI to a sample of 11-year-old children with internalizing problems through examining inter-rater reliability of the adjusted ACI, along with relationships between the AC aspects and aspects of mental health as symptoms of depression, symptoms of anxiety, social competence, besides general intelligence. Satisfactory inter-rater reliability was found, as well as consistent relationships between the AC aspects and the various aspects of mental health, a finding which coincides with previous research. The finding indicates that the attainment of the capacity to deal adaptively with affect is probably an important contributor to the development of adequate social competence and maybe in the prevention of psychopathology in children. The results indicate that the adjusted ACI and rating scales are useful tools in treatment planning with children at least from the age of 11 years. PMID:24941941

  15. Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

    PubMed Central

    2013-01-01

    Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations. PMID:23339280

  16. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis. PMID:27033694

  17. Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

    PubMed Central

    Roner, M R; Cox, D C

    1985-01-01

    Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell. Images PMID:3968718

  18. Persistence of DNA in carcasses, slime and avian feces may affect interpretation of environmental DNA data

    USGS Publications Warehouse

    Merkes, Christopher M.; McCalla, S. Grace; Jensen, Nathan R.; Gaikowski, Mark P.; Amberg, Jon J.

    2014-01-01

    The prevention of non-indigenous aquatic invasive species spreading into new areas is a goal of many resource managers. New techniques have been developed to survey for species that are difficult to capture with conventional gears that involve the detection of their DNA in water samples (eDNA). This technique is currently used to track the invasion of bigheaded carps (silver carp and bighead carp; Hypophthalmichthys molitrix and H. nobilis) in the Chicago Area Waterway System and Upper Mississippi River. In both systems DNA has been detected from silver carp without the capture of a live fish, which has led to some uncertainty about the source of the DNA. The potential contribution to eDNA by vectors and fomites has not been explored. Because barges move from areas with a high abundance of bigheaded carps to areas monitored for the potential presence of silver carp, we used juvenile silver carp to simulate the barge transport of dead bigheaded carp carcasses, slime residue, and predator feces to determine the potential of these sources to supply DNA to uninhabited waters where it could be detected and misinterpreted as indicative of the presence of live bigheaded carp. Our results indicate that all three vectors are feasible sources of detectable eDNA for at least one month after their deposition. This suggests that current monitoring programs must consider alternative vectors of DNA in the environment and consider alternative strategies to minimize the detection of DNA not directly released from live bigheaded carps.

  19. Does organizational culture affect out-patient DNA (did not attend) rates?

    PubMed

    Jackson, S

    1997-01-01

    Government interest in health service "did not attend" (DNA) rates was seen to occur by accident, following which efforts to reduce DNAs have tended to concentrate on operational rather than strategic issues. Considers the effect hospital culture has had on DNA rates from an organizational and patient perspective. Identifies some of the key cultural issues that impacted on DNA rates by utilizing observation and telephone survey research methods. Concludes that, in the main, the lack of customer-oriented organizational culture was seen to affect DNA rates adversely within one NHS provider trust. PMID:10179096

  20. Brown Planthopper Nudivirus DNA Integrated in Its Host Genome

    PubMed Central

    Cheng, Ruo-Lin; Xi, Yu; Lou, Yi-Han; Wang, Zhuo; Xu, Ji-Yu; Xu, Hai-Jun

    2014-01-01

    ABSTRACT The brown planthopper (BPH), Nilaparvata lugens (Hemiptera:Delphacidae), is one of the most destructive insect pests of rice crops in Asia. Nudivirus-like sequences were identified during the whole-genome sequencing of BPH. PCR examination showed that the virus sequences were present in all of the 22 BPH populations collected from East, Southeast, and South Asia. Thirty-two of the 33 nudivirus core genes were identified, including 20 homologues of baculovirus core genes. In addition, several gene clusters that were arranged collinearly with those of other nudiviruses were found in the partial virus genome. In a phylogenetic tree constructed using the supermatrix method, the original virus was grouped with other nudiviruses and was closely related to polydnavirus. Taken together, these data indicated that the virus sequences belong to a new member of the family Nudiviridae. More specifically, the virus sequences were integrated into the chromosome of its insect host during coevolution. This study is the first report of a large double-stranded circular DNA virus genome in a sap-sucking hemipteran insect. IMPORTANCE This is the first report of a large double-stranded DNA virus integrated genome in the planthopper, a plant sap-sucking hemipteran insect. It is an exciting addition to the evolutionary story of bracoviruses (polydnaviruses), nudiviruses, and baculoviruses. The results on the virus sequences integrated in the chromosomes of its insect host also represent a story of successful coevolution of an invertebrate virus and a plant sap-sucking insect. PMID:24574410

  1. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  2. T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.

    PubMed

    Ghedira, Rim; De Buck, Sylvie; Van Ex, Frédéric; Angenon, Geert; Depicker, Ann

    2013-12-01

    T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43-69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19-46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5-1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8-34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93-100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies. PMID:23975012

  3. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    PubMed Central

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  4. Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity

    PubMed Central

    Pal, Rahul; Hameed, Saif; Fatima, Zeeshan

    2015-01-01

    Multidrug resistance (MDR) acquired by Mycobacterium tuberculosis (MTB) through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs in Mycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR. PMID:26779346

  5. Exploration mode affects visuohaptic integration of surface orientation.

    PubMed

    Plaisier, Myrthe A; van Dam, Loes C J; Glowania, Catharina; Ernst, Marc O

    2014-01-01

    We experience the world mostly in a multisensory fashion using a combination of all of our senses. Depending on the modality we can select different exploration strategies for extracting perceptual information. For instance, using touch we can enclose an object in our hand to explore parts of the object in parallel. Alternatively, we can trace the object with a single finger to explore its parts in a serial fashion. In this study we investigated whether the exploration mode (parallel vs. serial) affects the way sensory signals are combined. To this end, participants visually and haptically explored surfaces that varied in roll angle and indicated which side of the surface was perceived as higher. In Experiment 1, the exploration mode was the same for both modalities (i.e., both parallel or both serial). In Experiment 2, we introduced a difference in exploration mode between the two modalities (visual exploration was parallel while haptic exploration was serial or vice versa). The results showed that visual and haptic signals were combined in a statistically optimal fashion only when the exploration modes were the same. In case of an asymmetry in the exploration modes across modalities, integration was suboptimal. This indicates that spatial-temporal discrepancies in the acquisition of information in the two senses (i.e., haptic and visual) can lead to the breakdown of sensory integration. PMID:25413627

  6. Integrated on-line system for DNA sequencing by capillary electrophoresis: From template to called bases

    SciTech Connect

    Ton, H.; Yeung, E.S.

    1997-02-15

    An integrated on-line prototype for coupling a microreactor to capillary electrophoresis for DNA sequencing has been demonstrated. A dye-labeled terminator cycle-sequencing reaction is performed in a fused-silica capillary. Subsequently, the sequencing ladder is directly injected into a size-exclusion chromatographic column operated at nearly 95{degree}C for purification. On-line injection to a capillary for electrophoresis is accomplished at a junction set at nearly 70{degree}C. High temperature at the purification column and injection junction prevents the renaturation of DNA fragments during on-line transfer without affecting the separation. The high solubility of DNA in and the relatively low ionic strength of 1 x TE buffer permit both effective purification and electrokinetic injection of the DNA sample. The system is compatible with highly efficient separations by a replaceable poly(ethylene oxide) polymer solution in uncoated capillary tubes. Future automation and adaptation to a multiple-capillary array system should allow high-speed, high-throughput DNA sequencing from templates to called bases in one step. 32 refs., 5 figs.

  7. Identification of host genes that affect acquisition of an integrative and conjugative element in Bacillus subtilis

    PubMed Central

    Johnson, Christopher M.; Grossman, Alan D.

    2014-01-01

    Summary Conjugation, a major type of horizontal gene transfer in bacteria, involves transfer of DNA from a donor to a recipient using donor-encoded conjugation machinery. Using a high throughput screen (Tn-seq), we identified genes in recipients that contribute to acquisition of the integrative and conjugative element ICEBs1 by Bacillus subtilis. We found that null mutations in some genes caused an increase, and others a decrease in conjugation efficiency. Some mutations affected conjugation only when present in recipients. Other mutations affected conjugation when present in donors or recipients. Most of the genes identified are known or predicted to affect the cell envelope. Several encode enzymes involved in phospholipid biosynthesis and one encodes a homolog of penicillin binding proteins. Two of the genes identified also affected conjugation of Tn916, indicating that their roles in conjugation may be general. We did not identify any genes in recipients that were essential for ICEBs1 conjugation, indicating that if there are such genes, then these are either essential for cell growth or redundant. Our results indicate that acquisition of ICEBs1, and perhaps other conjugative elements, is robust and not easily avoided by mutation and that several membrane-related functions affect the efficiency of conjugation. PMID:25069588

  8. Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis.

    PubMed

    Ambur, Ole Herman; Frye, Stephan A; Nilsen, Mariann; Hovland, Eirik; Tønjum, Tone

    2012-01-01

    Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID

  9. Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

    PubMed Central

    Ambur, Ole Herman; Frye, Stephan A.; Nilsen, Mariann; Hovland, Eirik; Tønjum, Tone

    2012-01-01

    Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID

  10. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility

    PubMed Central

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K.; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C.; Burgess, Shawn M.; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  11. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

    PubMed

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  12. Implicit Affective Cues and Attentional Tuning: An Integrative Review

    PubMed Central

    Friedman, Ronald S.; Förster, Jens

    2010-01-01

    A large and growing number of studies support the notion that arousing positive emotional states expand, and that arousing negative states constrict, the scope of attention on both the perceptual and conceptual levels. However, these studies have predominantly involved the manipulation or measurement of conscious emotional experiences (e.g., subjective feelings of happiness or anxiety). This raises the question: Do cues that are merely associated with benign versus threatening situations, but that do not elicit conscious feelings of positive or negative emotional arousal, independently expand or contract attentional scope? Integrating theoretical advances in affective neuroscience, positive psychology, and social cognition, it is proposed that rudimentary intero- and exteroceptive stimuli may indeed become associated with the onset of arousing positive or negative emotional states and/or with appraisals that the environment is benign or threatening and thereby come to moderate the scope of attention in the absence of conscious emotional experience. Specifically, implicit “benign situation” cues are posited to broaden, and implicit “threatening situation” cues to narrow, the range of both perceptual as well as conceptual attentional selection. An extensive array of research findings involving a diverse set of such implicit affective cues (e.g., enactment of approach and avoidance behaviors, incidental exposure to colors signaling safety versus danger) is marshaled in support of this proposition. Potential alternative explanations for and moderators of these attentional tuning effects, as well as their higher-level neuropsychological underpinnings, are also discussed along with prospective extensions to a range of other situational cues and domains of social cognitive processing. PMID:20804240

  13. How Does Guanine-Cytosine Base Pair Affect Excess-Electron Transfer in DNA?

    PubMed

    Lin, Shih-Hsun; Fujitsuka, Mamoru; Majima, Tetsuro

    2015-06-25

    Charge transfer and proton transfer in DNA have attracted wide attention due to their relevance in biological processes and so on. Especially, excess-electron transfer (EET) in DNA has strong relation to DNA repair. However, our understanding on EET in DNA still remains limited. Herein, by using a strongly electron-donating photosensitizer, trimer of 3,4-ethylenedioxythiophene (3E), and an electron acceptor, diphenylacetylene (DPA), two series of functionalized DNA oligomers were synthesized for investigation of EET dynamics in DNA. The transient absorption measurements during femtosecond laser flash photolysis showed that guanine:cytosine (G:C) base pair affects EET dynamics in DNA by two possible mechanisms: the excess-electron quenching by proton transfer with the complementary G after formation of C(•-) and the EET hindrance by inserting a G:C base pair as a potential barrier in consecutive thymines (T's). In the present paper, we provided useful information based on the direct kinetic measurements, which allowed us to discuss EET through oligonucleotides for the investigation of DNA damage/repair. PMID:26042867

  14. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements.

    PubMed

    Prior, Sara; Miousse, Isabelle R; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R; Allen, Antiño R; Raber, Jacob; Tackett, Alan J; Hauer-Jensen, Martin; Nelson, Gregory A; Koturbash, Igor

    2016-10-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2'-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5'-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. PMID:27419368

  15. Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

    PubMed Central

    Kemski, Megan M.; Stevens, Bryan; Rappleye, Chad A.

    2012-01-01

    Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border end of the T-DNA is largely preserved whereas the left border end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61% of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67% of T-DNA integrations are integrations at a single chromosomal site and 31% of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma. PMID:23332832

  16. Exogenous retroelement integration in sperm and embryos affects preimplantation development.

    PubMed

    Kitsou, C; Lazaros, L; Bellou, S; Vartholomatos, G; Sakaloglou, P; Hatzi, E; Markoula, S; Zikopoulos, K; Tzavaras, T; Georgiou, I

    2016-09-01

    Retroelement transcripts are present in male and female gametes, where they are typically regulated by methylation, noncoding RNAs and transcription factors. Such transcripts are required for occurrence of retrotransposition events, while failure of retrotransposition control may exert negative effects on cellular function and proliferation. In order to investigate the occurrence of retrotransposition events in mouse epididymal spermatozoa and to address the impact of uncontrolled retroelement RNA expression in early preimplantation embryos, we performed in vitro fertilization experiments using spermatozoa preincubated with plasmid vectors containing the human retroelements LINE-1, HERVK-10 or the mouse retroelement VL30, tagged with an enhanced green fluorescence (EGFP) gene-based cassette. Retrotransposition events in mouse spermatozoa and embryos were detected using PCR, FACS analysis and confocal microscopy. Our findings show that: (i) sperm cell incorporates exogenous retroelements and favors retrotransposition events, (ii) the inhibition of spermatozoa reverse transcriptase can decrease the retrotransposition frequency in sperm cells, (iii) spermatozoa can transfer exogenous human or mouse retroelements to the oocyte during fertilization and (iv) retroelement RNA overexpression affects embryo morphology and impairs preimplantation development. These findings suggest that the integration of exogenous retroelements in the sperm genome, as well as their transfer into the mouse oocyte, could give rise to new retrotransposition events and genetic alterations in mouse spermatozoa and embryos. PMID:27450800

  17. Turning the corner in fertility: high DNA integrity of boundary-following sperm.

    PubMed

    Eamer, Lise; Vollmer, Marion; Nosrati, Reza; San Gabriel, Maria C; Zeidan, Krista; Zini, Armand; Sinton, David

    2016-07-01

    We present a passive microfluidic sperm selection strategy that collects motile sperm based on their preference to follow boundaries and turn corners. Clinical assessment of selected human sperm from the device revealed a strong correlation between high DNA integrity and the tendency for sperm to follow boundaries. Human sperm with preference to follow boundaries on the left- or right-hand sides have higher (>51%) DNA integrity than straight swimmers and significantly higher (>67%) DNA integrity than sperm in raw semen. Boundary following behaviour offers a strategy to selecting sperm with the highest DNA integrity to improve the success rate of assisted reproduction. PMID:27241827

  18. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA.

    PubMed

    Choi, Ajin; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2014-01-01

    Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework. PMID:24052059

  19. How nanochannel confinement affects the DNA melting transition within the Poland-Scheraga model

    NASA Astrophysics Data System (ADS)

    Reiter-Schad, Michaela; Werner, Erik; Tegenfeldt, Jonas O.; Mehlig, Bernhard; Ambjörnsson, Tobias

    2015-09-01

    When double-stranded DNA molecules are heated, or exposed to denaturing agents, the two strands are separated. The statistical physics of this process has a long history and is commonly described in terms of the Poland-Scheraga (PS) model. Crucial to this model is the configurational entropy for a melted region (compared to the entropy of an intact region of the same size), quantified by the loop factor. In this study, we investigate how confinement affects the DNA melting transition, by using the loop factor for an ideal Gaussian chain. By subsequent numerical solutions of the PS model, we demonstrate that the melting temperature depends on the persistence lengths of single-stranded and double-stranded DNA. For realistic values of the persistence lengths, the melting temperature is predicted to decrease with decreasing channel diameter. We also demonstrate that confinement broadens the melting transition. These general findings hold for the three scenarios investigated: 1. homo-DNA, i.e., identical basepairs along the DNA molecule, 2. random sequence DNA, and 3. "real" DNA, here T4 phage DNA. We show that cases 2 and 3 in general give rise to broader transitions than case 1. Case 3 exhibits a similar phase transition as case 2 provided the random sequence DNA has the same ratio of AT to GC basepairs (A - adenine, T - thymine, G - guanine, C - cytosine). A simple analytical estimate for the shift in melting temperature is provided as a function of nanochannel diameter. For homo-DNA, we also present an analytical prediction of the melting probability as a function of temperature.

  20. Mutational analysis of the transcription factor IIIB-DNA target of Ty3 retroelement integration.

    PubMed

    Yieh, Lynn; Hatzis, Heather; Kassavetis, George; Sandmeyer, Suzanne B

    2002-07-19

    The Ty3 retrovirus-like element inserts preferentially at the transcription initiation sites of genes transcribed by RNA polymerase III. The requirements for transcription factor (TF) IIIC and TFIIIB in Ty3 integration into the two initiation sites of the U6 gene carried on pU6LboxB were previously examined. Ty3 integrates at low but detectable frequencies in the presence of TFIIIB subunits Brf1 and TATA-binding protein. Integration increases in the presence of the third subunit, Bdp1. TFIIIC is not essential, but the presence of TFIIIC specifies an orientation of TFIIIB for transcriptional initiation and directs integration to the U6 gene-proximal initiation site. In the current study, recombinant wild type TATA-binding protein, wild type and mutant Brf1, and Bdp1 proteins and highly purified TFIIIC were used to investigate the roles of specific protein domains in Ty3 integration. The amino-terminal half of Brf1, which contains a TFIIB-like repeat, contributed more strongly than the carboxyl-terminal half of Brf1 to Ty3 targeting. Each half of Bdp1 split at amino acid 352 enhanced integration. In the presence of TFIIIB and TFIIIC, the pattern of integration extended downstream by several base pairs compared with the pattern observed in vitro in the absence of TFIIIC and in vivo, suggesting that TFIIIC may not be present on genes targeted by Ty3 in vivo. Mutations in Bdp1 that affect its interaction with TFIIIC resulted in TFIIIC-independent patterns of Ty3 integration. Brf1 zinc ribbon and Bdp1 internal deletion mutants that are competent for polymerase III recruitment but defective in promoter opening were competent for Ty3 integration irrespective of the state of DNA supercoiling. These results extend the similarities between the TFIIIB domains required for transcription and Ty3 integration and also reveal requirements that are specific to transcription. PMID:11994300

  1. Integration and scaling of UV-B radiation effects on plants: from DNA to leaf.

    PubMed

    Suchar, Vasile Alexandru; Robberecht, Ronald

    2015-07-01

    A process-based model integrating the effects of UV-B radiation through epidermis, cellular DNA, and its consequences to the leaf expansion was developed from key parameters in the published literature. Enhanced UV-B radiation-induced DNA damage significantly delayed cell division, resulting in significant reductions in leaf growth and development. Ambient UV-B radiation-induced DNA damage significantly reduced the leaf growth of species with high relative epidermal absorbance at longer wavelengths and average/low pyrimidine cyclobutane dimers (CPD) photorepair rates. Leaf expansion was highly dependent on the number of CPD present in the DNA, as a result of UV-B radiation dose, quantitative and qualitative absorptive properties of epidermal pigments, and repair mechanisms. Formation of pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) has no effect on the leaf expansion. Repair mechanisms could not solely prevent the UV-B radiation interference with the cell division. Avoidance or effective shielding by increased or modified qualitative epidermal absorptance was required. Sustained increased UV-B radiation levels are more detrimental than short, high doses of UV-B radiation. The combination of low temperature and increased UV-B radiation was more significant in the level of UV-B radiation-induced damage than UV-B radiation alone. Slow-growing leaves were more affected by increased UV-B radiation than fast-growing leaves. PMID:26257869

  2. Integration and scaling of UV-B radiation effects on plants: from DNA to leaf

    PubMed Central

    Suchar, Vasile Alexandru; Robberecht, Ronald

    2015-01-01

    A process-based model integrating the effects of UV-B radiation through epidermis, cellular DNA, and its consequences to the leaf expansion was developed from key parameters in the published literature. Enhanced UV-B radiation-induced DNA damage significantly delayed cell division, resulting in significant reductions in leaf growth and development. Ambient UV-B radiation-induced DNA damage significantly reduced the leaf growth of species with high relative epidermal absorbance at longer wavelengths and average/low pyrimidine cyclobutane dimers (CPD) photorepair rates. Leaf expansion was highly dependent on the number of CPD present in the DNA, as a result of UV-B radiation dose, quantitative and qualitative absorptive properties of epidermal pigments, and repair mechanisms. Formation of pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) has no effect on the leaf expansion. Repair mechanisms could not solely prevent the UV-B radiation interference with the cell division. Avoidance or effective shielding by increased or modified qualitative epidermal absorptance was required. Sustained increased UV-B radiation levels are more detrimental than short, high doses of UV-B radiation. The combination of low temperature and increased UV-B radiation was more significant in the level of UV-B radiation-induced damage than UV-B radiation alone. Slow-growing leaves were more affected by increased UV-B radiation than fast-growing leaves. PMID:26257869

  3. Study of design parameters affecting the motion of DNA for nanoinjection

    NASA Astrophysics Data System (ADS)

    David, Regis A.; Jensen, Brian D.; Black, Justin L.; Burnett, Sandra H.; Howell, Larry L.

    2012-05-01

    This paper reports the effects of various parameters on the attraction and repulsion of DNA to and from a silicon lance. An understanding of DNA motion is crucial for a new approach to insert DNA, or other foreign microscopic matter, into a living cell. The approach, called nanoinjection, uses electrical forces to attract and repel the desired substance to a micromachined lance designed to pierce the cell membranes. We have developed mathematical models to predict the trajectory of DNA. The mathematical model allows investigation of the attraction/repulsion process by varying specific parameters. We find that the ground electrode placement, lance orientation and lance penetration significantly affect attraction or repulsion efficiency, while the gap, lance direction, lance tip width, lance tip half-angle and lance tip height do not.

  4. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies.

    PubMed

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  5. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    PubMed Central

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  6. DNA methylation affected by male sterile cytoplasm in rice (Oryza sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Male sterile cytoplasm plays an important role in hybrid rice and cytoplasmic effects are sufficiently documented. However, no reports are available on DNA methylation affected by male sterile cytoplasm in hybrid rice. We used a methylation sensitive amplified polymorphism (MSAP) technique to charac...

  7. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    PubMed

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  8. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

    PubMed Central

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M.

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  9. DNA transformations of Candida tropicalis with replicating and integrative vectors.

    PubMed

    Sanglard, D; Fiechter, A

    1992-12-01

    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats. PMID:1293885

  10. Vertically integrated analysis of human DNA. Final technical report

    SciTech Connect

    Olson, M.

    1997-10-01

    This project has been oriented toward improving the vertical integration of the sequential steps associated with the large-scale analysis of human DNA. The central focus has been on an approach to the preparation of {open_quotes}sequence-ready{close_quotes} maps, which is referred to as multiple-complete-digest (MCD) mapping, primarily directed at cosmid clones. MCD mapping relies on simple experimental steps, supported by advanced image-analysis and map-assembly software, to produce extremely accurate restriction-site and clone-overlap maps. We believe that MCD mapping is one of the few high-resolution mapping systems that has the potential for high-level automation. Successful automation of this process would be a landmark event in genome analysis. Once other higher organisms, paving the way for cost-effective sequencing of these genomes. Critically, MCD mapping has the potential to provide built-in quality control for sequencing accuracy and to make possible a highly integrated end product even if there are large numbers of discontinuities in the actual sequence.

  11. Activities of Human Immunodeficiency Virus (HIV) Integration Protein In vitro: Specific Cleavage and Integration of HIV DNA

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Craigie, Robert

    1991-02-01

    Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

  12. Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast.

    PubMed

    Maiorano, D; Cece, R; Badaracco, G

    1997-04-11

    We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro. It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin. In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid. We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S. cerevisiae. We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene. Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression. PMID:9161405

  13. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    PubMed

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. PMID:27315553

  14. Integrity and Biological Activity of DNA after UV Exposure

    NASA Astrophysics Data System (ADS)

    Lyon, Delina Y.; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M.

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m2s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

  15. Integrity and biological activity of DNA after UV exposure.

    PubMed

    Lyon, Delina Y; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m(2)s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity. PMID:20446869

  16. Affective instability: toward an integration of neuroscience and psychological perspectives.

    PubMed

    Koenigsberg, Harold W

    2010-02-01

    Affective instability is a prominent feature of a wide variety of psychiatric and neurological disorders, yet it has not been systematically studied. It encompasses a number of distinct phenomena, including: (1) frequent affective category shifts, (2) disturbances in affect intensity, (3) excessively rapid emotion rise-times, (4) delayed return to emotional baseline, (5) excessive reactivity to psychosocial cues, (6) endogenously driven, random, chaotic or rapid-cycling changes, and (7) overdramatic expression. To further clarify the construct of affective instability, this article examines the manifestations of affective instability in a range of psychiatric and neurologic disorders, reviews relevant neurobiological and psychological emotion regulatory processes, and considers the psychology of affective instability. PMID:20205499

  17. The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling

    PubMed Central

    Harami, Gábor M.; Nagy, Nikolett T.; Martina, Máté; Neuman, Keir C.; Kovács, Mihály

    2015-01-01

    DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom’s syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme. PMID:26067769

  18. Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress.

    PubMed

    Yildiz, Cengiz; Law, Napoleon; Ottaviani, Palma; Jarvi, Keith; McKerlie, Colin

    2010-11-01

    The first objective was to compare sperm quality following conventional manual sperm freezing (cryovials held 1, 2, 3, and 4 cm, respectively, above liquid nitrogen (LN(2)) for 10 min, resulting in cooling velocities of approximately -14.9, -10.1, -6.6, and -5.1 °C/min, respectively), and cooling velocities of -5, -20, -40, and -100 °C/min in a programmed automated freezer, for sperm recovered from CD-1, B6129SF1, and C57BL/6NCrlBR mice. Furthermore, using these strains, as well as 129S/SvPaslco, and DBA/2NCrlBR mice, the second objective was to determine the effects on DNA integrity of sperm exposed to hyposmotic (1 mOsm/L) and hyperosmotic (2400 mOsm/L) solutions, compared to an isosmotic control (300 mOsm/L). For freezing above LN(2) or in an automated freezer, 2 cm above LN(2) and -100 °C/min, respectively, were optimal (P < 0.05-0.01), with no significant differences between these two approaches for post-thaw progressive motility, DNA integrity, and in vitro rates of fertilization and blastocyst formation. Both manual and automated freezing techniques increased post-thaw sperm DNA fragmentation (P < 0.01); the DNA integrity of post-thaw sperm was significantly affected by cooling velocity and strain background. Relative to isosmotic controls, a hyposmotic solution was more deleterious (P < 0.05-0.01) to sperm DNA integrity than a hyperosmotic solution for CD-1, B6129SF1, C57BL/6, and DBA mice (there were strain-dependent differences). In conclusion, optimization of freezing distance and cooling velocity (manual and automated freezing, respectively) were significant factors for efficient cryopreservation and re-derivation of mice from frozen-thawed sperm. Additionally, osmotically-driven volume changes in mouse sperm increased DNA fragmentation, with susceptibility affected by background strain. PMID:20728931

  19. Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.

    PubMed

    Li, Hui; Swiercz, Rafal; Englander, Ella W

    2009-09-01

    Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death. PMID:19619136

  20. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

    PubMed Central

    Salvi, Samanta; Gurioli, Giorgia; Martignano, Filippo; Foca, Flavia; Gunelli, Roberta; Cicchetti, Giacomo; De Giorgi, Ugo; Zoli, Wainer; Calistri, Daniele; Casadio, Valentina

    2015-01-01

    Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations. PMID:26412928

  1. Factors affecting social integration of noninstitutionalized mentally retarded adults.

    PubMed

    Reiter, S; Levi, A M

    1980-07-01

    The social integration of noninstitutionalized moderately and mildly mentally retarded young adults was investigated. A group of moderately and mildly retarded adults (study group) was compared with a group of borderline retarded (control group) adults on employability, behavior at work, social integration and social skills, personality, and self-concept. Findings indicated that the study group was less well integrated at work and in society than was the control group and showed lack of social skills. The retarded adults who had nonretarded friends showed better social-educational skills than did the other subjects. Findings suggest that even retarded individuals who grow up in the community need help in order to become socially independent. The existence of a special social club for retarded adults was found to fulfill the functions of a sheltered framework. Participants in the club showed more positive self-concepts; however, the club did not seem to prepare them for social integration in the general community. PMID:7446566

  2. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  3. Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda)

    PubMed Central

    Ferri, Emanuele; Barbuto, Michela; Bain, Odile; Galimberti, Andrea; Uni, Shigehiko; Guerrero, Ricardo; Ferté, Hubert; Bandi, Claudio; Martin, Coralie; Casiraghi, Maurizio

    2009-01-01

    Background We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. Results DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. Conclusion An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes. PMID:19128479

  4. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  5. Variables affecting the academic and social integration of nursing students.

    PubMed

    Zeitlin-Ophir, Iris; Melitz, Osnat; Miller, Rina; Podoshin, Pia; Mesh, Gustavo

    2004-07-01

    This study attempted to analyze the variables that influence the academic integration of nursing students. The theoretical model presented by Leigler was adapted to the existing conditions in a school of nursing in northern Israel. The independent variables included the student's background; amount of support received in the course of studies; extent of outside family and social commitments; satisfaction with the school's facilities and services; and level of social integration. The dependent variable was the student's level of academic integration. The findings substantiated four central hypotheses, with the study model explaining approximately 45% of the variance in the dependent variable. Academic integration is influenced by a number of variables, the most prominent of which is the social integration of the student with colleagues and educational staff. Among the background variables, country of origin was found to be significant to both social and academic integration for two main groups in the sample: Israeli-born students (both Jewish and Arab) and immigrant students. PMID:15303587

  6. Integrating S-phase Checkpoint Signaling with Trans-Lesion Synthesis of Bulky DNA Adducts

    PubMed Central

    Barkley, Laura R.; Ohmori, Haruo; Vaziri, Cyrus

    2011-01-01

    Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression. PMID:17652783

  7. A new structural framework for integrating replication protein A into DNA processing machinery

    SciTech Connect

    Brosey, Chris; Yan, Chunli; Tsutakawa, Susan; Heller, William; Rambo, Robert; Tainer, John; Ivanov, Ivaylo; Chazin, Walter

    2013-01-17

    By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

  8. Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer.

    PubMed

    Kamel, Azza M; Teama, Salwa; Fawzy, Amal; El Deftar, Mervat

    2016-06-01

    Plasma DNA integrity index is increased in various malignancies including breast cancer, the most common cancer in women worldwide; early detection is crucial for successful treatment. Current screening methods fail to detect many cases of breast cancer at an early stage. In this study, we evaluated the level of plasma DNA integrity index in 260 females (95 with breast cancer, 95 with benign breast lesions, and 70 healthy controls) to verify its potential value in discriminating malignant from benign breast lesions. The criteria of the American Joint Committee on Cancer were used for staging of breast cancer patients. DNA integrity index was measured by real-time PCR. DNA integrity index was significantly higher in breast cancer than in benign breast patients and healthy subjects (P = <0.001). DNA integrity index is correlated with TNM stage. Given 100 % specificity, the highest sensitivity achieved in detecting cancer group was 85.3 % at 0.55 DNA integrity index cutoff. In conclusion, the plasma DNA integrity index may be a promising molecular diagnostic marker of malignancy in breast lesions. PMID:26684805

  9. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    SciTech Connect

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  10. DNA integrity of onion root cells under catechol influence.

    PubMed

    Petriccione, Milena; Forte, Valentina; Valente, Diego; Ciniglia, Claudia

    2013-07-01

    Catechol is a highly toxic organic pollutant, usually abundant in the waste effluents of industrial processes and agricultural activities. The environmental sources of catechol include pesticides, wood preservatives, tanning lotion, cosmetic creams, dyes, and synthetic intermediates. Genotoxicity of catechol at a concentration range 5 × 10(-1)-5 mM was evaluated by applying random amplified polymorphic DNA (RAPD) and time-lapse DNA laddering tests using onion (Allium cepa) root cells as the assay system. RAPD analysis revealed polymorphisms in the nucleotidic sequence of DNA that reflected the genotoxic potential of catechol to provoke point mutations, or deletions, or chromosomal rearrangements. Time-lapse DNA laddering test provided evidence that catechol provoked DNA necrosis and apoptosis. Acridine orange/ethidium bromide staining could distinguish apoptotic from necrotic cells in root cells of A. cepa. PMID:23307075

  11. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli.

    PubMed

    Kieser, T

    1984-07-01

    Based on the results of a systematic study of factors affecting plasmid yield and purity, a procedure suitable for the rapid screening for and isolation of covalently closed circular DNA from Streptomyces lividans and Escherichia coli was developed. The method consists of lysis of lysozyme-treated bacteria combined with alkaline denaturation of DNA at high temperature. Renaturation of CCC DNA and precipitation of single-stranded DNA together with protein is achieved by the addition of a minimal amount of phenol/chloroform. The screening procedure uses only a single tube and the samples can be analyzed by agarose gel electrophoresis about 30 min after lysis. Removal of phenol and further purification of the plasmid preparation is achieved by consecutive precipitations with isopropanol and spermine, followed by extraction with ethanol, producing samples suitable for restriction endonuclease digestion, ligation, and transformation of S. lividans protoplasts or competent E. coli cells in about 2 h. All steps of the procedure are explained in detail with information about the effects of changing parameters. This should help the experimenter to obtain reproducible results and may be useful if the method has to be adapted to new strains or plasmids. PMID:6387733

  12. Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

    2014-11-01

    Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

  13. DNA Hypomethylation Affects Cancer-Related Biological Functions and Genes Relevant in Neuroblastoma Pathogenesis

    PubMed Central

    Mayol, Gemma; Martín-Subero, José I.; Ríos, José; Queiros, Ana; Kulis, Marta; Suñol, Mariona; Esteller, Manel; Gómez, Soledad; Garcia, Idoia; de Torres, Carmen; Rodríguez, Eva; Galván, Patricia; Mora, Jaume; Lavarino, Cinzia

    2012-01-01

    Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3′ untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation. PMID:23144874

  14. Physical Activity Affects Brain Integrity in HIV + Individuals

    PubMed Central

    Ortega, Mario; Baker, Laurie M.; Vaida, Florin; Paul, Robert; Basco, Brian; Ances, Beau M.

    2015-01-01

    Prior research has suggested benefits of aerobic physical activity (PA) on cognition and brain volumes in HIV uninfected (HIV−) individuals, however, few studies have explored the relationships between PA and brain integrity (cognition and structural brain volumes) in HIV-infected (HIV +) individuals. Seventy HIV + individuals underwent neuropsychological testing, structural neuroimaging, laboratory tests, and completed a PA questionnaire, recalling participation in walking, running, and jogging activities over the last year. A PA engagement score of weekly metabolic equivalent (MET) hr of activity was calculated using a compendium of PAs. HIV + individuals were classified as physically active (any energy expended above resting expenditure, n = 22) or sedentary (n = 48). Comparisons of neuropsychological performance, grouped by executive and motor domains, and brain volumes were completed between groups. Physically active and sedentary HIV + individuals had similar demographic and laboratory values, but the active group had higher education (14.0 vs. 12.6 years, p = .034). Physically active HIV + individuals performed better on executive (p = .040, unadjusted; p = .043, adjusted) but not motor function (p = .17). In addition, among the physically active group the amount of physical activity (METs) positively correlated with executive (Pearson’s r = 0.45, p = 0.035) but not motor (r = 0.21; p = .35) performance. In adjusted analyses the physically active HIV + individuals had larger putamen volumes (p = .019). A positive relationship exists between PA and brain integrity in HIV + individuals. Results from the present study emphasize the importance to conduct longitudinal interventional investigation to determine if PA improves brain integrity in HIV + individuals. PMID:26581799

  15. Sperm global DNA methylation level: association with semen parameters and genome integrity.

    PubMed

    Montjean, D; Zini, A; Ravel, C; Belloc, S; Dalleac, A; Copin, H; Boyer, P; McElreavey, K; Benkhalifa, M

    2015-03-01

    Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity. PMID:25755112

  16. Blood DNA Yield but Not Integrity or Methylation Is Impacted After Long-Term Storage.

    PubMed

    Bulla, Alexandre; De Witt, Brian; Ammerlaan, Wim; Betsou, Fay; Lescuyer, Pierre

    2016-02-01

    Collection of human whole blood for genomic DNA extraction is part of numerous clinical studies. Since DNA extraction cannot always be performed at the time of sample collection, whole blood samples may be stored for years before being processed. The use of appropriate storage conditions is then critical to obtain DNA in sufficient quantity and of adequate quality in order to obtain reliable results from the subsequent molecular biological analyses. In this study, EDTA whole blood samples were collected from 8 healthy volunteers, and different durations (up to 1 year) and temperatures (room temperature, 4°C, -20°C, and -80°C) of storage were compared. The effect of the addition of a DNA preservative agent was also assessed before and after storage. DNA concentrations measured by UV spectrophotometry and spectrofluorometry were used to calculate DNA extraction yields and double-strand DNA ratios. DNA integrity was controlled by agarose gel electrophoresis and long-range polymerase chain reaction. The impact of storage conditions on DNA methylation was also evaluated. Results showed that certain storage conditions have a significant impact on the DNA extraction yield but little or no effect on DNA integrity and methylation. Storage of EDTA blood at -80°C guarantees high-quality DNA with a good yield. Higher DNA extraction yields were obtained with the addition of a DNA preservative agent before thawing EDTA blood stored at -20°C or -80°C. Long-term storage at room temperature in the presence of a DNA preservative agent also appeared to be a reliable procedure. PMID:26812548

  17. Sites of Retroviral DNA Integration: From Basic Research to Clinical Applications

    PubMed Central

    Serrao, Erik; Engelman, Alan N.

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of the viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with HIV-1 can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or AIDS patients on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  18. Sites of retroviral DNA integration: From basic research to clinical applications.

    PubMed

    Serrao, Erik; Engelman, Alan N

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  19. Opto-electronic DNA chip-based integrated card for clinical diagnostics.

    PubMed

    Marchand, Gilles; Broyer, Patrick; Lanet, Véronique; Delattre, Cyril; Foucault, Frédéric; Menou, Lionel; Calvas, Bernard; Roller, Denis; Ginot, Frédéric; Campagnolo, Raymond; Mallard, Frédéric

    2008-02-01

    Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip or lab-on-card systems. DNA chips, which provide multiparametric data, are privileged tools for genomic analysis. However, automation of molecular biology protocol and use of these DNA chips in fully integrated systems remains a great challenge. Simplicity of chip and/or card/instrument interfaces is amongst the most critical issues to be addressed. Indeed, current detection systems for DNA chip reading are often complex, expensive, bulky and even limited in terms of sensitivity or accuracy. Furthermore, for liquid handling in the lab-on-cards, many devices use complex and bulky systems, either to directly manipulate fluids, or to ensure pneumatic or mechanical control of integrated valves. All these drawbacks prevent or limit the use of DNA-chip-based integrated systems, for point-of-care testing or as a routine diagnostics tool. We present here a DNA-chip-based protocol integration on a plastic card for clinical diagnostics applications including: (1) an opto-electronic DNA-chip, (2) fluid handling using electrically activated embedded pyrotechnic microvalves with closing/opening functions. We demonstrate both fluidic and electric packaging of the optoelectronic DNA chip without major alteration of its electronical and biological functionalities, and fluid control using novel electrically activable pyrotechnic microvalves. Finally, we suggest a complete design of a card dedicated to automation of a complex biological protocol with a fully electrical fluid handling and DNA chip reading. PMID:17636395

  20. Aptamer-integrated DNA nanostructures for biosensing, bioimaging and cancer therapy.

    PubMed

    Meng, Hong-Min; Liu, Hui; Kuai, Hailan; Peng, Ruizi; Mo, Liuting; Zhang, Xiao-Bing

    2016-05-01

    The combination of nanostructures with biomolecules leading to the generation of functional nanosystems holds great promise for biotechnological and biomedical applications. As a naturally occurring biomacromolecule, DNA exhibits excellent biocompatibility and programmability. Also, scalable synthesis can be readily realized through automated instruments. Such unique properties, together with Watson-Crick base-pairing interactions, make DNA a particularly promising candidate to be used as a building block material for a wide variety of nanostructures. In the past few decades, various DNA nanostructures have been developed, including one-, two- and three-dimensional nanomaterials. Aptamers are single-stranded DNA or RNA molecules selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), with specific recognition abilities to their targets. Therefore, integrating aptamers into DNA nanostructures results in powerful tools for biosensing and bioimaging applications. Furthermore, owing to their high loading capability, aptamer-modified DNA nanostructures have also been altered to play the role of drug nanocarriers for in vivo applications and targeted cancer therapy. In this review, we summarize recent progress in the design of aptamers and related DNA molecule-integrated DNA nanostructures as well as their applications in biosensing, bioimaging and cancer therapy. To begin with, we first introduce the SELEX technology. Subsequently, the methodologies for the preparation of aptamer-integrated DNA nanostructures are presented. Then, we highlight their applications in biosensing and bioimaging for various targets, as well as targeted cancer therapy applications. Finally, we discuss several challenges and further opportunities in this emerging field. PMID:26954935

  1. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation

    SciTech Connect

    Kumar, Dayanidhi; Salian, Sujith Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumari, Sandhya; Challapalli, Srinivas; Chandraguthi, Shrinidhi Gururajarao; Jain, Navya; Krishnamurthy, Hanumanthappa; Kumar, Pratap; Adiga, Satish Kumar

    2014-07-15

    There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers. - Highlights: • Seminal plasma antioxidants were measured in men occupationally exposed to radiation. • Sperm chromatin integrity was significantly affected in the exposed group. • Glutathione and total antioxidant capacity was significantly higher in exposed group. • Sperm DNA damage in exposed subjects

  2. Disrupting Mitochondrial–Nuclear Coevolution Affects OXPHOS Complex I Integrity and Impacts Human Health

    PubMed Central

    Gershoni, Moran; Levin, Liron; Ovadia, Ofer; Toiw, Yasmin; Shani, Naama; Dadon, Sara; Barzilai, Nir; Bergman, Aviv; Atzmon, Gil; Wainstein, Julio; Tsur, Anat; Nijtmans, Leo; Glaser, Benjamin; Mishmar, Dan

    2014-01-01

    The mutation rate of the mitochondrial DNA (mtDNA), which is higher by an order of magnitude as compared with the nuclear genome, enforces tight mitonuclear coevolution to maintain mitochondrial activities. Interruption of such coevolution plays a role in interpopulation hybrid breakdown, speciation events, and disease susceptibility. Previously, we found an elevated amino acid replacement rate and positive selection in the nuclear DNA-encoded oxidative phosphorylation (OXPHOS) complex I subunit NDUFC2, a phenomenon important for the direct interaction of NDUFC2 with the mtDNA-encoded complex I subunit ND4. This finding underlines the importance of mitonuclear coevolution to physical interactions between mtDNA and nuclear DNA-encoded factors. Nevertheless, it remains unclear whether this interaction is important for the stability and activity of complex I. Here, we show that siRNA silencing of NDUFC2 reduced growth of human D-407 retinal pigment epithelial cells, significantly diminished mitochondrial membrane potential, and interfered with complex I integrity. Moreover, site-directed mutagenesis of a positively selected amino acid in NDUFC2 significantly interfered with the interaction of NDUFC2 with its mtDNA-encoded partner ND4. Finally, we show that a genotype combination involving this amino acid (NDUFC2 residue 46) and the mtDNA haplogroup HV likely altered susceptibility to type 2 diabetes mellitus in Ashkenazi Jews. Therefore, mitonuclear coevolution is important for maintaining mitonuclear factor interactions, OXPHOS, and for human health. PMID:25245408

  3. Disrupting mitochondrial-nuclear coevolution affects OXPHOS complex I integrity and impacts human health.

    PubMed

    Gershoni, Moran; Levin, Liron; Ovadia, Ofer; Toiw, Yasmin; Shani, Naama; Dadon, Sara; Barzilai, Nir; Bergman, Aviv; Atzmon, Gil; Wainstein, Julio; Tsur, Anat; Nijtmans, Leo; Glaser, Benjamin; Mishmar, Dan

    2014-10-01

    The mutation rate of the mitochondrial DNA (mtDNA), which is higher by an order of magnitude as compared with the nuclear genome, enforces tight mitonuclear coevolution to maintain mitochondrial activities. Interruption of such coevolution plays a role in interpopulation hybrid breakdown, speciation events, and disease susceptibility. Previously, we found an elevated amino acid replacement rate and positive selection in the nuclear DNA-encoded oxidative phosphorylation (OXPHOS) complex I subunit NDUFC2, a phenomenon important for the direct interaction of NDUFC2 with the mtDNA-encoded complex I subunit ND4. This finding underlines the importance of mitonuclear coevolution to physical interactions between mtDNA and nuclear DNA-encoded factors. Nevertheless, it remains unclear whether this interaction is important for the stability and activity of complex I. Here, we show that siRNA silencing of NDUFC2 reduced growth of human D-407 retinal pigment epithelial cells, significantly diminished mitochondrial membrane potential, and interfered with complex I integrity. Moreover, site-directed mutagenesis of a positively selected amino acid in NDUFC2 significantly interfered with the interaction of NDUFC2 with its mtDNA-encoded partner ND4. Finally, we show that a genotype combination involving this amino acid (NDUFC2 residue 46) and the mtDNA haplogroup HV likely altered susceptibility to type 2 diabetes mellitus in Ashkenazi Jews. Therefore, mitonuclear coevolution is important for maintaining mitonuclear factor interactions, OXPHOS, and for human health. PMID:25245408

  4. Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas

    PubMed Central

    Xu, Bo; Chotewutmontri, Sasithorn; Wolf, Stephan; Klos, Ursula; Schmitz, Martina; Dürst, Matthias; Schwarz, Elisabeth

    2013-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the

  5. Employees’ Organizational Identification and Affective Organizational Commitment: An Integrative Approach

    PubMed Central

    Stinglhamber, Florence; Marique, Géraldine; Caesens, Gaëtane; Desmette, Donatienne; Hansez, Isabelle; Hanin, Dorothée; Bertrand, Françoise

    2015-01-01

    Although several studies have empirically supported the distinction between organizational identification (OI) and affective commitment (AC), there is still disagreement regarding how they are related. Precisely, little attention has been given to the direction of causality between these two constructs and as to why they have common antecedents and outcomes. This research was designed to fill these gaps. Using a cross-lagged panel design with two measurement times, Study 1 examined the directionality of the relationship between OI and AC, and showed that OI is positively related to temporal change in AC, confirming the antecedence of OI on AC. Using a cross-sectional design, Study 2 investigated the mediating role of OI in the relationship between three work experiences (i.e., perceived organizational support, leader-member exchange, and job autonomy) and AC, and found that OI partially mediates the influence of work experiences on AC. Finally, Study 3 examined longitudinally how OI and AC combine in the prediction of actual turnover, and showed that AC totally mediates the relationship between OI and turnover. Overall, these findings suggest that favorable work experiences operate via OI to increase employees' AC that, in turn, decreases employee turnover. PMID:25875086

  6. Employees' organizational identification and affective organizational commitment: an integrative approach.

    PubMed

    Stinglhamber, Florence; Marique, Géraldine; Caesens, Gaëtane; Desmette, Donatienne; Hansez, Isabelle; Hanin, Dorothée; Bertrand, Françoise

    2015-01-01

    Although several studies have empirically supported the distinction between organizational identification (OI) and affective commitment (AC), there is still disagreement regarding how they are related. Precisely, little attention has been given to the direction of causality between these two constructs and as to why they have common antecedents and outcomes. This research was designed to fill these gaps. Using a cross-lagged panel design with two measurement times, Study 1 examined the directionality of the relationship between OI and AC, and showed that OI is positively related to temporal change in AC, confirming the antecedence of OI on AC. Using a cross-sectional design, Study 2 investigated the mediating role of OI in the relationship between three work experiences (i.e., perceived organizational support, leader-member exchange, and job autonomy) and AC, and found that OI partially mediates the influence of work experiences on AC. Finally, Study 3 examined longitudinally how OI and AC combine in the prediction of actual turnover, and showed that AC totally mediates the relationship between OI and turnover. Overall, these findings suggest that favorable work experiences operate via OI to increase employees' AC that, in turn, decreases employee turnover. PMID:25875086

  7. DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast.

    PubMed

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A(Cnp1) at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A(Cnp1) occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A(Cnp1) at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A(Cnp1) in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A(Cnp1) nucleosomes. PMID:23516381

  8. DNA Topoisomerase III Localizes to Centromeres and Affects Centromeric CENP-A Levels in Fission Yeast

    PubMed Central

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-ACnp1 nucleosomes. PMID:23516381

  9. Human cytomegalovirus RL13 protein interacts with host NUDT14 protein affecting viral DNA replication.

    PubMed

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanping; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-03-01

    The interaction between the host and human cytomegalovirus (HCMV) is important in determining the outcome of a viral infection. The HCMV RL13 gene product exerts independent, inhibitory effects on viral growth in fibroblasts and epithelial cells. At present, there are few reports on the interactions between the HCMV RL13 protein and human host proteins. The present study provided direct evidence for the specific interaction between HCMV RL13 and host nucleoside diphosphate linked moiety X (nudix)‑type motif 14 (NUDT14), a UDP‑glucose pyrophosphatase, using two‑hybrid screening, an in vitro glutathione S‑transferase pull‑down assay, and co‑immunoprecipitation in human embryonic kidney HEK293 cells. Additionally, the RL13 protein was shown to co‑localize with the NUDT14 protein in the HEK293 cell membrane and cytoplasm, demonstrated using fluorescence confocal microscopy. Decreasing the expression level of NUDT14 via NUDT14‑specific small interfering RNAs increased the number of viral DNA copies in the HCMV‑infected cells. However, the overexpression of NUDT14 in a stably expressing cell line did not affect viral DNA levels significantly in the HCMV infected cells. Based on the known functions of NUDT14, the results of the present study suggested that the interaction between the RL13 protein and NUDT14 protein may be involved in HCMV DNA replication, and that NUDT14 may offer potential in the modulation of viral infection. PMID:26781650

  10. Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

    PubMed Central

    Barrett, Angela N.; Zimmermann, Bernhard G.; Wang, Darrell; Holloway, Andrew; Chitty, Lyn S.

    2011-01-01

    Objective Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. Methods Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples. Results Total cell-free DNA quantity increased significantly with time in samples stored in K3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes. Conclusion When samples can be processed within eight hours of blood draw, K3EDTA tubes can be used. Prolonged transfer times in K3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests. PMID:21998643

  11. Nuclear DNA content affects the productivity of conifer forests by altering hydraulic architecture

    NASA Astrophysics Data System (ADS)

    Alday, Josu; Resco de Dios, Víctor

    2014-05-01

    Predictions of future global climate rely on feedbacks between terrestrial vegetation and the global carbon cycle, but the exact mechanisms underlying this relationship are still being discussed. One of the key knowledge gaps lies on the scaling of cellular processes to the ecosystem level. Here we examine whether an under-explored plant trait, inter-specific variation in the bulk amount of DNA in unreplicated somatic cells (2C DNA content), can explain inter-specific variation in the maximum productivity of conifer forests. We expected 2C DNA content to be negatively related to conifer productivity because: 1) it is positively correlated with cell volume (which, in turn, potentially affects structural features such as leaf mass area, a strong predictor of photosynthetic capacity); 2) it is positively correlated with stomatal size (with larger stomata leading to lower overall stomatal conductance and, by extension, lower CO2 uptake); and 3) larger genome sizes may reduce P availability in RNA (which has been hypothesized to slow growth). We present the results of regression and independent contrasts in different monospecific forests encompassing a 52º latitudinal gradient, each being dominated by 1 of 35 different conifer species. Contrary to expectations, we observed a positive correlation between genome size and maximum Gross Primary Productivity (R2 = 0.47) and also between genome size maximum tree height (R2 = 0.27). This correlation was apparently driven by the effects of genome size on stem hydraulics, since 2C DNA was positively correlated with wood density (R2 = 0.40) and also with resistance to cavitation (P50, R2 = 0.28). That is, increased genome sizes have a positive effect on the productivity of conifer forests by affecting the vascular tissues to increase their capacity for water transport. Our results shed a new light on the evolution of the vascular system of conifer forests and how they affect ecosystem productivity, and indicate the potential to

  12. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration

    PubMed Central

    Jones, Nathan D.; Lopez Jr, Miguel A.; Hanne, Jeungphill; Peake, Mitchell B.; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E.

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3′-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3′-hydroxyls into the target DNA separated by 4–6 bp. Host DNA repair restores the resulting 5′-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  13. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

    PubMed

    Jones, Nathan D; Lopez, Miguel A; Hanne, Jeungphill; Peake, Mitchell B; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  14. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  15. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

  16. A new structural framework for integrating replication protein A into DNA processing machinery

    SciTech Connect

    Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E; Heller, William T; Rambo, Robert P; Tainer, John A; Ivanov, Ivaylo; Chazin, Walter J

    2013-01-01

    By coupling the protection and organization of ssDNA with the recruitment and alignment of DNA processing factors, Replication Protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA manages to coordinate the biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA s DNA binding activity, combining small-angle x-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA s DNA-binding core. It has been long held that RPA engages ssDNA in three stages, but our data reveal that RPA undergoes two rather than three transitions as it binds ssDNA. In contrast to previous models, RPA is more compact when fully engaged on 20-30 nucleotides of ssDNA than when DNA-free, and there is no evidence for significant population of a highly compacted structure in the initial 8-10 nucleotide binding mode. These results provide a new framework for understanding the integration of ssDNA into DNA processing machinery and how binding partners may manipulate RPA architecture to gain access to the substrate.

  17. The Tip of the Tail Needle Affects the Rate of DNA Delivery by Bacteriophage P22

    PubMed Central

    Leavitt, Justin C.; Gogokhia, Lasha; Gilcrease, Eddie B.; Bhardwaj, Anshul; Cingolani, Gino; Casjens, Sherwood R.

    2013-01-01

    The P22-like bacteriophages have short tails. Their virions bind to their polysaccharide receptors through six trimeric tailspike proteins that surround the tail tip. These short tails also have a trimeric needle protein that extends beyond the tailspikes from the center of the tail tip, in a position that suggests that it should make first contact with the host’s outer membrane during the infection process. The base of the needle serves as a plug that keeps the DNA in the virion, but role of the needle during adsorption and DNA injection is not well understood. Among the P22-like phages are needle types with two completely different C-terminal distal tip domains. In the phage Sf6-type needle, unlike the other P22-type needle, the distal tip folds into a “knob” with a TNF-like fold, similar to the fiber knobs of bacteriophage PRD1 and Adenovirus. The phage HS1 knob is very similar to that of Sf6, and we report here its crystal structure which, like the Sf6 knob, contains three bound L-glutamate molecules. A chimeric P22 phage with a tail needle that contains the HS1 terminal knob efficiently infects the P22 host, Salmonella enterica, suggesting the knob does not confer host specificity. Likewise, mutations that should abrogate the binding of L-glutamate to the needle do not appear to affect virion function, but several different other genetic changes to the tip of the needle slow down potassium release from the host during infection. These findings suggest that the needle plays a role in phage P22 DNA delivery by controlling the kinetics of DNA ejection into the host. PMID:23951045

  18. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  19. Concentration of carp edema virus (CEV) DNA in koi tissues affected by koi sleepy disease (KSD).

    PubMed

    Adamek, Mikolaj; Jung-Schroers, Verena; Hellmann, John; Teitge, Felix; Bergmann, Sven Michael; Runge, Martin; Kleingeld, Dirk Willem; Way, Keith; Stone, David Michael; Steinhagen, Dieter

    2016-05-26

    Carp edema virus (CEV), the causative agent of 'koi sleepy disease' (KSD), appears to be spreading worldwide and to be responsible for losses in koi, ornamental varieties of the common carp Cyprinus carpio. Clinical signs of KSD include lethargic behaviour, swollen gills, sunken eyes and skin alterations and can easily be mistaken for other diseases, such as infection with cyprinid herpesvirus 3 (CyHV-3). To improve the future diagnosis of CEV infection and to provide a tool to better explore the relationship between viral load and clinical disease, we developed a specific quantitative PCR (qPCR) for strains of the virus known to infect koi carp. In samples from several clinically affected koi, CEV-specific DNA was present in a range from 1 to 2,046,000 copies, with a mean of 129,982 copies and a median of 45 copies per 250 ng of isolated DNA, but virus DNA could not be detected in all clinically affected koi. A comparison of the newly developed qPCR, which is based on a dual-labelled probe, to an existing end-point PCR procedure revealed higher specificity and sensitivity of the qPCR and demonstrated that the new protocol could improve CEV detection in koi. In addition to improved diagnosis, the newly developed qPCR test would be a useful research tool. For example, studies on the pathobiology of CEV could employ controlled infection experiments in which the development of clinical signs could be examined in parallel with a quantitative determination of virus load. PMID:27225208

  20. Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

    PubMed Central

    Prinsen, C F; Veerkamp, J H

    1998-01-01

    We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect. PMID:9425108

  1. Combinative exposure effect of radio frequency signals from CDMA mobile phones and aphidicolin on DNA integrity.

    PubMed

    Tiwari, R; Lakshmi, N K; Surender, V; Rajesh, A D V; Bhargava, S C; Ahuja, Y R

    2008-01-01

    The aim of present study is to assess DNA integrity on the effect of exposure to a radio frequency (RF) signal from Code Division Multiple Access (CDMA) mobile phones. Whole blood samples from six healthy male individuals were exposed for RF signals from a CDMA mobile phone for 1 h. Alkaline comet assay was performed to assess the DNA damage. The combinative exposure effect of the RF signals and APC at two concentrations on DNA integrity was studied. DNA repair efficiency of the samples was also studied after 2 h of exposure. The RF signals and APC (0.2 microg/ml) alone or in synergism did not have any significant DNA damage as compared to sham exposed. However, univariate analysis showed that DNA damage was significantly different among combinative exposure of RF signals and APC at 0.2 microg/ml (p < 0.05) and at 2 microg/ml (p < 0.02). APC at 2 microg/ml concentration also showed significant damage levels (p < 0.05) when compared to sham exposed. DNA repair efficiency also varied in a significant way in combinative exposure sets (p < 0.05). From these results, it appears that the repair inhibitor APC enhances DNA breaks at 2 microg/ml concentration and that the damage is possibly repairable. Thus, it can be inferred that the in vitro exposure to RF signals induces reversible DNA damage in synergism with APC. PMID:19037791

  2. Integration of DNA marker information into breeding value predictions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calves from seven breeds including 20 herds were genotyped with a reduced DNA marker panel for weaning weight. The marker panel used was derived using USMARC Cycle VII animals. The results from the current study suggest marker effects are not robust across breeds and that methodology exists to integ...

  3. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites.

    PubMed

    Serrao, Erik; Cherepanov, Peter; Engelman, Alan N

    2016-01-01

    Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients. PMID:27023428

  4. New insights into the transition pathway from nonspecific to specific complex of DNA with Escherichia coli integration host factor.

    PubMed

    Vivas, Paula; Kuznetsov, Serguei V; Ansari, Anjum

    2008-05-15

    To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by approximately 180 degrees . We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (Kuznetsov, S. V.; Sugimura, S.; Vivas, P.; Crothers, D. M.; Ansari, A. Proc. Natl. Acad. Sci. USA 2006, 103, 18515). Here, we probe the effect of varying [KCl], which affects the stability of the complex, on this rate-limiting step. We find that below approximately 250 mM KCl, the observed relaxation kinetics are from the unimolecular bending/unbending of DNA, and the relaxation rate kr is independent of [KCl]. Above approximately 300 mM KCl, dissociation of the IHF-DNA complex becomes significant, and the observed relaxation process includes contributions from the association/dissociation step, with kr decreasing with increasing [KCl]. The DNA bending step occurs with a positive activation enthalpy, despite the large negative enthalpy change reported for the specific IHF-DNA complex (Holbrook, J. A.; Tsodikov, O. V.; Saecker, R. M.; Record, M. T., Jr. J. Mol. Biol. 2001, 310, 379). Our conclusion from these studies is that in the uphill climb to the transition state, the DNA is kinked, but with no release of ions, as indicated by the salt-independent behavior of k(r) at low [KCl]. Any release of ions in the unimolecular process, together with conformational changes in the protein-DNA complex that facilitate

  5. Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner.

    PubMed

    Prados, Julien; Stenz, Ludwig; Somm, Emmanuel; Stouder, Christelle; Dayer, Alexandre; Paoloni-Giacobino, Ariane

    2015-01-01

    Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms. PMID:26244509

  6. Prenatal Exposure to DEHP Affects Spermatogenesis and Sperm DNA Methylation in a Strain-Dependent Manner

    PubMed Central

    Somm, Emmanuel; Stouder, Christelle; Dayer, Alexandre; Paoloni-Giacobino, Ariane

    2015-01-01

    Di-(2-ethylhexyl)phtalate (DEHP) is a plasticizer with endocrine disrupting properties found ubiquitously in the environment and altering reproduction in rodents. Here we investigated the impact of prenatal exposure to DEHP on spermatogenesis and DNA sperm methylation in two distinct, selected, and sequenced mice strains. FVB/N and C57BL/6J mice were orally exposed to 300 mg/kg/day of DEHP from gestation day 9 to 19. Prenatal DEHP exposure significantly decreased spermatogenesis in C57BL/6J (fold-change = 0.6, p-value = 8.7*10-4), but not in FVB/N (fold-change = 1, p-value = 0.9). The number of differentially methylated regions (DMRs) by DEHP-exposure across the entire genome showed increased hyper- and decreased hypo-methylation in C57BL/6J compared to FVB/N. At the promoter level, three important subsets of genes were massively affected. Promoters of vomeronasal and olfactory receptors coding genes globally followed the same trend, more pronounced in the C57BL/6J strain, of being hyper-methylated in DEHP related conditions. In contrast, a large set of micro-RNAs were hypo-methylated, with a trend more pronounced in the FVB/N strain. We additionally analyze both the presence of functional genetic variations within genes that were associated with the detected DMRs and that could be involved in spermatogenesis, and DMRs related with the DEHP exposure that affected both strains in an opposite manner. The major finding in this study indicates that prenatal exposure to DEHP can decrease spermatogenesis in a strain-dependent manner and affects sperm DNA methylation in promoters of large sets of genes putatively involved in both sperm chemotaxis and post-transcriptional regulatory mechanisms. PMID:26244509

  7. DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity

    PubMed Central

    Gonzalez-Huici, Victor; Szakal, Barnabas; Urulangodi, Madhusoodanan; Psakhye, Ivan; Castellucci, Federica; Menolfi, Demis; Rajakumara, Eerappa; Fumasoni, Marco; Bermejo, Rodrigo; Jentsch, Stefan; Branzei, Dana

    2014-01-01

    DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability. PMID:24473148

  8. A novel T-DNA integration in rice involving two interchromosomal translocations.

    PubMed

    Majhi, Bharat Bhusan; Shah, Jasmine M; Veluthambi, Karuppannan

    2014-06-01

    A male sterile transgenic rice plant TC-19 harboured a novel T-DNA integration in chromosome 8 with two interchromosomal translocations of 6.55 kb chromosome 3 and 29.8 kb chromosome 9 segments. We report a complex Agrobacterium T-DNA integration in rice (Oryza sativa) associated with two interchromosomal translocations. The T-DNA-tagged rice mutant TC-19, which harboured a single copy of the T-DNA, displayed male sterile phenotype in the homozygous condition. Analysis of the junctions between the T-DNA ends and the rice genome by genome walking showed that the right border is flanked by a chromosome 3 sequence and the left border is flanked by a chromosome 9 sequence. Upon further walking on chromosome 3, a chromosome 3/chromosome 8 fusion was detected. Genome walking from the opposite end of the chromosome 8 break point revealed a chromosome 8/chromosome 9 fusion. Our findings revealed that the T-DNA, together with a 6.55-kb region of chromosome 3 and a 29.8-kb region of chromosome 9, was translocated to chromosome 8. Southern blot analysis of the homozygous TC-19 mutant revealed that the native sequences of chromosome 3 and 9 were restored but the disruption of chromosome 8 in the first intron of the gene Os08g0152500 was not restored. The integration of the complex T-DNA in chromosome 8 caused male sterility. PMID:24487649

  9. SMARCAL1 maintains telomere integrity during DNA replication.

    PubMed

    Poole, Lisa A; Zhao, Runxiang; Glick, Gloria G; Lovejoy, Courtney A; Eischen, Christine M; Cortez, David

    2015-12-01

    The SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) DNA translocase is one of several related enzymes, including ZRANB3 (zinc finger, RAN-binding domain containing 3) and HLTF (helicase-like transcription factor), that are recruited to stalled replication forks to promote repair and restart replication. These enzymes can perform similar biochemical reactions such as fork reversal; however, genetic studies indicate they must have unique cellular activities. Here, we present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells using the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as elevated telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome-maintenance activity in bulk chromosomal replication because it does not require interaction with replication protein A. Finally, this telomere-maintenance function is not shared by ZRANB3 or HLTF. Our results provide the first identification, to our knowledge, of an endogenous source of replication stress that requires SMARCAL1 for resolution and define differences between members of this class of replication fork-repair enzymes. PMID:26578802

  10. Nutri-epigenomic Studies Related to Neural Tube Defects: Does Folate Affect Neural Tube Closure Via Changes in DNA Methylation?

    PubMed

    Rochtus, Anne; Jansen, Katrien; Van Geet, Chris; Freson, Kathleen

    2015-01-01

    Neural tube defects (NTDs), affecting 1-2 per 1000 pregnancies, are severe congenital malformations that arise from the failure of neurulation during early embryonic development. The methylation hypothesis suggests that folate prevents NTDs by stimulating cellular methylation reactions. Folate is central to the one-carbon metabolism that produces pyrimidines and purines for DNA synthesis and for the generation of the methyldonor S-adenosyl-methionine. This review focuses on the relation between the folate-mediated one-carbon metabolism, DNA methylation and NTDs. Studies will be discussed that investigated global or locus-specific DNA methylation differences in patients with NTDs. Folate deficiency may increase NTD risk by decreasing DNA methylation, but to date, human studies vary widely in study design in terms of analyzing different clinical subtypes of NTDs, using different methylation quantification assays and using DNA isolated from diverse types of tissues. Some studies have focused mainly on global DNA methylation differences while others have quantified specific methylation differences for imprinted genes, transposable elements and DNA repair enzymes. Findings of global DNA hypomethylation and LINE-1 hypomethylation suggest that epigenetic alterations may disrupt neural tube closure. However, current research does not support a linear relation between red blood cell folate concentration and DNA methylation. Further studies are required to better understand the interaction between folate, DNA methylation changes and NTDs. PMID:26349489

  11. Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis.

    PubMed

    Ahmed, Wareed; Menon, Shruti; Karthik, Pullela V; Nagaraja, Valakunja

    2015-02-01

    The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (TopoI) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. TopoI is essential for Mtb survival. However, the necessity of TopoI or other relaxases in Msm has not been investigated. To recognize the importance of TopoI for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of TopoI in Msm. The TopoI-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in TopoI level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the TopoI-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of TopoI in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in TopoI level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects. PMID:25516959

  12. Mutations affecting sensitivity of the cellular slime mold Dictyostelium discoideum to DNA-damaging agents.

    PubMed

    Bronner, C E; Welker, D L; Deering, R A

    1992-09-01

    We describe 22 new mutants of D. discoideum that are sensitive to DNA damage. These mutants were isolated on the basis of sensitivity to either temperature, gamma-rays, or 4-nitroquinolone-1-oxide (4NQO). The doses of gamma-rays, ultraviolet light (UV), and 4NQO required to reduce the survival of colony-forming ability of these mutants to 10% (D10) range from 2% to 100% of the D10s for the nonmutant, parent strains. For most of the mutants, those which are very sensitive to one agent are very sensitive to all agents tested and those which are moderately sensitive to one agent, are moderately sensitive to all agents tested. One mutant is sensitive only to 4NQO. Linkage relationships have been examined for 13 of these mutants. This linkage information was used to design complementation tests to determine allelism with previously characterized complementation groups affecting sensitivity to radiation. 4 of the new mutants fall within previously identified complementation groups and 3 new complementation groups have been identified (radJ, radK and radL). Other new loci probably also exist among these new mutants. This brings the number of characterized mutants of D. discoideum which are sensitive to DNA-damaging agents to 33 and the number of assigned complementation groups to 11. PMID:1380652

  13. Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules

    SciTech Connect

    Folger, K.R.; Wong, E.A.; Wahl, G.; Capecchi, M.R.

    1982-11-01

    The authors examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk/sup -/ and RAT-2tk/sup -/ cells to the TK/sup +/ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, the authors were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA.

  14. 3-base periodicity in coding DNA is affected by intercodon dinucleotides

    PubMed Central

    Sánchez, Joaquín

    2011-01-01

    All coding DNAs exhibit 3-base periodicity (TBP), which may be defined as the tendency of nucleotides and higher order n-tuples, e.g. trinucleotides (triplets), to be preferentially spaced by 3, 6, 9 etc, bases, and we have proposed an association between TBP and clustering of same-phase triplets. We here investigated if TBP was affected by intercodon dinucleotide tendencies and whether clustering of same-phase triplets was involved. Under constant protein sequence intercodon dinucleotide frequencies depend on the distribution of synonymous codons. So, possible effects were revealed by randomly exchanging synonymous codons without altering protein sequences to subsequently document changes in TBP via frequency distribution of distances (FDD) of DNA triplets. A tripartite positive correlation was found between intercodon dinucleotide frequencies, clustering of same-phase triplets and TBP. So, intercodon C|A (where “|” indicates the boundary between codons) was more frequent in native human DNA than in the codon-shuffled sequences; higher C|A frequency occurred along with more frequent clustering of C|AN triplets (where N jointly represents A, C, G and T) and with intense CAN TBP. The opposite was found for C|G, which was less frequent in native than in shuffled sequences; lower C|G frequency occurred together with reduced clustering of C|GN triplets and with less intense CGN TBP. We hence propose that intercodon dinucleotides affect TBP via same-phase triplet clustering. A possible biological relevance of our findings is briefly discussed. PMID:21814388

  15. An integrated encyclopedia of DNA elements in the human genome.

    PubMed

    2012-09-01

    The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. PMID:22955616

  16. An Integrated Encyclopedia of DNA Elements in the Human Genome

    PubMed Central

    2012-01-01

    Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

  17. Integrated digital error suppression for improved detection of circulating tumor DNA.

    PubMed

    Newman, Aaron M; Lovejoy, Alexander F; Klass, Daniel M; Kurtz, David M; Chabon, Jacob J; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V; Say, Carmen; Zhou, Li; Carter, Justin N; West, Robert B; Sledge, George W; Shrager, Joseph B; Loo, Billy W; Neal, Joel W; Wakelee, Heather A; Diehn, Maximilian; Alizadeh, Ash A

    2016-05-01

    High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by about threefold, and synergize when combined to yield ∼15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to non-small cell lung cancer (NSCLC) patients, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and >99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, our approach allowed monitoring of NSCLC ctDNA down to 4 in 10(5) cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

  18. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform

    PubMed Central

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-01-01

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ~36% for Chinese hamster ovary cells) and bacterial cells (up to ~45% for Salmonella typhimurium) that is comparable to the widely-used chemical lysis. The DNA extraction efficiency has dependence on both electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedure. PMID:23061629

  19. Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae)

    PubMed Central

    Montagna, Matteo; Mereghetti, Valeria; Lencioni, Valeria; Rossaro, Bruno

    2016-01-01

    Rapid and efficient DNA-based tools are recommended for the evaluation of the insect biodiversity of high-altitude streams. In the present study, focused principally on larvae of the genus Diamesa Meigen 1835 (Diptera: Chironomidae), the congruence between morphological/molecular delimitation of species as well as performances in taxonomic assignments were evaluated. A fragment of the mitochondrial cox1 gene was obtained from 112 larvae, pupae and adults (Diamesinae, Orthocladiinae and Tanypodinae) that were collected in different mountain regions of the Alps and Apennines. On the basis of morphological characters 102 specimens were attributed to 16 species, and the remaining ten specimens were identified to the genus level. Molecular species delimitation was performed using: i) distance-based Automatic Barcode Gap Discovery (ABGD), with no a priori assumptions on species identification; and ii) coalescent tree-based approaches as the Generalized Mixed Yule Coalescent model, its Bayesian implementation and Bayesian Poisson Tree Processes. The ABGD analysis, estimating an optimal intra/interspecific nucleotide distance threshold of 0.7%-1.4%, identified 23 putative species; the tree-based approaches, identified between 25–26 entities, provided nearly identical results. All species belonging to zernyi, steinboecki, latitarsis, bertrami, dampfi and incallida groups, as well as outgroup species, are recovered as separate entities, perfectly matching the identified morphospecies. In contrast, within the cinerella group, cases of discrepancy arose: i) the two morphologically separate species D. cinerella and D. tonsa are neither monophyletic nor diagnosable exhibiting low values of between-taxa nucleotide mean divergence (0.94%); ii) few cases of larvae morphological misidentification were observed. Head capsule color is confirmed to be a valid character able to discriminate larvae of D. zernyi, D. tonsa and D. cinerella, but it is here better defined as a color

  20. Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae).

    PubMed

    Montagna, Matteo; Mereghetti, Valeria; Lencioni, Valeria; Rossaro, Bruno

    2016-01-01

    Rapid and efficient DNA-based tools are recommended for the evaluation of the insect biodiversity of high-altitude streams. In the present study, focused principally on larvae of the genus Diamesa Meigen 1835 (Diptera: Chironomidae), the congruence between morphological/molecular delimitation of species as well as performances in taxonomic assignments were evaluated. A fragment of the mitochondrial cox1 gene was obtained from 112 larvae, pupae and adults (Diamesinae, Orthocladiinae and Tanypodinae) that were collected in different mountain regions of the Alps and Apennines. On the basis of morphological characters 102 specimens were attributed to 16 species, and the remaining ten specimens were identified to the genus level. Molecular species delimitation was performed using: i) distance-based Automatic Barcode Gap Discovery (ABGD), with no a priori assumptions on species identification; and ii) coalescent tree-based approaches as the Generalized Mixed Yule Coalescent model, its Bayesian implementation and Bayesian Poisson Tree Processes. The ABGD analysis, estimating an optimal intra/interspecific nucleotide distance threshold of 0.7%-1.4%, identified 23 putative species; the tree-based approaches, identified between 25-26 entities, provided nearly identical results. All species belonging to zernyi, steinboecki, latitarsis, bertrami, dampfi and incallida groups, as well as outgroup species, are recovered as separate entities, perfectly matching the identified morphospecies. In contrast, within the cinerella group, cases of discrepancy arose: i) the two morphologically separate species D. cinerella and D. tonsa are neither monophyletic nor diagnosable exhibiting low values of between-taxa nucleotide mean divergence (0.94%); ii) few cases of larvae morphological misidentification were observed. Head capsule color is confirmed to be a valid character able to discriminate larvae of D. zernyi, D. tonsa and D. cinerella, but it is here better defined as a color gradient

  1. Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.

    PubMed

    Woolley, A T; Hadley, D; Landre, P; deMello, A J; Mathies, R A; Northrup, M A

    1996-12-01

    Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems. PMID:8946790

  2. Effect of cooled storage on quality and DNA integrity of Asian elephant (Elephas maximus) spermatozoa.

    PubMed

    Imrat, P; Mahasawangkul, S; Gosálvez, J; Suthanmapinanth, P; Sombutputorn, P; Jansittiwate, S; Thongtip, N; Pinyopummin, A; Colenbrander, B; Holt, W V; Stout, T A E

    2012-01-01

    Artificial insemination (AI) is a potentially useful tool for breeding captive elephants because it facilitates efforts to minimise inbreeding. However, cooled storage of elephant semen markedly reduces fertility. This study compared the effects on semen-quality parameters, including sperm DNA fragmentation, of storing elephant semen at 4°C or 15°C in a commonly-used diluent (TEST) or a diluent developed to protect against sperm DNA damage (BullMax). Storing elephant semen for >24 h in either extender at either temperature resulted in decreases in sperm motility, viability, acrosome integrity and DNA integrity (P < 0.05); the decrease in motility was especially rapid. A subjective impression of circular sperm movement in TEST was confirmed by a higher curvilinear velocity and amplitude of lateral head displacement, but lower straight-line velocity and linearity than in BullMax. Initial percentages of spermatozoa with fragmented DNA (%SDF) did not differ between extenders or temperatures, but the rate of increase in %SDF during a 48-h incubation at 37°C was higher in TEST than in BullMax (P < 0.05). In conclusion, BullMax allows more linear movement and better preserves DNA stability of stored elephant spermatozoa than TEST. Sperm DNA stability during incubation at 37°C is a promising, discriminative parameter for selecting semen storage conditions of bulls for elephant AI. PMID:22951013

  3. The structure of adenovirus type 12 DNA integration sites in the hamster cell genome.

    PubMed Central

    Knoblauch, M; Schröer, J; Schmitz, B; Doerfler, W

    1996-01-01

    Foreign DNA can integrate into the genomes of mammalian cells, and this process plays major roles in viral oncogenesis and in the generation of transgenic organisms and will be important in evolving regimens for human somatic gene therapy. In the present study, the insertion sites of adenovirus type 12 (Ad12) DNA genomes have been analyzed in detail in the Ad12-transformed hamster cell line T637, its revertants, which have lost most of the >20 Ad12 genome equivalents integrated chromosomally in cell line T637, and in the Ad12-induced tumor T191. Some of these junction sites have been molecularly cloned, and the nucleotide sequences at the sites of transition between viral and cellular DNAs have been determined. The sites of linkage between the hamster cellular and the foreign (viral) DNA are characterized by the frequent occurrence of patch homologies between the recombination partners. The cellular junction sites investigated here are not transcriptionally active. One of the cellular DNA sequences abutting the right Ad12 DNA terminus in cell line T637 (os2) is represented only once in the hamster genome and has a strikingly low abundance of 5'-CG-3' dinucleotide sequences. One 5'-GCGC-3' sequence close to the Ad12 DNA integration site is heavily methylated in normal cells, Ad12-transformed cells, and Ad12-induced tumor cells. The second such sequence is more remote from the junction site, is partly methylated in BHK21 hamster cells, and shows differences in methylation in different Ad12-transformed cell lines. This site is unmethylated in liver DNA. The cellular DNA sequence at the site of Ad12 linkage in the tumor T191 exhibits homologies to highly repetitive sequences of the Alu family and to an origin of hamster DNA replication containing an Alu element. A number of junction sites between Ad12 DNA and hamster or mouse DNA in Ad12-transformed cell lines or Ad12-induced tumor cell lines, investigated here and previously, are characterized by stem-loop structures

  4. An integrated microfluidic system for bovine DNA purification and digital PCR detection.

    PubMed

    Tian, Qingchang; Mu, Ying; Xu, Yanan; Song, Qi; Yu, Bingwen; Ma, Congcong; Jin, Wei; Jin, Qinhan

    2015-12-15

    In this paper, we described an integrated modularized microfluidic system that contained two distinct functional modules, one for nucleic acids (NA) extraction and the other for digital PCR (dPCR), allowing for detecting the bovine DNA in ovine tissue. PMID:26364950

  5. Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death

    PubMed Central

    Sun, Luxi; Tan, Rong; Xu, Jianquan; LaFace, Justin; Gao, Ying; Xiao, Yanchun; Attar, Myriam; Neumann, Carola; Li, Guo-Min; Su, Bing; Liu, Yang; Nakajima, Satoshi; Levine, Arthur S.; Lan, Li

    2015-01-01

    Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage. PMID:26082495

  6. An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules.

    PubMed

    Lam, Liza; Sakakihara, Shouichi; Ishizuka, Koji; Takeuchi, Shoji; Noji, Hiroyuki

    2007-12-01

    A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically. The critical parameters for electrostretching of encapsulated DNA were investigated and optimum stretching was achieved by using 1.5 kHz pulses with electric field strength in the order of 10(3) V cm(-1) in 7% linear polyacrylamide (LPA) solution. LPA was adopted to minimize the adverse effects of ionic thermal agitation on molecular dielectrophoretic elongation in the microchamber. In our experiments, as the fragments were not immobilized throughout the entire protocol, it was found from repeated tests that digestion always occurred, producing the expected number of cleaved fragments. This versatile microchamber approach realized direct observation of these biological reactions on real-time basis at a single-molecule level. Furthermore, with the employment of porous polyacrylamide gel, the effective manipulation of DNA assays and the ability to combine conventionally independent bioanalytical processes have been demonstrated. PMID:18030395

  7. Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways

    PubMed Central

    Giordano, L; Deceglie, S; d'Adamo, P; Valentino, M L; La Morgia, C; Fracasso, F; Roberti, M; Cappellari, M; Petrosillo, G; Ciaravolo, S; Parente, D; Giordano, C; Maresca, A; Iommarini, L; Del Dotto, V; Ghelli, A M; Salomao, S R; Berezovsky, A; Belfort, R; Sadun, A A; Carelli, V; Loguercio Polosa, P; Cantatore, P

    2015-01-01

    Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that

  8. DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity

    PubMed Central

    Lawrence, Katherine S.; Chau, Thinh; Engebrecht, JoAnne

    2015-01-01

    Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved. PMID:25898113

  9. Pedagogical Factors Affecting Integration of Computers in Mathematics Instruction in Secondary Schools in Kenya

    ERIC Educational Resources Information Center

    Wanjala, Martin M. S.; Aurah, Catherine M.; Symon, Koros C.

    2015-01-01

    The paper reports findings of a study which sought to examine the pedagogical factors that affect the integration of computers in mathematics instruction as perceived by teachers in secondary schools in Kenya. This study was based on the Technology Acceptance Model (TAM). A descriptive survey design was used for this study. Stratified and simple…

  10. The Views of Mathematics Teachers on the Factors Affecting the Integration of Technology in Mathematics Courses

    ERIC Educational Resources Information Center

    Kaleli-Yilmaz, Gül

    2015-01-01

    The aim of this study was to determine the views of mathematics teachers on the factors that affect the integration of technology in mathematic courses. It is a qualitative case study. The sample size of the study is 10 teachers who are receiving postgraduate education in a university in Turkey. The current study was conducted in three stages. At…

  11. An Investigation of Relationships between Internal and External Factors Affecting Technology Integration in Classrooms

    ERIC Educational Resources Information Center

    Hur, Jung Won; Shannon, David; Wolf, Sara

    2016-01-01

    Various factors affecting technology integration have been identified, but little research has examined the relationships between factors, especially internal and external ones, and whether they directly or indirectly influenced each other. To fill this research gap, this study examined the significance and relationships of five factors…

  12. Emotional Language Processing: How Mood Affects Integration Processes during Discourse Comprehension

    ERIC Educational Resources Information Center

    Egidi, Giovanna; Nusbaum, Howard C.

    2012-01-01

    This research tests whether mood affects semantic processing during discourse comprehension by facilitating integration of information congruent with moods' valence. Participants in happy, sad, or neutral moods listened to stories with positive or negative endings during EEG recording. N400 peak amplitudes showed mood congruence for happy and sad…

  13. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  14. Genome Sequencing of Autism-Affected Families Reveals Disruption of Putative Noncoding Regulatory DNA

    PubMed Central

    Turner, Tychele N.; Hormozdiari, Fereydoun; Duyzend, Michael H.; McClymont, Sarah A.; Hook, Paul W.; Iossifov, Ivan; Raja, Archana; Baker, Carl; Hoekzema, Kendra; Stessman, Holly A.; Zody, Michael C.; Nelson, Bradley J.; Huddleston, John; Sandstrom, Richard; Smith, Joshua D.; Hanna, David; Swanson, James M.; Faustman, Elaine M.; Bamshad, Michael J.; Stamatoyannopoulos, John; Nickerson, Deborah A.; McCallion, Andrew S.; Darnell, Robert; Eichler, Evan E.

    2016-01-01

    We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism. For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES). We integrated multiple CNV and SNV analyses and extensive experimental validation to identify additional candidate mutations in eight families. We report that compared to control individuals, probands showed a significant (p = 0.03) enrichment of de novo and private disruptive mutations within fetal CNS DNase I hypersensitive sites (i.e., putative regulatory regions). This effect was only observed within 50 kb of genes that have been previously associated with autism risk, including genes where dosage sensitivity has already been established by recurrent disruptive de novo protein-coding mutations (ARID1B, SCN2A, NR3C2, PRKCA, and DSCAM). In addition, we provide evidence of gene-disruptive CNVs (in DISC1, WNT7A, RBFOX1, and MBD5), as well as smaller de novo CNVs and exon-specific SNVs missed by exome sequencing in neurodevelopmental genes (e.g., CANX, SAE1, and PIK3CA). Our results suggest that the detection of smaller, often multiple CNVs affecting putative regulatory elements might help explain additional risk of simplex autism. PMID:26749308

  15. Genome Sequencing of Autism-Affected Families Reveals Disruption of Putative Noncoding Regulatory DNA.

    PubMed

    Turner, Tychele N; Hormozdiari, Fereydoun; Duyzend, Michael H; McClymont, Sarah A; Hook, Paul W; Iossifov, Ivan; Raja, Archana; Baker, Carl; Hoekzema, Kendra; Stessman, Holly A; Zody, Michael C; Nelson, Bradley J; Huddleston, John; Sandstrom, Richard; Smith, Joshua D; Hanna, David; Swanson, James M; Faustman, Elaine M; Bamshad, Michael J; Stamatoyannopoulos, John; Nickerson, Deborah A; McCallion, Andrew S; Darnell, Robert; Eichler, Evan E

    2016-01-01

    We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism. For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES). We integrated multiple CNV and SNV analyses and extensive experimental validation to identify additional candidate mutations in eight families. We report that compared to control individuals, probands showed a significant (p = 0.03) enrichment of de novo and private disruptive mutations within fetal CNS DNase I hypersensitive sites (i.e., putative regulatory regions). This effect was only observed within 50 kb of genes that have been previously associated with autism risk, including genes where dosage sensitivity has already been established by recurrent disruptive de novo protein-coding mutations (ARID1B, SCN2A, NR3C2, PRKCA, and DSCAM). In addition, we provide evidence of gene-disruptive CNVs (in DISC1, WNT7A, RBFOX1, and MBD5), as well as smaller de novo CNVs and exon-specific SNVs missed by exome sequencing in neurodevelopmental genes (e.g., CANX, SAE1, and PIK3CA). Our results suggest that the detection of smaller, often multiple CNVs affecting putative regulatory elements might help explain additional risk of simplex autism. PMID:26749308

  16. Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)

    PubMed Central

    2012-01-01

    Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants. PMID:23006412

  17. The effects of pyridaben pesticide on the DNA integrity of sperms and early in vitro embryonic development in mice

    PubMed Central

    Ebadi Manas, Ghodrat; Hasanzadeh, Shapour; Najafi, Golamreza; Parivar, Kazem; Yaghmaei, Parichehr

    2013-01-01

    Background: Pyridaben, a pyridazinone derivative, is a new acaricide and insecticide for control of mites and some insects such as white flies, aphids and thrips. Objective: This study was designed to elucidate how pyridaben can affect the sperms' morphological parameters, its DNA integrity, and to estimate the effect of various quantities of pyridaben on in vitro fertilization rate. Materials and Methods: In this study, 80 adult male Balb/C strain mice were used. Animals were divided into control and two test groups. Control group received distilled water. The test group was divided into two subgroups, viz, high dose (212 mg/kg/day) and low dose (53 mg/kg/day) and they received the pyridaben, orally for duration of 45 days. The spermatozoa were obtained from caudae epididymides on day 45 in all groups. Sperm viability, protamin compression (nuclear maturity), DNA double-strand breaks, and in vitro fertilizing (IVF) ability were examined. Results: The pyridaben treatment provoked a significant decrease in sperm population and viability in epididymides. The data obtained from this experiment revealed that, the pyridaben brings about negative impact on the sperm maturation and DNA integrity in a time-dependent manner, which consequently caused a significant (p<0.05) reduction in IVF capability. Embryo developing arrest was significantly (p<0.05) higher in treated than the control group. Conclusion: Theses results confirmed that, the pyridaben is able to induce DNA damage and chromatin abnormalities in spermatozoa which were evident by low IVF rate. This article extracted from Ph.D. thesis. (Ghodrat Ebadi Mans) PMID:24639796

  18. Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

    PubMed

    Gual-Frau, Josep; Abad, Carlos; Amengual, María J; Hannaoui, Naim; Checa, Miguel A; Ribas-Maynou, Jordi; Lozano, Iris; Nikolaou, Alexandros; Benet, Jordi; García-Peiró, Agustín; Prats, Juan

    2015-09-01

    Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 μg vitamin B9, 1 μg vitamin B12, 10 mg zinc, 50 μg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment. PMID:26090928

  19. MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status

    PubMed Central

    Attimonelli, M.; Altamura, N.; Benne, R.; Brennicke, A.; Cooper, J. M.; D’Elia, D.; Montalvo, A. de; Pinto, B. de; De Robertis, M.; Golik, P.; Knoop, V.; Lanave, C.; Lazowska, J.; Licciulli, F.; Malladi, B. S.; Memeo, F.; Monnerot, M.; Pasimeni, R.; Pilbout, S.; Schapira, A. H. V.; Sloof, P.; Saccone, C.

    2000-01-01

    MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbase.pl . The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme. PMID:10592207

  20. DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion.

    PubMed

    Suhasini, Avvaru N; Sommers, Joshua A; Yu, Stephen; Wu, Yuliang; Xu, Ting; Kelman, Zvi; Kaplan, Daniel L; Brosh, Robert M

    2012-06-01

    DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions. PMID:22500020

  1. Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

    NASA Astrophysics Data System (ADS)

    Sherman, Paula A.; Fyfe, James A.

    1990-07-01

    The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

  2. The Impact of Affect on Out-Group Judgments Depends on Dominant Information-Processing Styles: Evidence From Incidental and Integral Affect Paradigms.

    PubMed

    Isbell, Linda M; Lair, Elicia C; Rovenpor, Daniel R

    2016-04-01

    Two studies tested the affect-as-cognitive-feedback model, in which positive and negative affective states are not uniquely associated with particular processing styles, but rather serve as feedback about currently accessible processing styles. The studies extend existing work by investigating (a) both incidental and integral affect, (b) out-group judgments, and (c) downstream consequences. We manipulated processing styles and either incidental (Study 1) or integral (Study 2) affect and measured perceptions of out-group homogeneity. Positive (relative to negative) affect increased out-group homogeneity judgments when global processing was primed, but under local priming, the effect reversed (Studies 1 and 2). A similar interactive effect emerged on attributions, which had downstream consequences for behavioral intentions (Study 2). These results demonstrate that both incidental and integral affect do not directly produce specific processing styles, but rather influence thinking by providing feedback about currently accessible processing styles. PMID:26984013

  3. DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity.

    PubMed

    Wang, Y; Sun, H; Wang, J; Wang, H; Meng, L; Xu, C; Jin, M; Wang, B; Zhang, Y; Zhang, Y; Zhu, T

    2016-01-01

    EZH2 is a histone methyltransferase whose functions in stem cells and tumor cells are well established. Accumulating evidence shows that EZH2 has critical roles in T cells and could be a promising therapeutic target for several immune diseases. To further reveal the novel functions of EZH2 in human T cells, protein co-immunoprecipitation combined mass spectrometry was conducted and several previous unknown EZH2-interacting proteins were identified. Of them, we focused on a DNA damage responsive protein, Ku80, because of the limited knowledge regarding EZH2 in the DNA damage response. Then, we demonstrated that instead of being methylated by EZH2, Ku80 bridges the interaction between the DNA-dependent protein kinase (DNA-PK) complex and EZH2, thus facilitating EZH2 phosphorylation. Moreover, EZH2 histone methyltransferase activity was enhanced when Ku80 was knocked down or DNA-PK activity was inhibited, suggesting DNA-PK-mediated EZH2 phosphorylation impairs EZH2 histone methyltransferase activity. On the other hand, EZH2 inhibition increased the DNA damage level at the late phase of T-cell activation, suggesting EZH2 involved in genomic integrity maintenance. In conclusion, our study is the first to demonstrate that EZH2 is phosphorylated by the DNA damage responsive complex DNA-PK and regulates DNA damage-mediated T-cell apoptosis, which reveals a novel functional crosstalk between epigenetic regulation and genomic integrity. PMID:27468692

  4. DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity

    PubMed Central

    Wang, Y; Sun, H; Wang, J; Wang, H; Meng, L; Xu, C; Jin, M; Wang, B; Zhang, Y; Zhang, Y; Zhu, T

    2016-01-01

    EZH2 is a histone methyltransferase whose functions in stem cells and tumor cells are well established. Accumulating evidence shows that EZH2 has critical roles in T cells and could be a promising therapeutic target for several immune diseases. To further reveal the novel functions of EZH2 in human T cells, protein co-immunoprecipitation combined mass spectrometry was conducted and several previous unknown EZH2-interacting proteins were identified. Of them, we focused on a DNA damage responsive protein, Ku80, because of the limited knowledge regarding EZH2 in the DNA damage response. Then, we demonstrated that instead of being methylated by EZH2, Ku80 bridges the interaction between the DNA-dependent protein kinase (DNA-PK) complex and EZH2, thus facilitating EZH2 phosphorylation. Moreover, EZH2 histone methyltransferase activity was enhanced when Ku80 was knocked down or DNA-PK activity was inhibited, suggesting DNA-PK-mediated EZH2 phosphorylation impairs EZH2 histone methyltransferase activity. On the other hand, EZH2 inhibition increased the DNA damage level at the late phase of T-cell activation, suggesting EZH2 involved in genomic integrity maintenance. In conclusion, our study is the first to demonstrate that EZH2 is phosphorylated by the DNA damage responsive complex DNA-PK and regulates DNA damage-mediated T-cell apoptosis, which reveals a novel functional crosstalk between epigenetic regulation and genomic integrity. PMID:27468692

  5. Super DNAging-New insights into DNA integrity, genome stability and telomeres in the oldest old.

    PubMed

    Franzke, Bernhard; Neubauer, Oliver; Wagner, Karl-Heinz

    2015-01-01

    Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to "averagely" aged humans. These inverse observations in nonagenarians (90-99 years), centenarians (100-109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in "super old" humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in "successful agers". There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching "super old" age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere

  6. Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas

    PubMed Central

    2013-01-01

    Background High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear. Results We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability. Conclusions Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas. PMID:24345474

  7. Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA.

    PubMed

    Lowe, Brenda A; Shiva Prakash, N; Way, Melissa; Mann, Michael T; Spencer, T Michael; Boddupalli, Raghava S

    2009-12-01

    Transgene copy number is an important criterion for determining the utility of transgenic events. Single copy integration events are highly desirable when the objective is to produce marker free plants through segregation or when it is necessary to introgress different transgenes into commercial cultivars from different transgenic events. In contrast multi-copy events are advocated by several authors for higher expression of the transgene. Till recently, it was thought that employment of the particle gun for transformation results in the production of a high proportion of multi-copy events often with complex integration pattern when compared to Agrobacterium-mediated transformation. However, it has been demonstrated that usage of cassette DNA for bombardment in place of whole plasmids would result in simple insertion pattern of the transgenes. While investigating the effect of varying the cassette DNA amount on stable transformation, the frequency of occurrence of low copy events was observed to increase when lower doses of cassette DNA was employed for bombardment. Large scale experimentation with rigorous statistical analysis performed to verify the above observations employing Helium gun and the Electric discharge gun for gene delivery confirmed the above observations. Helium gun experiments involving production of more than 1,600 corn events consistently yielded single copy events at higher frequencies at lower cassette DNA load (46% at 2.5 ng/shot) as compared to higher cassette DNA load (29% at 25 ng/shot) across 18 independent experiments. Results were nearly identical with the Electric discharge particle gun device where single copy events were recovered at frequencies of 54% at 2.5 ng cassettes DNA per shot as compared to 18% at 25 ng cassette DNA per shot. The transformation frequency declined from 41 to 34% (Helium gun) and from 48 to 31% (Electric discharge gun) with reduction in cassette DNA quantity from 25 to 2.5 ng per shot. This reduction in the

  8. Benefits of Selenium Supplementation on Leukocyte DNA Integrity Interact with Dietary Micronutrients: A Short Communication

    PubMed Central

    Karunasinghe, Nishi; Zhu, Shuotun; Ferguson, Lynnette R.

    2016-01-01

    A male cohort from New Zealand has previously shown variability in Selenium (Se) supplementation effects on measured biomarkers. The current analysis is to understand the reasons for variability of the H2O2-induced DNA damage recorded after Se supplementation. We have looked at the variation of demographic, lifestyle, medication, genetic and dietary factors and biomarkers measured at baseline and post-supplementation in these two extreme subgroups A and B. Group A showed increased H2O2-induced DNA damage and group B showed decreased damage after Se supplementation. We have also considered correlations of biomarkers and dietary factors in the complete dataset. The glutathione peroxidase (GPx) activity and DNA damage were significantly lower at post-supplementation in Group B compared to Group A. Post-supplementation, Group B showed a significant reduction in the GPx activity, while Group A showed a significant increase in DNA damage compared to baseline levels. Dietary methionine intake was significantly higher and folate intake was significantly lower in Group B compared to Group A. Se supplementation significantly increased the caspase-cleaved keratin 18 levels in both groups, indicating increased apoptotic potential of this supplement. Parameter correlation with the complete dataset showed dietary methionine to have a significant negative correlation with H2O2-induced DNA damage post-supplementation. The data suggest that Se supplementation is beneficial for the leukocyte DNA integrity only in interaction with the dietary methionine and folate intake. PMID:27128937

  9. Nutriomes and personalised nutrition for DNA damage prevention, telomere integrity maintenance and cancer growth control.

    PubMed

    Fenech, Michael F

    2014-01-01

    DNA damage at the base sequence and chromosome level is a fundamental cause of developmental and degenerative diseases. Multiple micronutrients and their interactions with the inherited and/or acquired genome determine DNA damage and genomic instability rates. The challenge is to identify for each individual the combination of micronutrients and their doses (i.e. the nutriome) that optimises genome stability, including telomere integrity and functionality and DNA repair. Using nutrient array systems with high-content analysis diagnostics of DNA damage, cell death and cell growth, it is possible to define, on an individual basis, the optimal nutriome for DNA damage prevention and cancer growth control. This knowledge can also be used to improve culture systems for cells used in therapeutics such as stem cells to ensure that they are not genetically aberrant when returned to the body. Furthermore, this information could be used to design dietary patterns that deliver the micronutrient combinations and concentrations required for preventing DNA damage by micronutrient deficiency or excess. Using this approach, new knowledge could be obtained to identify the dietary restrictions and/or supplementations required to control specific cancers, which is particularly important given that reliable validated advice is not yet available for those diagnosed with cancer. PMID:24114494

  10. Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

    PubMed

    Yao, Dongbao; Song, Tingjie; Sun, Xianbao; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures. PMID:26485090

  11. Early life trauma is associated with altered white matter integrity and affective control.

    PubMed

    Corbo, Vincent; Amick, Melissa A; Milberg, William P; McGlinchey, Regina E; Salat, David H

    2016-08-01

    Early life trauma (ELT) has been shown to impair affective control and attention well into adulthood. Neuroimaging studies have further shown that ELT was associated with decreased white matter integrity in the prefrontal areas in children and adults. However, no study to date has looked at the relationship between white matter integrity and affective control in individuals with and without a history of ELT. To examine this, we tested 240 Veterans with (ELT N = 80) and without (NoELT N = 160) a history of childhood sexual abuse, physical abuse or family violence. Affective control was measured with the Affective Go/No-Go (AGN) and attention was indexed with the Test of Variable Attention (TOVA). White matter integrity was measured using fractional anisotropy (FA). Results showed greater number of errors on the AGN in ELT compared to NoELT. There was no difference on the TOVA. While there were no mean differences in FA, there was an interaction between FA and reaction time to positive stimuli on the AGN where the ELT group showed a positive relationship between FA and reaction time in right frontal and prefrontal areas, whereas the NoELT group showed a negative or no association between FA and reaction time. This suggests that ELT may be associated with a distinct brain-behavior relationship that could be related to other determinants of FA than those present in healthy adults. PMID:27214523

  12. Integrating Learning Styles and Personality Traits into an Affective Model to Support Learner's Learning

    NASA Astrophysics Data System (ADS)

    Leontidis, Makis; Halatsis, Constantin

    The aim of this paper is to present a model in order to integrate the learning style and the personality traits of a learner into an enhanced Affective Style which is stored in the learner’s model. This model which can deal with the cognitive abilities as well as the affective preferences of the learner is called Learner Affective Model (LAM). The LAM is used to retain learner’s knowledge and activities during his interaction with a Web-based learning environment and also to provide him with the appropriate pedagogical guidance. The proposed model makes use of an ontological approach in combination with the Bayesian Network model and contributes to the efficient management of the LAM in an Affective Module.

  13. Speed matters: How subtle changes in DNA end resection rate affect repair

    PubMed Central

    Huertas, Pablo; Cruz-García, Andrés

    2015-01-01

    The contribution of BRCA1 (breast cancer 1) to the repair of broken DNA is well established, but its real role at the molecular level is less well understood. By developing a new high-resolution, single-molecule technique, we have now shown that BRCA1 accelerates the processing of DNA breaks that subsequently engage in homologous recombination. PMID:27308460

  14. Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

    PubMed Central

    Risseeuw, E; Franke-van Dijk, M E; Hooykaas, P J

    1996-01-01

    Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector. PMID:8816506

  15. Urine Cell-Free DNA Integrity as a Marker for Early Prostate Cancer Diagnosis: A Pilot Study

    PubMed Central

    Casadio, Valentina; Calistri, Daniele; Salvi, Samanta; Gunelli, Roberta; Carretta, Elisa; Amadori, Dino; Silvestrini, Rosella; Zoli, Wainer

    2013-01-01

    Circulating cell-free DNA has been recognized as an accurate marker for the diagnosis of prostate cancer, whereas the role of urine cell-free DNA (UCF DNA) has never been evaluated in this setting. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. We thus verified the potential role of UCF DNA integrity for early prostate cancer diagnosis. UCF DNA was isolated from 29 prostate cancer patients and 25 healthy volunteers. Sequences longer than 250 bp (c-Myc, BCAS1, and HER2) were quantified by real-time PCR to verify UCF DNA integrity. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.7959 (95% CI 0.6729–0.9188). At the best cut-off value of 0.04 ng/μL, UCF DNA integrity analysis showed a sensitivity of 0.79 (95% CI 0.62–0.90) and a specificity of 0.84 (95% CI 0.65–0.94). These preliminary findings indicate that UCF DNA integrity could be a promising noninvasive marker for the early diagnosis of prostate cancer and pave the way for further research into this area. PMID:23509700

  16. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

    PubMed

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-03-01

    The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication

  17. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

    PubMed Central

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-01-01

    The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

  18. Mutations that affect production of branched RNA-linked msDNA in Myxococcus xanthus.

    PubMed Central

    Dhundale, A; Furuichi, T; Inouye, M; Inouye, S

    1988-01-01

    A deletion mutation of the gene (msd-msr) for the branched RNA-linked msDNA of Myxococcus xanthus was constructed by replacing the chromosomal 0.7-kilobase (kb) SmaI-XhoI fragment encompassing msd-msr with a 1.4-kb fragment carrying a gene for kanamycin resistance. It was found that this deletion strain (delta msSX) could not produce msDNA, although it still contained another species of msDNA, mrDNA (msDNA, reduced size). No apparent differences between delta msSX and the wild-type strain were observed in terms of cell growth, morphogenesis, fruiting-body formation, or motility. Both a deletion mutation at the region 100 base pairs upstream of msd and an insertion mutation at a site 500 base pairs upstream of msd showed a significant reduction of msDNA production, indicating that there is a cis- or trans-acting positive element in this region. When the 3.5-kb BamHI fragment carrying msd-msr from Stigmatella aurantiaca was inserted into the M. xanthus chromosome, the S. aurantiaca msDNA was found to be produced in M. xanthus. Images PMID:2461359

  19. Integration of hepatitis B virus DNA in chromosome-specific satellite sequences

    SciTech Connect

    Shaul, Y.; Garcia, P.D.; Schonberg, S.; Rutter, W.J.

    1986-09-01

    The authors previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. They report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence.

  20. Integrated biochip for PCR-based DNA amplification and detection on capacitive biosensors

    NASA Astrophysics Data System (ADS)

    Moschou, D.; Vourdas, N.; Filippidou, M. K.; Tsouti, V.; Kokkoris, G.; Tsekenis, G.; Zergioti, I.; Chatzandroulis, S.; Tserepi, A.

    2013-05-01

    Responding to an increasing demand for LoC devices to perform bioanalytical protocols for disease diagnostics, the development of an integrated LoC device consisting of a μPCR module integrated with resistive microheaters and a biosensor array for disease diagnostics is presented. The LoC is built on a Printed Circuit Board (PCB) platform, implementing both the amplification of DNA samples and DNA detection/identification on-chip. The resistive microheaters for PCR and the wirings for the sensor read-out are fabricated by means of standard PCB technology. The microfluidic network is continuous-flow, designed to perform 30 PCR cycles with heated zones at constant temperatures, and is built onto the PCB utilizing commercial photopatternable polyimide layers. Following DNA amplification, the product is driven in a chamber where a Si-based biosensor array is placed for DNA detection through hybridization. The sensor array is tested for the detection of mutations of the KRAS gene, responsible for colon cancer.

  1. Integrative Processing of Touch and Affect in Social Perception: An fMRI Study.

    PubMed

    Ebisch, Sjoerd J H; Salone, Anatolia; Martinotti, Giovanni; Carlucci, Leonardo; Mantini, Dante; Perrucci, Mauro G; Saggino, Aristide; Romani, Gian Luca; Di Giannantonio, Massimo; Northoff, Georg; Gallese, Vittorio

    2016-01-01

    Social perception commonly employs multiple sources of information. The present study aimed at investigating the integrative processing of affective social signals. Task-related and task-free functional magnetic resonance imaging was performed in 26 healthy adult participants during a social perception task concerning dynamic visual stimuli simultaneously depicting facial expressions of emotion and tactile sensations that could be either congruent or incongruent. Confounding effects due to affective valence, inhibitory top-down influences, cross-modal integration, and conflict processing were minimized. The results showed that the perception of congruent, compared to incongruent stimuli, elicited enhanced neural activity in a set of brain regions including left amygdala, bilateral posterior cingulate cortex (PCC), and left superior parietal cortex. These congruency effects did not differ as a function of emotion or sensation. A complementary task-related functional interaction analysis preliminarily suggested that amygdala activity depended on previous processing stages in fusiform gyrus and PCC. The findings provide support for the integrative processing of social information about others' feelings from manifold bodily sources (sensory-affective information) in amygdala and PCC. Given that the congruent stimuli were also judged as being more self-related and more familiar in terms of personal experience in an independent sample of participants, we speculate that such integrative processing might be mediated by the linking of external stimuli with self-experience. Finally, the prediction of task-related responses in amygdala by intrinsic functional connectivity between amygdala and PCC during a task-free state implies a neuro-functional basis for an individual predisposition for the integrative processing of social stimulus content. PMID:27242474

  2. Integrative Processing of Touch and Affect in Social Perception: An fMRI Study

    PubMed Central

    Ebisch, Sjoerd J. H.; Salone, Anatolia; Martinotti, Giovanni; Carlucci, Leonardo; Mantini, Dante; Perrucci, Mauro G.; Saggino, Aristide; Romani, Gian Luca; Di Giannantonio, Massimo; Northoff, Georg; Gallese, Vittorio

    2016-01-01

    Social perception commonly employs multiple sources of information. The present study aimed at investigating the integrative processing of affective social signals. Task-related and task-free functional magnetic resonance imaging was performed in 26 healthy adult participants during a social perception task concerning dynamic visual stimuli simultaneously depicting facial expressions of emotion and tactile sensations that could be either congruent or incongruent. Confounding effects due to affective valence, inhibitory top–down influences, cross-modal integration, and conflict processing were minimized. The results showed that the perception of congruent, compared to incongruent stimuli, elicited enhanced neural activity in a set of brain regions including left amygdala, bilateral posterior cingulate cortex (PCC), and left superior parietal cortex. These congruency effects did not differ as a function of emotion or sensation. A complementary task-related functional interaction analysis preliminarily suggested that amygdala activity depended on previous processing stages in fusiform gyrus and PCC. The findings provide support for the integrative processing of social information about others’ feelings from manifold bodily sources (sensory-affective information) in amygdala and PCC. Given that the congruent stimuli were also judged as being more self-related and more familiar in terms of personal experience in an independent sample of participants, we speculate that such integrative processing might be mediated by the linking of external stimuli with self-experience. Finally, the prediction of task-related responses in amygdala by intrinsic functional connectivity between amygdala and PCC during a task-free state implies a neuro-functional basis for an individual predisposition for the integrative processing of social stimulus content. PMID:27242474

  3. Parameters affecting temporal resolution of Time Resolved Integrative Optical Neutron Detector (TRION)

    NASA Astrophysics Data System (ADS)

    Mor, I.; Vartsky, D.; Dangendorf, V.; Bar, D.; Feldman, G.; Goldberg, M. B.; Tittelmeier, K.; Bromberger, B.; Brandis, M.; Weierganz, M.

    2013-11-01

    The Time-Resolved Integrative Optical Neutron (TRION) detector was developed for Fast Neutron Resonance Radiography (FNRR), a fast-neutron transmission imaging method that exploits characteristic energy-variations of the total scattering cross-section in the En = 1-10 MeV range to detect specific elements within a radiographed object. As opposed to classical event-counting time of flight (ECTOF), it integrates the detector signal during a well-defined neutron Time of Flight window corresponding to a pre-selected energy bin, e.g., the energy-interval spanning a cross-section resonance of an element such as C, O and N. The integrative characteristic of the detector permits loss-free operation at very intense, pulsed neutron fluxes, at a cost however, of recorded temporal resolution degradation This work presents a theoretical and experimental evaluation of detector related parameters which affect temporal resolution of the TRION system.

  4. Integrated urban and rural water affairs management reform in China: Affecting factors

    NASA Astrophysics Data System (ADS)

    Shen, Dajun; Liu, Bin

    The institutional evolution is often induced by some factors. This paper intends to analyze the affecting factors in integrated urban and rural water affairs management reform in China. The integrated urban and rural water affairs management reform is to restructure the governmental organizational setting in water management by forms of water affair bureau or re-designing functions of current water resources bureau to incorporate part or all functions of resources management, service regulation and environment management in water sector. The analyses selected some natural and socio-economic factors. The results point out that the integrated urban and rural water affairs management reform is a factor-induced institutional evolution. The factors promoting this reform include occasional drought events, higher central water investment percentage; but the data from the urban sector do not provide the support to the reform.

  5. DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology

    PubMed Central

    Grun, Casey; Werfel, Justin; Zhang, David Yu; Yin, Peng

    2015-01-01

    Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, we present software implementing a three ‘tier’ design process: a high-level visual programming language is used to describe systems, a molecular compiler builds a DNA implementation and nucleotide sequences are generated and optimized. Additionally, our software includes tools for analysing and ‘debugging’ the designs in silico, and for importing/exporting designs to other commonly used software systems. The software we present is built on many existing pieces of software, but is integrated into a single package—accessible using a Web-based interface at http://molecular-systems.net/workbench. We hope that the deep integration between tools and the flexibility of this design process will lead to better experimental results, fewer experimental design iterations and the development of more complex DNA nanosystems. PMID:26423437

  6. Study on optics integrated manufacture technology based on Windows DNA-OM

    NASA Astrophysics Data System (ADS)

    Yu, Min; Yang, Li; Wang, Yong-jan

    2006-02-01

    As the shortcoming and insufficiency of CNC systems applied in optics manufacturing, a design and application based on Windows DNA-OM is presented. After introducing the prime principles and logic structures of Windows DNA-OM, a specific solution of optics integrated manufacture based on this framework is discussed in detail. In this solution, the OPC(OLE for Process Control) technology based on COM(Component Object Model) of Microsoft company is used for CNC equipment in the workshop, and this could provide a general interface for communication of hetero-structure CNC equipment of workshop and so all hetero-structure CNC can be accessed in a simple way. The experience and knowledge of optic manufacturing is stored and integrated in Windows DNA-OM Data part, this data part can guide optics manufacture. By completing this data part, an optics manufacture expert system can be realized. Then the prime characteristics of the integrated manufacture system are given. Finally this system is proved to has a good flexibility and opening, ease realizing, low cost and high feasibility.

  7. DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology.

    PubMed

    Grun, Casey; Werfel, Justin; Zhang, David Yu; Yin, Peng

    2015-10-01

    Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, we present software implementing a three 'tier' design process: a high-level visual programming language is used to describe systems, a molecular compiler builds a DNA implementation and nucleotide sequences are generated and optimized. Additionally, our software includes tools for analysing and 'debugging' the designs in silico, and for importing/exporting designs to other commonly used software systems. The software we present is built on many existing pieces of software, but is integrated into a single package—accessible using a Web-based interface at http://molecular-systems.net/workbench. We hope that the deep integration between tools and the flexibility of this design process will lead to better experimental results, fewer experimental design iterations and the development of more complex DNA nanosystems. PMID:26423437

  8. A novel EDA glove based on textile-integrated electrodes for affective computing.

    PubMed

    Lanatà, Antonio; Valenza, Gaetano; Scilingo, Enzo Pasquale

    2012-11-01

    This paper reports on performance evaluation of a preliminary system prototype based on a fabric glove, with integrated textile electrodes placed at the fingertips, able to acquire and process the electrodermal response (EDR) to discriminate affective states. First, textile electrodes have been characterized in terms of voltage-current characteristics and trans-surface electric impedance. Next, signal quality of EDR acquired simultaneously from textile and standard electrodes was comparatively evaluated. Finally, a dedicated experiment in which 35 subjects were enrolled, aiming at discriminating different affective states using only EDR was designed and realized. A new set of features extracted from non-linear methods were used, improving remarkably successful recognition rates. Results are, indeed, very satisfactory and promising in the field of affective computing. PMID:22711069

  9. Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules.

    PubMed

    Szpechcinski, Adam; Rudzinski, Piotr; Kupis, Wlodzimierz; Langfort, Renata; Orlowski, Tadeusz; Chorostowska-Wynimko, Joanna

    2016-05-01

    Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. The analysis of cell-free DNA (cfDNA) in blood may greatly aid the early detection of lung cancer by evaluating cancer-related alterations. The plasma cfDNA levels and integrity were analysed in 65 non-small cell lung cancer (NSCLC) patients, 28 subjects with benign lung tumours, and 16 healthy controls using real-time PCR. The NSCLC patients demonstrated significantly higher mean plasma cfDNA levels compared with those with benign tumours (P = 0.0009) and healthy controls (P < 0.0001). The plasma cfDNA integrity in healthy individuals was significantly different than that found in patients with NSCLC or benign lung tumours (P < 0.0003). In ROC curve analysis, plasma cfDNA levels >2.8 ng/ml provided 86.4% sensitivity and 61.4% specificity in discriminating NSCLC from benign lung pathologies and healthy controls. cfDNA integrity showed better discriminatory power (91% sensitivity, 68.2% specificity). These data demonstrate that plasma cfDNA concentration and integrity analyses can significantly differentiate between NSCLC and benign lung tumours. The diagnostic capacity of the quantitative cfDNA assay is comparable to the values presented by conventional imaging modalities used in clinical practice. PMID:26854716

  10. Integration of Noninvasive DNA Testing for Aneuploidy into Prenatal Care: What Has Happened Since the Rubber Met the Road?

    PubMed Central

    Bianchi, Diana W.; Wilkins-Haug, Louise

    2016-01-01

    BACKGROUND Over the past 2 years, noninvasive prenatal testing (NIPT), which uses massively parallel sequencing to align and count DNA fragments floating in the plasma of pregnant women, has become integrated into prenatal care. Professional societies currently recommend offering NIPT as an advanced screen to pregnant women at high risk for fetal aneuploidy, reserving invasive diagnostic procedures for those at the very highest risk. CONTENT In this review, we summarize the available information on autosomal and sex chromosome aneuploidy detection. Clinical performance in CLIA-certified, College of American Pathology–accredited laboratories appears to be equivalent to prior clinical validation studies, with high sensitivities and specificities and very high negative predictive values. The main impact on clinical care has been a reduction in invasive procedures. Test accuracy is affected by the fetal fraction, the percentage of fetal DNA in the total amount of circulating cell-free DNA. Fetal fraction is in turn affected by maternal body mass index, gestational age, type of aneuploidy, singleton vs multiples, and mosaicism. Three studies comparing NIPT to serum or combined screening for autosomal aneuploidy all show that NIPT has significantly lower false-positive rates (approximately 0.1%), even in all-risk populations. A significant number of the discordant positive cases have underlying biological reasons, including confined placental mosaicism, maternal mosaicism, cotwin demise, or maternal malignancy. SUMMARY NIPT performs well as an advanced screen for whole chromosome aneuploidy. Economic considerations will likely dictate whether its use can be expanded to all risk populations and whether it can be applied routinely for the detection of subchromosome abnormalities. PMID:24255077

  11. Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA

    PubMed Central

    Sha, Y.; Melcher, U.; Davis, R. E.; Fletcher, J.

    1995-01-01

    Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by

  12. Chemometric method of spectra analysis leading to isolation of lysozyme and CtDNA spectra affected by osmolytes.

    PubMed

    Bruździak, Piotr; Rakowska, Paulina W; Stangret, Janusz

    2012-11-01

    In this paper we present a chemometric method of analysis leading to isolation of Fourier transform infrared (FT-IR) spectra of biomacromolecules (HEW lysozyme, ctDNA) affected by osmolytes (trimethylamine-N-oxide and N,N,N-trimethylglycine, respectively) in aqueous solutions. The method is based on the difference spectra method primarily used to characterize the structure of solvent affected by solute. The cyclical usage of factor analysis allows precise information to be obtained on the shape of "affected spectra" of analyzed biomacromolecules. "Affected spectra" of selected biomacromolecules give valuable information on their structure in the presence of the osmolytes in solution, as well as on the level of perturbation in dependence of osmolyte concentration. The method also gives a possibility of insight into the mechanism of interaction in presented types of systems. It can be easily adapted to various chemical and biochemical problems where vibrational or ultraviolet-visible (UV-Vis) spectroscopy is used. PMID:23146186

  13. DNA polymerase kappa deficiency does not affect somatic hypermutation in mice.

    PubMed

    Schenten, Dominik; Gerlach, Valerie L; Guo, Caixia; Velasco-Miguel, Susana; Hladik, Christa L; White, Charles L; Friedberg, Errol C; Rajewsky, Klaus; Esposito, Gloria

    2002-11-01

    Somatic hypermutation (SH) in B cells undergoing T cell-dependent immune responses generates high-affinity antibodies that provide protective immunity. Most current models of SH postulate the introduction of a nick into the DNA and subsequent replication-independent, error-prone short-patch synthesis by one or more DNA polymerases. The Pol kappa (DinB1) gene encodes a specialized mammalian DNA polymerase called DNA polymerase kappa (pol kappa), a member of the recently discovered Y family of DNA polymerases. The mouse PolK gene is expressed at high levels in the seminiferous tubules of the testis and in the adrenal cortex, and at lower levels in most other cells of the body including B lymphocytes. In vitro studies showed that pol kappa can act as an error-prone polymerase, although they failed to ascribe a clear function to this enzyme. The ability of pol kappa to generate mutations when extending primers on undamaged DNA templates identifies this enzyme as a potential candidate for the introduction of nucleotide changes in the immunoglobulin (Ig) genes during the process of SH. Here we show that pol kappa-deficient mice are viable, fertile and able to mount a normal immune response to the antigen (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (NP-GC). They also mutate their Ig genes normally. However, pol kappa-deficient embryonic fibroblasts are abnormally sensitive to killing following exposure to ultraviolet (UV) radiation, suggesting a role of pol kappa in translesion DNA synthesis. PMID:12555660

  14. Agents that reverse UV-induced immune suppression and photocarcinogenesis affect DNA repair

    PubMed Central

    Sreevidya, Coimbatore S.; Fukunaga, Atsushi; Khaskhely, Noor M.; Masaki, Taro; Ono, Ryusuke; Nishigori, Chikako; Ullrich, Stephen E.

    2010-01-01

    UV exposure induces skin cancer, in part by inducing immune suppression. Repairing DNA damage, neutralizing the activity of cis-urocanic acid (cis-UCA), and reversing oxidative stress abrogates UV-induced immune suppression and skin cancer induction, suggesting the DNA, UCA and lipid photo-oxidation serves as UV photoreceptors. What is not clear is whether signaling through each of these different photoreceptors activates independent pathways to induce biological effects or whether there is a common checkpoint where these pathways converge. Here we show that agents known to reverse photocarcinogenesis and photoimmune suppression, such as platelet activating factor (PAF) and serotonin (5-HT) receptor antagonists regulate DNA repair. Pyrimidine dimer repair was accelerated in UV-irradiated mice injected with PAF and 5-HT receptor antagonists. Nucleotide excision repair, as measured by unscheduled DNA synthesis, was accelerated by PAF and 5-HT receptor antagonists. Injecting PAF and 5-HT receptor antagonists into UV-irradiated Xeroderma pigmentosum complementation group A (XPA) deficient mice, which lack the enzymes responsible for nucleotide excision repair, did not accelerate photoproduct repair. Similarly, UV-induced formation of 8-oxo-deoxyguanosine (8-oxo-dG) was reduced by PAF and 5-HT receptor antagonists. We conclude that PAF and 5-HT receptor antagonists accelerate DNA repair caused by UV radiation, which prevents immune suppression and interferes with photocarcinogenesis. PMID:19829299

  15. Multiple factors affect immunogenicity of DNA plasmid HIV vaccines in human clinical trials

    PubMed Central

    Jin, Xia; Morgan, Cecilia; Yu, Xuesong; DeRosa, Stephen; Tomaras, Georgia D.; Montefiori, David C.; Kublin, James; Corey, Larry; Keefer, Michael C.

    2015-01-01

    Plasmid DNA vaccines have been licensed for use in domesticated animals because of their excellent immunogenicity, but none have yet been licensed for use in humans. Here we report a retrospective analysis of 1218 healthy human volunteers enrolled in 10 phase I clinical trials in which DNA plasmids encoding HIV antigens were administered. Elicited T-cell immune responses were quantified by validated intracellular cytokine staining (ICS) stimulated with HIV peptide pools. HIV-specific binding and neutralizing antibody activities were also analyzed using validated assays. Results showed that, in the absence of adjuvants and boosting with alternative vaccines, DNA vaccines elicited CD8+ and CD4+ T-cell responses in an average of 13.3% (95% CI: 9.8% to 17.8%) and 37.7% (95% CI: 31.9% to 43.8%) of vaccine recipients, respectively. Three vaccinations (versus 2) improved the proportion of subjects with antigen-specific CD8+ responses (p=0.02), as did increased DNA dosage (p=0.007). Furthermore, female gender and participants having a lower Body Mass Index were independently associated with higher CD4+ T-cell response rate (p=0.001 and p=0.008, respectively). These vaccines elicited minimal neutralizing and binding antibody responses. These findings of the immunogenicity of HIV DNA vaccines in humans can provide guidance for future clinical trials. PMID:25820067

  16. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17.

    PubMed Central

    Walz, C; Schlehofer, J R

    1992-01-01

    Parvoviruses are known to interfere with cellular transformation and carcinogenesis. Since infecting adeno-associated virus (AAV) frequently integrates its DNA into the cellular genome, we analyzed whether this integration influences the transformed phenotype of the human tumor cell line HeLa. Analysis of three independent HeLa cell clones with integrated AAV DNA (HA-3x, HA-16, and HA-28) revealed the following phenotypic changes of these cells: (i) reduced growth rate, (ii) increased serum requirement, (iii) reduced capacity for colony formation in soft agar, (iv) reduced cloning efficiency on plastic, (v) elevated sensitivity to genotoxic agents (N-methyl-N'-nitro-N-nitrosoguanidine, 7,12-dimethylbenz[a]anthracene, human tumor necrosis factor alpha, UV irradiation [256 nm], and heat [42 degrees C]), and (vi) reduced sensitivity to the cytolytic effect of parvovirus H-1. Reduced growth rate and enhanced sensitivity to gamma irradiation were also observed in vivo when tumors from AAV DNA-containing HeLa cells were transplanted into nude mice. This alteration of the biological properties of HeLa cells was independent of the number of AAV genomes integrated, the physical structure of integrated AAV DNA, and the transcription of AAV genes. Integration of AAV DNA was found to occur preferentially on the long arm of chromosome 17 in the three HeLa cell clones analyzed. These findings demonstrate that genomic integration of AAV DNA can alter the biological properties of human tumor cells. Images PMID:1313913

  17. Integrating Negative Affect Measures in a Measurement Model: Assessing the Function of Negative Affect as Interference to Self-Regulation

    ERIC Educational Resources Information Center

    Magno, Carlo

    2010-01-01

    The present study investigated the composition of negative affect and its function as inhibitory to thought processes such as self-regulation. Negative affect in the present study were composed of anxiety, worry, thought suppression, and fear of negative evaluation. These four factors were selected based on the criteria of negative affect by…

  18. ARTIE: An Integrated Environment for the Development of Affective Robot Tutors

    PubMed Central

    Imbernón Cuadrado, Luis-Eduardo; Manjarrés Riesco, Ángeles; De La Paz López, Félix

    2016-01-01

    Over the last decade robotics has attracted a great deal of interest from teachers and researchers as a valuable educational tool from preschool to highschool levels. The implementation of social-support behaviors in robot tutors, in particular in the emotional dimension, can make a significant contribution to learning efficiency. With the aim of contributing to the rising field of affective robot tutors we have developed ARTIE (Affective Robot Tutor Integrated Environment). We offer an architectural pattern which integrates any given educational software for primary school children with a component whose function is to identify the emotional state of the students who are interacting with the software, and with the driver of a robot tutor which provides personalized emotional pedagogical support to the students. In order to support the development of affective robot tutors according to the proposed architecture, we also provide a methodology which incorporates a technique for eliciting pedagogical knowledge from teachers, and a generic development platform. This platform contains a component for identiying emotional states by analysing keyboard and mouse interaction data, and a generic affective pedagogical support component which specifies the affective educational interventions (including facial expressions, body language, tone of voice,…) in terms of BML (a Behavior Model Language for virtual agent specification) files which are translated into actions of a robot tutor. The platform and the methodology are both adapted to primary school students. Finally, we illustrate the use of this platform to build a prototype implementation of the architecture, in which the educational software is instantiated with Scratch and the robot tutor with NAO. We also report on a user experiment we carried out to orient the development of the platform and of the prototype. We conclude from our work that, in the case of primary school students, it is possible to identify, without

  19. ARTIE: An Integrated Environment for the Development of Affective Robot Tutors.

    PubMed

    Imbernón Cuadrado, Luis-Eduardo; Manjarrés Riesco, Ángeles; De La Paz López, Félix

    2016-01-01

    Over the last decade robotics has attracted a great deal of interest from teachers and researchers as a valuable educational tool from preschool to highschool levels. The implementation of social-support behaviors in robot tutors, in particular in the emotional dimension, can make a significant contribution to learning efficiency. With the aim of contributing to the rising field of affective robot tutors we have developed ARTIE (Affective Robot Tutor Integrated Environment). We offer an architectural pattern which integrates any given educational software for primary school children with a component whose function is to identify the emotional state of the students who are interacting with the software, and with the driver of a robot tutor which provides personalized emotional pedagogical support to the students. In order to support the development of affective robot tutors according to the proposed architecture, we also provide a methodology which incorporates a technique for eliciting pedagogical knowledge from teachers, and a generic development platform. This platform contains a component for identiying emotional states by analysing keyboard and mouse interaction data, and a generic affective pedagogical support component which specifies the affective educational interventions (including facial expressions, body language, tone of voice,…) in terms of BML (a Behavior Model Language for virtual agent specification) files which are translated into actions of a robot tutor. The platform and the methodology are both adapted to primary school students. Finally, we illustrate the use of this platform to build a prototype implementation of the architecture, in which the educational software is instantiated with Scratch and the robot tutor with NAO. We also report on a user experiment we carried out to orient the development of the platform and of the prototype. We conclude from our work that, in the case of primary school students, it is possible to identify, without

  20. DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint

    PubMed Central

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K.; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress. PMID:26884712

  1. SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase

    PubMed Central

    Ahn, Jang-Won; Kim, Sunjik; Na, Wooju; Baek, Su-Jin; Kim, Jeong-Hwan; Min, Keehong; Yeom, Jeonghun; Kwak, Hoyun; Jeong, Sunjoo; Lee, Cheolju; Kim, Seon-Young; Choi, Cheol Yong

    2015-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase. PMID:26068472

  2. Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study.

    PubMed

    Yagci, Artay; Murk, William; Stronk, Jill; Huszar, Gabor

    2010-01-01

    During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection. PMID:20133967

  3. Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence.

    PubMed

    Kunnimalaiyaan, M; Lodha, M L; Sreekumar, K R

    1992-11-01

    Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome. PMID:24425601

  4. Folate supplementation differently affects uracil content in DNA in the mouse colon and liver

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High folate intake may increase the risk of cancer, especially in the elderly. The present study examined the effects of ageing and dietary folate on uracil misincorporation into DNA, which has a mutagenic effect, in the mouse colon and liver. Old (18 months; n 42) and young (4 months; n 42) male C5...

  5. Do DNA barcoding delimitation methods affect our view of stream biodiversity?

    EPA Science Inventory

    How we delimit molecular operational taxonomic units (MOTUs) is an important aspect in the use of DNA barcoding for bioassessment. Four delimitation methods were examined to gain an understanding of their relative strengths at organizing data from 5300 specimens collected during ...

  6. Suberoylanilide Hydroxyamic Acid Modification of Chromatin Architecture Affects DNA Break Formation and Repair

    SciTech Connect

    Singh, Sheetal; Le Hongan; Shih, S.-J.; Ho, Bay; Vaughan, Andrew T.

    2010-02-01

    Purpose: Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. Methods and Materials: The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. Results: SAHA acts as radiosensitizer at 1 {mu}M, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 +- 0.05; MCF7 - 1.16 +- 0.09 and TK6 - 1.17 +- 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. Conclusions: SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival.

  7. Integrated adenovirus type 12 DNA in the transformed hamster cell line T637: sequence arrangements at the termini of viral DNA and mode of amplification.

    PubMed Central

    Eick, D; Doerfler, W

    1982-01-01

    Approximately 20 to 22 copies of adenovirus type 12 (Ad12) DNA per cell were integrated into the genome of the cell line T637. Only a few of these copies seemed to remain intact and colinear with virion DNA. All other persisting viral genomes exhibited deletions or inversions or both in the right-hand part of Ad12 DNA. Spontaneously arising morphological revertants of T637 cells has lost viral DNA. In most of the revertant cell lines only the intact or a part of the intact viral genome was preserved; other revertant cell lines has lost all viral DNA. In three other Ad12-transformed hamster cell lines, HA12/7, A2497-3, and CLAC3 (Stabel et al., J. Virol. 36:22-40, 1980), major rearrangements at the right end of the integrated Ad12 DNA were not found. These studies were performed to investigate the phenomena of amplification, rearrangements, and deletions of Ad12 DNA in hamster cells. Images PMID:6283150

  8. Micromachined pipettes integrated in a flow channel for single DNA molecule study by optical trapping

    NASA Astrophysics Data System (ADS)

    Rusu, Cristina R.; van't Oever, Ronny; de Boer, Meint J.; Jansen, Henri V.; Berenschot, Erwin; Elwenspoek, Miko C.; Bennink, Martin L.; Kanger, Johannes S.; de Grooth, Bart G.; Greve, Jan; Brugger, Juergen P.; van den Berg, Albert

    2000-03-01

    We have developed a micromachined flow cell consisting of a flow channel integrated with micropipettes. The flow cell is used in combination with an optical trap set-up (optical tweezers) to study mechanical and structural properties of (lambda) -DNA molecules. The flow cell was realized using silicon micromachining including the so-called buried channel technology to fabricate the micropipettes, the wet etching of glass to create the flow channel, and the powder blasting of glass to create the fluid connections. The volume of the flow cell is 2 (mu) l. The pipettes have a length of 130 micrometer, a width of 5 - 10 micrometer, a round opening of 1 micron and can be processed with different shapes. Using this flow cell we stretched single molecules ((lambda) -DNA) showing typical force-extension curves also found with conventional techniques.

  9. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective

    PubMed Central

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaitre, Jean-Marc; De Vos, John

    2013-01-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that “genome-friendly” reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address. PMID:23317057

  10. DNA Methylation of Lipid-Related Genes Affects Blood Lipid Levels

    PubMed Central

    Pfeiffer, Liliane; Wahl, Simone; Pilling, Luke C.; Reischl, Eva; Sandling, Johanna K.; Kunze, Sonja; Holdt, Lesca M.; Kretschmer, Anja; Schramm, Katharina; Adamski, Jerzy; Klopp, Norman; Illig, Thomas; Hedman, Åsa K.; Roden, Michael; Hernandez, Dena G.; Singleton, Andrew B.; Thasler, Wolfgang E.; Grallert, Harald; Gieger, Christian; Herder, Christian; Teupser, Daniel; Meisinger, Christa; Spector, Timothy D.; Kronenberg, Florian; Prokisch, Holger; Melzer, David; Peters, Annette; Deloukas, Panos; Ferrucci, Luigi; Waldenberger, Melanie

    2016-01-01

    Background Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction. Methods and Results Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=−0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06–1.25). Conclusions Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases. PMID:25583993

  11. Microelectrophoresis devices with integrated fluorescence detectors and reactors for high-throughput DNA sequencing

    NASA Astrophysics Data System (ADS)

    Soper, Steven A.; Ford, Sean M.; Davies, Jack; Williams, Daryl C.; Cheng, Benxu; Klopf, J. Michael; Calderon, Gina M.; Saile, Volker

    1997-05-01

    This work describes the development of micro-devices for high-throughput DNA sequencing applications. Basically, two research efforts will be discussed; (1) fabrication and characterization of micro-reactors to prepare Sanger chain terminated DNA sequencing fragments on a nanoliter scale and; (2) x-ray photolithography of PMMA substrates for the high aspect ratio preparation of electrophoresis devices. The micro-reactor consisted of a 5'-biotinylated catfish olfactory gene, which was amplified by PCR, and attached to the interior wall of an aminoalkylisilane derivatized fused- silica capillary tube via a streptavidin/biotin linkage. Coverage of the interior capillary wall with biotinylated DNA averaged 77 percent. Stability of the anchored template under pressure and electroosmotic rinsing was favorable, requiring approximately 150 h of continuous rinsing to reduce the coverage by only 50 percent. The capillary micro- reactor was placed inside an air thermocycler to control temperature during Sanger ddNTP chain extension and directly coupled to a capillary separation column filled with a LPA solution via low dead volume capillary interlocks. The complimentary DNA fragments generated in the reactor were heat denatured from the immobilized template and directly injected onto a gel-filled capillary using electropumping for size fractionation and detection using NIR-LIF analysis. The total amount of termination fragments in the 31 nL reactor volume was estimated to be 5.2 X 1013 moles and sequencing was shown to produce read lengths on the order to 400 bases. Work will also be described concerning the development of micro-electrophoresis devices in x-ray sensitive photoresists using LIGA techniques. An electrophoresis device with an integrated fluorescence detector was constructed for the high resolution separation of DNA oligonucleotides. The choice of substrate for the electrophoresis was PMMA, due to its intrinsic low electroosmotic flow. Using x-ray lithography in

  12. Glycation of Ribonuclease A affects its enzymatic activity and DNA binding ability.

    PubMed

    Dinda, Amit Kumar; Tripathy, Debi Ranjan; Dasgupta, Swagata

    2015-11-01

    Prolonged non-enzymatic glycation of proteins results in the formation of advanced glycation end products (AGEs) that cause several diseases. The glycation of Ribonuclease A (RNase A) at pH 7.4 and 37 °C with ribose, glucose and fructose has been monitored by UV-vis, fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization spectroscopy-time of flight (MALDI-TOF) methods. The enzymatic activity and DNA binding ability of glycated RNase A was also investigated by an agarose gel-based assay. A precipitation assay examined the ribonucleolytic activity of the glycated enzyme. An increase in incubation time resulted in the formation of high molecular weight AGEs with a decrease in ribonucleolytic activity. Ribose exhibits the highest potency as a glycating agent and showed the greatest reduction in the ribonucleolytic activity of the enzyme. Interestingly, glycated RNase A was unable to bind with the ribonuclease inhibitor (RI) and DNA. The glycated form of the protein was also found to be ineffective in DNA melting unlike native RNase A. PMID:26365067

  13. Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

    SciTech Connect

    Elstein, K.H.; Thomas, D.J.; Zucker, R.M.

    1995-10-01

    Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we observed a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 {mu}M tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 {mu}M dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. 37 refs., 3 figs., 1 tab.

  14. Cisplatin-induced DNA damage activates replication checkpoint signaling components that differentially affect tumor cell survival.

    PubMed

    Wagner, Jill M; Karnitz, Larry M

    2009-07-01

    Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents. Here we show that Rad9 and ATR play critical roles in helping tumor cells survive cisplatin treatment. However, depleting Chk1 with small interfering RNA or inhibiting Chk1 with 3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide (AZD7762) did not sensitize these cells to cisplatin, oxaliplatin, or carboplatin. Moreover, when Rad18, Rad51, BRCA1, BRCA2, or FancD2 was disabled, Chk1 depletion did not further sensitize the cells to cisplatin. In fact, Chk1 depletion reversed the sensitivity seen when Rad18 was disabled. Collectively, these studies suggest that the pharmacological manipulation of Chk1 may not be an effective strategy to sensitize tumors to platinating agents. PMID:19403702

  15. How absent negativity relates to affect and motivation: an integrative relief model

    PubMed Central

    Deutsch, Roland; Smith, Kevin J. M.; Kordts-Freudinger, Robert; Reichardt, Regina

    2015-01-01

    The present paper concerns the motivational underpinnings and behavioral correlates of the prevention or stopping of negative stimulation – a situation referred to as relief. Relief is of great theoretical and applied interest. Theoretically, it is tied to theories linking affect, emotion, and motivational systems. Importantly, these theories make different predictions regarding the association between relief and motivational systems. Moreover, relief is a prototypical antecedent of counterfactual emotions, which involve specific cognitive processes compared to factual or mere anticipatory emotions. Practically, relief may be an important motivator of addictive and phobic behaviors, self destructive behaviors, and social influence. In the present paper, we will first provide a review of conflicting conceptualizations of relief. We will then present an integrative relief model (IRMO) that aims at resolving existing theoretical conflicts. We then review evidence relevant to distinctive predictions regarding the moderating role of various procedural features of relief situations. We conclude that our integrated model results in a better understanding of existing evidence on the affective and motivational underpinnings of relief, but that further evidence is needed to come to a more comprehensive evaluation of the viability of IRMO. PMID:25806008

  16. Theta phase coherence in affective picture processing reveals dysfunctional sensory integration in psychopathic offenders.

    PubMed

    Tillem, Scott; Ryan, Jonathan; Wu, Jia; Crowley, Michael J; Mayes, Linda C; Baskin-Sommers, Arielle

    2016-09-01

    Psychopathic offenders are described as emotionally cold, displaying deficits in affective responding. However, research demonstrates that many of the psychopathy-related deficits are moderated by attention, such that under conditions of high attentional and perceptual load psychopathic offenders display deficits in affective responses, but do not in conditions of low load. To date, most studies use measures of defensive reflex (i.e., startle) and conditioning manipulations to examine the impact of load on psychopathy-related processing, but have not examined more direct measures of attention processing. In a sample of adult male offenders, the present study examined time-frequency EEG phase coherence in response to a picture-viewing paradigm that manipulated picture familiarity to assess neural changes in processing based on perceptual demands. Results indicated psychopathy-related differences in the theta response, an index of readiness to perceive and integrate sensory information. These data provide further evidence that psychopathic offenders have disrupted integration of sensory information. PMID:27373371

  17. How absent negativity relates to affect and motivation: an integrative relief model.

    PubMed

    Deutsch, Roland; Smith, Kevin J M; Kordts-Freudinger, Robert; Reichardt, Regina

    2015-01-01

    The present paper concerns the motivational underpinnings and behavioral correlates of the prevention or stopping of negative stimulation - a situation referred to as relief. Relief is of great theoretical and applied interest. Theoretically, it is tied to theories linking affect, emotion, and motivational systems. Importantly, these theories make different predictions regarding the association between relief and motivational systems. Moreover, relief is a prototypical antecedent of counterfactual emotions, which involve specific cognitive processes compared to factual or mere anticipatory emotions. Practically, relief may be an important motivator of addictive and phobic behaviors, self destructive behaviors, and social influence. In the present paper, we will first provide a review of conflicting conceptualizations of relief. We will then present an integrative relief model (IRMO) that aims at resolving existing theoretical conflicts. We then review evidence relevant to distinctive predictions regarding the moderating role of various procedural features of relief situations. We conclude that our integrated model results in a better understanding of existing evidence on the affective and motivational underpinnings of relief, but that further evidence is needed to come to a more comprehensive evaluation of the viability of IRMO. PMID:25806008

  18. The epididymal sperm viability, motility and DNA integrity in dead mice maintained at 4-6oC

    PubMed Central

    Golshan Iranpour, Farhad; Rezazadeh Valojerdi, Mojtaba

    2013-01-01

    Background: When male animals die, spermatozoa within the body of animal will be degenerated. Because of unique chromatin structure of sperm, maybe this degeneration is different from other cells. However there is not any research which considered directly the integrity of sperm DNA by keeping the cadaver in refrigerator. Objective: The aim of this study was to assess viability, total motility and DNA integrity of sperm cells after death. Materials and Methods:In this experimental study, 24 male Swiss white mice were killed by cervical dislocation and then kept in refrigerator (4-6oC) for up to 12 days. On the 0 (immediately after death as control group), 1st, 2nd, 3rd, 5th, 7th, 10th and the 12th days after death cauda epididymides were removed and squeezed in Ham’s F10 medium. The proportion of viable, motile and double stranded DNA spermatozoa was examined. Viability and DNA integrity of sperm cells were examined consecutively by eosin nigrosin and acridine orange stainings. Results:The data obtained from this study showed that viability and total motility of sperm cells were significantly decreased during 12 days after death (p<0.001). In contrast with viability and motility, DNA integrity was without significant changes (even 12 days after death). Conclusion:This study suggests that integrity of sperm DNA would not change even after 12 days after death if the cadaver kept in refrigerator. PMID:24639746

  19. Integration of constrained electrical and seismic tomographies to study the landslide affecting the cathedral of Agrigento

    NASA Astrophysics Data System (ADS)

    Capizzi, P.; Martorana, R.

    2014-08-01

    The Cathedral of Saint Gerland, located on the top of the hill of Agrigento, is an important historical church, which dates back to the Arab-Norman period (XI century). Unfortunately throughout its history the Cathedral and the adjacent famous Archaeological Park of the ‘Valley of the Temples’ have been affected by landslides. In this area the interleaving of calcarenites, silt, sand and clay is complicated by the presence of dislocated rock blocks and cavities and by a system of fractures partly filled with clay or water. Integrated geophysical surveys were carried out on the north side of the hill, on which the Cathedral of Agrigento is founded, to define lithological structures involved in the failure process. Because of the landslide, the cathedral has been affected by fractures, which resulted in the overall instability of the structure. Along each of four footpaths a combination of 2D electrical resistivity tomographies (ERT) and 2D seismic refraction tomographies (SRT) was performed. Moreover, along two of these footpaths microtremor (HVSR) and surface wave soundings (MASW) were carried out to reconstruct 2D sections of shear waves velocity. Furthermore a 3D electrical resistivity tomography was carried out in a limited area characterized by gentle slopes. After a preliminary phase, in which the data were processed independently, a subsequent inversion of seismic and electrical data was constrained with stratigraphic information obtained from geognostic continuous core boreholes located along the geophysical lines. This process allowed us to significantly increase the robustness of the geophysical models. The acquired data were interpolated to construct 3D geophysical models of the electrical resistivity and of the P-wave velocity. The interpolation algorithm took into account the average direction and immersion of geological strata. Results led to a better understanding of the complexity of the subsoil in the investigated area. The use of integrated

  20. Integrating cognitive and affective dimensions of pain experience into health professions education

    PubMed Central

    Murinson, Beth B; Mezei, Lina; Nenortas, Elizabeth

    2011-01-01

    Pain is prevalent in clinical settings, and yet it is relatively under-represented in the education of most students in the health professions. Because pain includes both sensory-discriminative and affective features, teaching students about pain presents unique challenges and opportunities. The present article describes the evolution of a new blueprint for clinical excellence that, among other competencies, incorporates a need for the emotional development of clinical trainees. The framework has been applied to the development and implementation of two new courses in pain. The first course is designed to provide a comprehensive foundation of medical knowledge regarding pain, while integratively introducing students to the affective dimensions of pain. The second course is designed to enhance students’ appreciation for the protean effects of pain through use of the humanities to represent medical experience. It is concluded that, to be most effective, fostering the emotional development of trainees in the health professions necessitates the incorporation of affect-focused learning objectives, educational tasks and assessment methods. PMID:22184551

  1. Impact of a novel homozygous mutation in nicotinamide nucleotide transhydrogenase on mitochondrial DNA integrity in a case of familial glucocorticoid deficiency

    PubMed Central

    Fujisawa, Yasuko; Napoli, Eleonora; Wong, Sarah; Song, Gyu; Yamaguchi, Rie; Matsui, Toshiharu; Nagasaki, Keisuke; Ogata, Tsutomu; Giulivi, Cecilia

    2014-01-01

    Background Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder that is characterized by isolated glucocorticoid deficiency. Recently, mutations in the gene encoding for the mitochondrial nicotinamide nucleotide transhydrogenase (NNT) have been identified as a causative gene for FGD; however, no NNT activities have been reported in FGD patients carrying NNT mutations. Methods Clinical, biochemical and molecular analyses of lymphocytes from FDG homozygous and heterozygous carriers for the F215S NNT mutation were performed. Results In this study, we described an FGD-affected Japanese patient carrying a novel NNT homozygous mutation (c.644T>C; F215S) with a significant loss-of-function (NNT activity = 31% of healthy controls) in peripheral blood cells' mitochondria. The NNT activities of the parents, heterozygous for the mutation, were 61% of the controls. Conclusions Our results indicated that (i) mitochondrial biogenesis (citrate synthase activity) and/or mtDNA replication (mtDNA copy number) were affected at ≤ 60% NNT activity because these parameters were affected in individuals carrying either one or both mutated alleles; and (ii) other outcomes (mtDNA deletions, protein tyrosine nitration, OXPHOS capacity) were affected at ≤ 30% NNT activity as also observed in murine cerebellar mitochondria from C57BL/6J (NNT−/−) vs. C57BL/6JN (NNT+/+) substrains. General significance By studying a family affected with a novel point mutation in the NNT gene, a gene–dose response was found for various mitochondrial outcomes providing for novel insights into the role of NNT in the maintenance of mtDNA integrity beyond that described for preventing oxidative stress. PMID:26309815

  2. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  3. Class I HDACs Affect DNA Replication, Repair, and Chromatin Structure: Implications for Cancer Therapy

    PubMed Central

    Stengel, Kristy R.

    2015-01-01

    Abstract Significance: The contribution of epigenetic alterations to cancer development and progression is becoming increasingly clear, prompting the development of epigenetic therapies. Histone deacetylase inhibitors (HDIs) represent one of the first classes of such therapy. Two HDIs, Vorinostat and Romidepsin, are broad-spectrum inhibitors that target multiple histone deacetylases (HDACs) and are FDA approved for the treatment of cutaneous T-cell lymphoma. However, the mechanism of action and the basis for the cancer-selective effects of these inhibitors are still unclear. Recent Advances: While the anti-tumor effects of HDIs have traditionally been attributed to their ability to modify gene expression after the accumulation of histone acetylation, recent studies have identified the effects of HDACs on DNA replication, DNA repair, and genome stability. In addition, the HDIs available in the clinic target multiple HDACs, making it difficult to assign either their anti-tumor effects or their associated toxicities to the inhibition of a single protein. However, recent studies in mouse models provide insights into the tissue-specific functions of individual HDACs and their involvement in mediating the effects of HDI therapy. Critical Issues: Here, we describe how altered replication contributes to the efficacy of HDAC-targeted therapies as well as discuss what knowledge mouse models have provided to our understanding of the specific functions of class I HDACs, their potential involvement in tumorigenesis, and how their disruption may contribute to toxicities associated with HDI treatment. Future Directions: Impairment of DNA replication by HDIs has important therapeutic implications. Future studies should assess how best to exploit these findings for therapeutic gain. Antioxid. Redox Signal. 23, 51–65. PMID:24730655

  4. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

    PubMed Central

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  5. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction.

    PubMed

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-Ichi

    2016-01-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing. PMID:27499264

  6. Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach

    PubMed Central

    Lee, Mark N; Roy, Matthew; Ong, Shao-En; Mertins, Philipp; Villani, Alexandra-Chloé; Li, Weibo; Dotiwala, Farokh; Sen, Jayita; Doench, John G; Orzalli, Megan H; Kramnik, Igor; Knipe, David M; Lieberman, Judy; Carr, Steven A; Hacohen, Nir

    2013-01-01

    The innate immune system senses viral DNA that enters mammalian cells, or in aberrant situations self-DNA, and triggers type I interferon production. Here we present an integrative approach that combines quantitative proteomics, genomics and small molecule perturbations to identify genes involved in this pathway. We silenced 809 candidate genes, measured the response to dsDNA and connected resulting hits with the known signaling network. We identified ABCF1 as a critical protein that associates with dsDNA and the DNA-sensing components HMGB2 and IFI204. We also found that CDC37 regulates the stability of the signaling molecule TBK1 and that chemical inhibition of the CDC37-HSP90 interaction and several other pathway regulators potently modulates the innate immune response to DNA and retroviral infection. PMID:23263557

  7. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction

    PubMed Central

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-ichi

    2016-01-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing. PMID:27499264

  8. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction

    NASA Astrophysics Data System (ADS)

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-Ichi

    2016-08-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.

  9. Effects of mercuric chloride on antioxidant system and DNA integrity of the crab Charybdis japonica

    NASA Astrophysics Data System (ADS)

    Zhang, Hongxia; Pan, Luqing; Miao, Jingjing; Xu, Chaoqun

    2009-12-01

    Mercury (Hg) is one of the commonly encountered heavy metals, which is widespread in inshore sediments of China. In order to investigate the toxicity of Hg on marine invertebrates, we studied the effects of the divalent mercuricion (Hg2+) (at two final concentrations of 0.0025 and 0.0050 mg L-1, prepared with HgCl2) on metallothionein (MT) content, DNA integrity (DNA strand breaks) and catalase (CAT) in the gills and hepatopancreas, antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the hemolymph, gills and hepatopancreas of the portunid crab Charybdis japonica for an experiment period up to 15 d. The results indicated that MT was significantly induced after 3 d, with a positive correlation with Hg2+ dose and time in the hepatopancreas and a negative correlation with Hg2+ dose and time in the gills. While CAT in the hemolymph was not detected, it increased in the hepatopancreas during the entire experiment; SOD and GPx in the three tissues were stimulated after 12 h, both attained peak value and then reduced during the experimental period. Meanwhile, DNA strand breaks were all induced significantly after 12 h. These results suggested the detoxification strategies against Hg2+ in three tissues of C. japonica.

  10. An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues

    PubMed Central

    Guo, Jia; Yu, Lin; Turro, Nicholas J.; Ju, Jingyue

    2010-01-01

    via click chemistry is unambiguously identified with this chip-SBS system. The second generation (G-2) SBS system was developed based on the concept that the closer the structures of the added nucleotide and the primer are to their natural counterparts, the more faithfully the polymerase would incorporate the nucleotide. In this approach, the polymerase reaction is performed with the combination of 3′-capped nucleotide reversible terminators (NRTs) and cleavable fluorescent dideoxynucleotides (ddNTPs). By sacrificing a small amount of the primers permanently terminated by ddNTPs, the majority of the primers extended by the reversible terminators are reverted to the natural ones after each sequencing cycle. We have also developed the 3′-capped nucleotide reversible terminators to solve the problem of deciphering the homopolymeric regions of the template in conventional pyrosequencing. The 3′-capping moiety on the DNA extension product temporarily terminates the polymerase reaction, which allows only one nucleotide to be incorporated during each sequencing cycle. Thus, the number of nucleotides in the homopolymeric regions are unambiguously determined using the 3′-capped NRTs. It has been established that millions of DNA templates can be immobilized on a chip surface through a variety of approaches. Therefore, the integration of these high-density DNA chips with the molecular-level SBS approaches described in this Account is expected to generate a high-throughput and accurate DNA sequencing system with wide applications in biological research and health care. PMID:20121268

  11. Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure

    PubMed Central

    Yang, Darren; Boyer, Benjamin; Prévost, Chantal; Danilowicz, Claudia; Prentiss, Mara

    2015-01-01

    RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results. PMID:26384422

  12. Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure.

    PubMed

    Yang, Darren; Boyer, Benjamin; Prévost, Chantal; Danilowicz, Claudia; Prentiss, Mara

    2015-12-01

    RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results. PMID:26384422

  13. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

    PubMed

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanpin; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis. PMID:27240978

  14. Physical Factors Affecting Plasmid DNA Compaction in Stearylamine-Containing Nanoemulsions Intended for Gene Delivery

    PubMed Central

    Silva, André Leandro; Júnior, Francisco Alexandrino; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-01-01

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. PMID:24281666

  15. DNA Replication Licensing Affects Cell Proliferation or Endoreplication in a Cell Type–Specific Manner

    PubMed Central

    del Mar Castellano, María; Boniotti, María Beatrice; Caro, Elena; Schnittger, Arp; Gutierrez, Crisanto

    2004-01-01

    In eukaryotic cells, the function of DNA replication licensing components (Cdc6 and Cdt1, among others) is crucial for cell proliferation and genome stability. However, little is known about their role in whole organisms and whether licensing control interfaces with differentiation and developmental programs. Here, we study Arabidopsis thaliana CDT1, its regulation, and the consequences of overriding licensing control. The availability of AtCDT1 is strictly regulated at two levels: (1) at the transcription level, by E2F and growth-arresting signals, and (2) posttranscriptionally, by CDK phosphorylation, a step that is required for its proteasome-mediated degradation. We also show that CDC6 and CDT1 are key targets for the coordination of cell proliferation, differentiation, and development. Indeed, altered CDT1 or CDC6 levels have cell type–specific effects in developing Arabidopsis plants: in leaf cells competent to divide, cell proliferation is stimulated, whereas in cells programmed to undergo differentiation-associated endoreplication rounds, extra endocycles are triggered. Thus, we propose that DNA replication licensing control is critical for the proper maintenance of proliferative potential, developmental programs, and morphogenetic patterns. PMID:15316110

  16. Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery.

    PubMed

    Silva, André Leandro; Alexandrino, Francisco; Verissimo, Lourena Mafra; Agnez-Lima, Lucymara Fassarella; Egito, Lucila Carmem Monte; de Oliveira, Anselmo Gomes; do Egito, Eryvaldo Socrates Tabosa

    2012-01-01

    Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. PMID:24281666

  17. Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

    PubMed Central

    Talebi, Ali Reza; Khalili, Mohammad Ali; Vahidi, Serajodin; Ghasemzadeh, Jalal; Tabibnejad, Nasim

    2013-01-01

    Objectives To evaluate the effect of cord injury on (1) sperm parameters and (2) DNA chromatin status. Design Case–control study. Setting Data were collected from men referred to Research and Clinical Center for Infertility, Yazd, Iran. Participants Thirty infertile men with the presence of any level of spinal cord injury (SCI) were compared with 30 healthy donors with definite fertility and normal sperm parameters. Interventions Not applicable. Outcome measures Sperm chromatin integrity was assessed using aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. The rate of apoptotic spermatozoa was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. Results Sperm concentration, motility, and morphology in men with SCI were significantly decreased compared with control group (P < 0.05). In addition, with regard to cytochemical staining and TUNEL test, the rate of reacted spermatozoa was increased significantly in SCI group when compared with the controls (P < 0.05). The majority of AB, TB, AO, and CMA3-reacted spermatozoa were higher than the “cut-off” value in men with SCI, as were the number of apoptotic spermatozoa stained with TUNEL. Conclusion Results showed that SCI disturbs sperm parameters, nuclear maturity, and DNA integrity of spermatozoa. Therefore, the production of spermatozoa with less condensed chromatin and more apoptotic rate increases after cord injury and this may be one possible cause of infertility following SCI. PMID:23809529

  18. Outer domains of integrase within retroviral intasomes are dispensible for catalysis of DNA integration.

    PubMed

    Li, Min; Lin, Shiqiang; Craigie, Robert

    2016-02-01

    Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these "missing domains" required for function or are they dispensable? We have devised a strategy to assemble "hetero-intasomes" in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes. PMID:26537415

  19. Integrative DNA, RNA, and Protein Evidence Connects TREML4 to Coronary Artery Calcification

    PubMed Central

    Sen, Shurjo K.; Boelte, Kimberly C.; Barb, Jennifer J.; Joehanes, Roby; Zhao, XiaoQing; Cheng, Qi; Adams, Lila; Teer, Jamie K.; Accame, David S.; Chowdhury, Soma; Singh, Larry N.; Kavousi, Maryam; Peyser, Patricia A.; Quigley, Laura; Priel, Debra Long; Lau, Karen; Kuhns, Douglas B.; Yoshimura, Teizo; Johnson, Andrew D.; Hwang, Shih-Jen; Chen, Marcus Y.; Arai, Andrew E.; Green, Eric D.; Mullikin, James C.; Kolodgie, Frank D.; O’Donnell, Christopher J.; Virmani, Renu; Munson, Peter J.; McVicar, Daniel W.; Biesecker, Leslie G.

    2014-01-01

    Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease. PMID:24975946

  20. Factors Affecting Definitions of and Approaches to Integrative Medicine: A Mixed Methods Study Examining China's Integrative Medicine Development

    PubMed Central

    Zhang, Weijun; Pritzker, Sonya E.; Hui, Ka-Kit

    2015-01-01

    Aim. This study identifies existing definitions and approaches among China's integrative medicine (IM) experts and examines relationships with key characteristics distinguishing individual experts. Methods. Snowball sampling was used to select 73 IM experts for semistructured interviews. In this mixed methods study, we first identified definitions and approaches through analyzing core statements. Four key factors, including age, education, practice type, and working environment, were then chosen to evaluate the associations with the definitions. Results. Four unique definitions were identified, including IM as a “new medicine” (D1), as a combination of western medicine (WM) and Chinese medicine (CM) (D2), as a modernization of CM (D3), and as a westernization of CM (D4). D4 was mostly supported by those working in WM organizations, while D3 was more prominent from individuals working in CM organizations (P = 0.00004). More than 64% clinicians had D2 while only 1 (5.9%) nonclinician had D2. Only 1 clinician (1.8%) had D4 while almost 30% nonclinicians had D4 (P = 0.0001). Among nonclinicians working in WM organizations, 83.3% of them had D4 (P = 0.001). Conclusion. Findings indicate that institutional structure and practice type are factors affecting IM approaches. These results carry implications for the ways in which western countries move forward with the definition and implementation of IM. PMID:25792999

  1. Factors Affecting Definitions of and Approaches to Integrative Medicine: A Mixed Methods Study Examining China's Integrative Medicine Development.

    PubMed

    Zhang, Weijun; Pritzker, Sonya E; Hui, Ka-Kit

    2015-01-01

    Aim. This study identifies existing definitions and approaches among China's integrative medicine (IM) experts and examines relationships with key characteristics distinguishing individual experts. Methods. Snowball sampling was used to select 73 IM experts for semistructured interviews. In this mixed methods study, we first identified definitions and approaches through analyzing core statements. Four key factors, including age, education, practice type, and working environment, were then chosen to evaluate the associations with the definitions. Results. Four unique definitions were identified, including IM as a "new medicine" (D1), as a combination of western medicine (WM) and Chinese medicine (CM) (D2), as a modernization of CM (D3), and as a westernization of CM (D4). D4 was mostly supported by those working in WM organizations, while D3 was more prominent from individuals working in CM organizations (P = 0.00004). More than 64% clinicians had D2 while only 1 (5.9%) nonclinician had D2. Only 1 clinician (1.8%) had D4 while almost 30% nonclinicians had D4 (P = 0.0001). Among nonclinicians working in WM organizations, 83.3% of them had D4 (P = 0.001). Conclusion. Findings indicate that institutional structure and practice type are factors affecting IM approaches. These results carry implications for the ways in which western countries move forward with the definition and implementation of IM. PMID:25792999

  2. A Simple Sperm DNA Toroid Integrity Test and Risk of Miscarriage

    PubMed Central

    Chan, Philip J.; Orzylowska, Eliza M.; Corselli, Johannah U.; Jacobson, John D.; Wei, Albert K.

    2015-01-01

    Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N = 35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6 ± 1.7%, mean ± SEM) when compared with the miscarriage (51.2 ± 6.6%) or nonpregnant groups (60.9 ± 4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R = 0.32, P = 0.04) and pregnancy outcome (pregnant cases 73.6 ± 1.7% versus nonpregnant 58.0 ± 3.9%, P = 0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment. PMID:25649376

  3. An integrated lateral flow assay for effective DNA amplification and detection at the point of care.

    PubMed

    Choi, Jane Ru; Hu, Jie; Gong, Yan; Feng, Shangsheng; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-05-10

    Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future. PMID:27010033

  4. Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes

    PubMed Central

    Chen Wongworawat, Yan; Filippova, Maria; Williams, Vonetta M; Filippov, Valery; Duerksen-Hughes, Penelope J

    2016-01-01

    Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis. PMID:27186429

  5. Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes.

    PubMed

    Chen Wongworawat, Yan; Filippova, Maria; Williams, Vonetta M; Filippov, Valery; Duerksen-Hughes, Penelope J

    2016-01-01

    Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis. PMID:27186429

  6. The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

    PubMed Central

    Koch, S; Freytag von Loringhoven, A; Kahmann, R; Hofschneider, P H; Koshy, R

    1984-01-01

    Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations. Images PMID:6091042

  7. A speaker's gesture style can affect language comprehension: ERP evidence from gesture-speech integration.

    PubMed

    Obermeier, Christian; Kelly, Spencer D; Gunter, Thomas C

    2015-09-01

    In face-to-face communication, speech is typically enriched by gestures. Clearly, not all people gesture in the same way, and the present study explores whether such individual differences in gesture style are taken into account during the perception of gestures that accompany speech. Participants were presented with one speaker that gestured in a straightforward way and another that also produced self-touch movements. Adding trials with such grooming movements makes the gesture information a much weaker cue compared with the gestures of the non-grooming speaker. The Electroencephalogram was recorded as participants watched videos of the individual speakers. Event-related potentials elicited by the speech signal revealed that adding grooming movements attenuated the impact of gesture for this particular speaker. Thus, these data suggest that there is sensitivity to the personal communication style of a speaker and that affects the extent to which gesture and speech are integrated during language comprehension. PMID:25688095

  8. Mixing positive and negative valence: Affective-semantic integration of bivalent words.

    PubMed

    Kuhlmann, Michael; Hofmann, Markus J; Briesemeister, Benny B; Jacobs, Arthur M

    2016-01-01

    Single words have affective and aesthetic properties that influence their processing. Here we investigated the processing of a special case of word stimuli that are extremely difficult to evaluate, bivalent noun-noun-compounds (NNCs), i.e. novel words that mix a positive and negative noun, e.g. 'Bombensex' (bomb-sex). In a functional magnetic resonance imaging (fMRI) experiment we compared their processing with easier-to-evaluate non-bivalent NNCs in a valence decision task (VDT). Bivalent NNCs produced longer reaction times and elicited greater activation in the left inferior frontal gyrus (LIFG) than non-bivalent words, especially in contrast to words of negative valence. We attribute this effect to a LIFG-grounded process of semantic integration that requires greater effort for processing converse information, supporting the notion of a valence representation based on associations in semantic networks. PMID:27491491

  9. Mixing positive and negative valence: Affective-semantic integration of bivalent words

    PubMed Central

    Kuhlmann, Michael; Hofmann, Markus J.; Briesemeister, Benny B.; Jacobs, Arthur M.

    2016-01-01

    Single words have affective and aesthetic properties that influence their processing. Here we investigated the processing of a special case of word stimuli that are extremely difficult to evaluate, bivalent noun-noun-compounds (NNCs), i.e. novel words that mix a positive and negative noun, e.g. ‘Bombensex’ (bomb-sex). In a functional magnetic resonance imaging (fMRI) experiment we compared their processing with easier-to-evaluate non-bivalent NNCs in a valence decision task (VDT). Bivalent NNCs produced longer reaction times and elicited greater activation in the left inferior frontal gyrus (LIFG) than non-bivalent words, especially in contrast to words of negative valence. We attribute this effect to a LIFG-grounded process of semantic integration that requires greater effort for processing converse information, supporting the notion of a valence representation based on associations in semantic networks. PMID:27491491

  10. An Integrative Process Approach on Judgment and Decision Making: The Impact of Arousal, Affect, Motivation, and Cognitive Ability

    ERIC Educational Resources Information Center

    Roets, Arne; Van Hiel, Alain

    2011-01-01

    This article aims to integrate the findings from various research traditions on human judgment and decision making, focusing on four process variables: arousal, affect, motivation, and cognitive capacity/ability. We advocate a broad perspective referred to as the integrative process approach (IPA) of decision making, in which these process…

  11. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology.

    PubMed

    Liu, Ying; Lu, Zheming; Xu, Ruiping; Ke, Yang

    2016-02-01

    Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 (n = 101), HPV18 (n = 7), and HPV58 (n = 9). We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma. PMID:26735580

  12. Smoking and polymorphisms in xenobiotic metabolism and DNA repair genes are additive risk factors affecting bladder cancer in Northern Tunisia.

    PubMed

    Rouissi, Kamel; Ouerhani, Slah; Hamrita, Bechr; Bougatef, Karim; Marrakchi, Raja; Cherif, Mohamed; Ben Slama, Mohamed Riadh; Bouzouita, Mohamed; Chebil, Mohamed; Ben Ammar Elgaaied, Amel

    2011-12-01

    Cancer epidemiology has undergone marked development since the nineteen-fifties. One of the most spectacular and specific contributions was the demonstration of the massive effect of smoking and genetic polymorphisms on the occurrence of bladder cancer. The tobacco carcinogens are metabolized by various xenobiotic metabolizing enzymes, such as the super-families of N-acetyltransferases (NAT) and glutathione S-transferases (GST). DNA repair is essential to an individual's ability to respond to damage caused by tobacco carcinogens. Alterations in DNA repair genes may affect cancer risk by influencing individual susceptibility to this environmental exposure. Polymorphisms in NAT2, GST and DNA repair genes alter the ability of these enzymes to metabolize carcinogens or to repair alterations caused by this process. We have conducted a case-control study to assess the role of smoking, slow NAT2 variants, GSTM1 and GSTT1 null, and XPC, XPD, XPG nucleotide excision-repair (NER) genotypes in bladder cancer development in North Tunisia. Taken alone, each gene unless NAT2 did not appear to be a factor affecting bladder cancer susceptibility. For the NAT2 slow acetylator genotypes, the NAT2*5/*7 diplotype was found to have a 7-fold increased risk to develop bladder cancer (OR = 7.14; 95% CI: 1.30-51.41). However, in tobacco consumers, we have shown that Null GSTM1, Wild GSTT1, Slow NAT2, XPC (CC) and XPG (CC) are genetic risk factors for the disease. When combined together in susceptible individuals compared to protected individuals these risk factors give an elevated OR (OR = 61). So, we have shown a strong cumulative effect of tobacco and different combinations of studied genetic risk factors which lead to a great susceptibility to bladder cancer. PMID:21647780

  13. The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

    PubMed

    Evenson, Donald P

    2016-06-01

    Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of

  14. Association of a DNA virus with grapevines affected by red blotch disease in California.

    PubMed

    Al Rwahnih, Maher; Dave, Ashita; Anderson, Michael M; Rowhani, Adib; Uyemoto, Jerry K; Sudarshana, Mysore R

    2013-10-01

    In the Napa Valley of California, vineyards of 'Cabernet Franc' (CF) clone 214, 'Cabernet Sauvignon' clone 337, and 'Zinfandel' clone 1A (Z1A) with grapevines exhibiting foliar symptoms of red blotches, marginal reddening, and red veins that were accompanied by reduced sugar accumulation in fruit at harvest were initially suspected to be infected with leafroll-associated viruses. However, reverse-transcription polymerase chain reaction (PCR) tests were negative for all known leafroll-associated viruses, with the exception of Grapevine leafroll-associated virus 2 in Z1A. Metagenomic analysis of cDNA libraries obtained from double-stranded RNA enriched nucleic acid (NA) preparations from bark scrapings of dormant canes on an Illumina platform revealed sequences having a distant relationship with members of the family Geminiviridae. Sequencing of products obtained by PCR assays using overlapping primers and rolling circle amplification (RCA) confirmed the presence of a single circular genome of 3,206 nucleotides which was nearly identical to the genome of a recently reported Grapevine cabernet franc-associated virus found in declining grapevines in New York. We propose to call this virus "Grapevine red blotch-associated virus" (GRBaV) to describe its association with grapevine red blotch disease. Primers specific to GRBaV amplified a product of expected size (557 bp) from NA preparations obtained from petioles of several diseased source vines. Chip bud inoculations successfully transmitted GRBaV to test plants of CF, as confirmed by PCR analysis. This is the first report of a DNA virus associated with red blotch disease of grapevines in California. PMID:23656312

  15. Tales from scales: old DNA yields insights into contemporary evolutionary processes affecting fishes.

    PubMed

    Quinn, Thomas P; Seamons, Todd R

    2009-06-01

    Salmon and trout populations are suffering declines in abundance and diversity over much of their range around the Atlantic and Pacific rims as a consequence of many factors. One method of dealing with the decline has been to produce them in hatcheries but the wisdom of this approach has been hotly debated (e.g. Hilborn & Winton 1993; Waples 1999; Brannon et al. 2004). One concern is that domesticated hatchery strains will interbreed with locally adapted wild fish; but how do we study the genetic effects if the introgression might have occurred in the past? Hansen (2002) used DNA isolated from archived scales from brown trout, Salmo trutta (Fig. 1), to show that domesticated trout had, to varying degrees, genetically introgressed with wild, native trout in two Danish rivers. Extending that study, Hansen et al. (2009) have examined DNA from brown trout scales in six Danish rivers collected during historical (1927-1956) and contemporary (2000-2006) periods and from two hatchery source populations, to assess the effects of stocking nonlocal strains of hatchery trout and declining abundance on genetic diversity. Using 21 microsatellite loci, they revealed that genetic change occurred between the historic and contemporary time periods. Many populations appeared to have some low level of introgression from hatchery stocks and two populations apparently experienced high levels of introgression. Hansen et al. (2009) also showed that population structure persists in contemporary populations despite apparent admixture and migration among populations, providing evidence that the locally adapted populations have struggled against and, to some extent, resisted being overwhelmed by repeated introductions of and interbreeding with non-native, hatchery-produced conspecifics. PMID:19457205

  16. Interoceptive dysfunction: toward an integrated framework for understanding somatic and affective disturbance in depression.

    PubMed

    Harshaw, Christopher

    2015-03-01

    Depression is characterized by disturbed sleep and eating, a variety of other nonspecific somatic symptoms, and significant somatic comorbidities. Why there is such close association between cognitive and somatic dysfunction in depression is nonetheless poorly understood. An explosion of research in the area of interoception-the perception and interpretation of bodily signals-over the last decade nonetheless holds promise for illuminating what have until now been obscure links between the social, cognitive-affective, and somatic features of depression. This article reviews rapidly accumulating evidence that both somatic signaling and interoception are frequently altered in depression. This includes comparative studies showing vagus-mediated effects on depression-like behaviors in rodent models as well as studies in humans indicating both dysfunction in the neural substrates for interoception (e.g., vagus, insula, anterior cingulate cortex) and reduced sensitivity to bodily stimuli in depression. An integrative framework for organizing and interpreting this evidence is put forward which incorporates (a) multiple potential pathways to interoceptive dysfunction; (b) interaction with individual, gender, and cultural differences in interoception; and (c) a developmental psychobiological systems perspective, emphasizing likely differential susceptibility to somatic and interoceptive dysfunction across the lifespan. Combined with current theory and evidence, it is suggested that core symptoms of depression (e.g., anhedonia, social deficits) may be products of disturbed interoceptive-exteroceptive integration. More research is nonetheless needed to fully elucidate the relationship between mind, body, and social context in depression. PMID:25365763

  17. Interoceptive Dysfunction: Toward An Integrated Framework for Understanding Somatic and Affective Disturbance in Depression

    PubMed Central

    Harshaw, Christopher

    2014-01-01

    Depression is characterized by disturbed sleep and eating, a variety of other, nonspecific somatic symptoms, and significant somatic comorbidities. Why there is such close association between cognitive and somatic dysfunction in depression is nonetheless poorly understood. An explosion of research in the area of interoception—the perception and interpretation of bodily signals—over the last decade nonetheless holds promise for illuminating what have until now been obscure links between the social, cognitive-affective, and somatic features of depression. This paper reviews rapidly accumulating evidence that both somatic signaling and interoception are frequently altered in depression. This includes comparative studies showing vagus-mediated effects on depression-like behaviors in rodent models as well as studies in humans indicating both dysfunction in the neural substrates for interoception (e.g., vagus, insula, anterior cingulate cortex) and reduced sensitivity to bodily stimuli in depression. An integrative framework for organizing and interpreting this evidence is put forward which incorporates (a) multiple potential pathways to interoceptive dysfunction; (b) interaction with individual, gender, and cultural differences in interoception; and (c) a developmental psychobiological systems perspective, emphasizing likely differential susceptibility to somatic and interoceptive dysfunction across the lifespan. Combined with current theory and evidence, it is suggested that core symptoms of depression (e.g., anhedonia, social deficits) may be products of disturbed interoceptive-exteroceptive integration. More research is nonetheless needed to fully elucidate the relationship between mind, body, and social context in depression. PMID:25365763

  18. The Effect of Computer-Assisted Learning Integrated with Metacognitive Prompts on Students' Affective Skills

    NASA Astrophysics Data System (ADS)

    Tatar, Nilgün; Akpınar, Ercan; Feyzioğlu, Eylem Yıldız

    2012-12-01

    The purpose of this study is to investigate the effect of computer-assisted learning integrated with metacognitive prompts on elementary students' affective skills on the subject of electricity. The researchers developed educational software to enable students to easily and comprehensively learn the concepts in the subject of electricity. A case study method was used. Eighteen students from the seventh grade (12-13 years) participated in the study. Students' views on their performances while using educational software and the impact of the software on their affective skills towards the subject of electricity were examined. Data were collected by open-ended questions in the educational software. According to the research results, there were students who had negative attitudes and perceptions before starting to learn about the subject of electricity. Interactive activities, animations, and visual experiments in the educational software were effective in overcoming the students' negative attitudes and perceptions about the subject. Besides, students who assessed their own performances during the learning process believed themselves to be more successful over time. In the light of the research results, some suggestions are made for future studies.

  19. Cardio-visual integration modulates the subjective perception of affectively neutral stimuli.

    PubMed

    Azevedo, Ruben T; Ainley, Vivien; Tsakiris, Manos

    2016-01-01

    Interoception, which refers to the perception of internal body signals, has been consistently associated with emotional processing and with the sense of self. However, its influence on the subjective appraisal of affectively neutral and body-unrelated stimuli is still largely unknown. Across two experiments we sought to investigate this issue by asking participants to detect changes in the flashing rhythm of a simple stimulus (a circle) that could either be pulsing synchronously with their own heartbeats or following the pattern of another person's heart. While overall task performance did not vary as a function of cardio-visual synchrony, participants were better at identifying trials in which no change occurred when the flashes were synchronous with their own heartbeats. This study adds to the growing body of research indicating that we use our body as a reference point when perceiving the world; and extends this view by focusing on the role that signals coming from inside the body, such as heartbeats, may play in this referencing process. Specifically we show that private interoceptive sensations can be combined with affectively neutral information unrelated to the self to influence the processing of a multisensory percept. Results are discussed in terms of both standard multisensory integration processes and predictive coding theories. PMID:26620928

  20. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

    PubMed Central

    Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

    2015-01-01

    The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway. PMID:26074930

  1. Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration.

    PubMed Central

    Nam, J; Matthysse, A G; Gelvin, S B

    1997-01-01

    We show that among ecotypes of Arabidopsis, there is considerable variation in their susceptibility to crown gall disease. Differences in susceptibility are heritable and, in one ecotype, segregate as a single major contributing locus. In several ecotypes, recalcitrance to tumorigenesis results from decreased binding of Agrobacterium to inoculated root explants. The recalcitrance of another ecotype occurs at a late step in T-DNA transfer. Transient expression of a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is deficient in crown gall tumorigenesis, transformation to kanamycin resistance, and stable GUS expression. This ecotype is also more sensitive to gamma radiation than is a susceptible ecotype. DNA gel blot analysis showed that after infection by Agrobacterium, less T-DNA was integrated into the genome of the recalcitrant ecotype than was integrated into the genome of a highly susceptible ecotype. PMID:9090878

  2. Acridine Orange and Flow Cytometry: Which Is Better to Measure the Effect of Varicocele on Sperm DNA Integrity?

    PubMed Central

    Mohammed, Essam-Elden M.; Mosad, Eman; Zahran, Asmaa M.; Hameed, Diaa A.; Taha, Emad A.; Mohamed, Mohamed A.

    2015-01-01

    We evaluated the effect of varicocelectomy on semen parameters and levels of sperm DNA damage in infertile men. A total of 75 infertile men with varicocele and 40 fertile men (controls) were included in this study. Semen analysis and sperm DNA damage expressed as the DNA fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before and 6 months after varicocelectomy. The patients were also followed up for 1 year for pregnancy outcome. Semen parameters were significantly lower in varicocele patients compared to controls (P < 0.05). Mean percentages of sperm DNA fragmentation and sperm DNA chromatin condensation in patients were significantly higher than those in controls (P < 0.05). After varicocelectomy, sperm DNA fragmentation improved significantly, whereas sperm chromatin condensation was not significantly changed. In 15 out of 75 varicocele patients, clinical pregnancy was diagnosed; those with positive pregnancy outcome had significant improvement in sperm count, progressive sperm motility, and sperm DNA fragmentation, but there was no significant difference in sperm DNA condensation compared to negative pregnancy outcome patients. We concluded from this study that acridine orange stain is more reliable method than flow cytometry in the evaluation of sperm DNA integrity after varicocelectomy. PMID:26681938

  3. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.

    PubMed

    Hisano, Yu; Sakuma, Tetsushi; Nakade, Shota; Ohga, Rie; Ota, Satoshi; Okamoto, Hitoshi; Yamamoto, Takashi; Kawahara, Atsuo

    2015-01-01

    The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish. PMID:25740433

  4. Automated and integrated system for high-throughput DNA genotyping directly from blood.

    PubMed

    Zhang, N; Tan, H; Yeung, E S

    1999-03-15

    An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1, and TPOX, are coamplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the sample for polymerase chain reaction (PCR) without any pretreatment. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The instrumentation is compatible with future expansion to hundreds of capillaries to achieve even higher throughput. PMID:10093495

  5. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

    PubMed

    Marx, Hans; Mecklenbräuker, Astrid; Gasser, Brigitte; Sauer, Michael; Mattanovich, Diethard

    2009-12-01

    The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers. PMID:19799640

  6. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  7. Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

    PubMed

    Suzumori, Nobuhiro; Ebara, Takeshi; Yamada, Takahiro; Samura, Osamu; Yotsumoto, Junko; Nishiyama, Miyuki; Miura, Kiyonori; Sawai, Hideaki; Murotsuki, Jun; Kitagawa, Michihiro; Kamei, Yoshimasa; Masuzaki, Hideaki; Hirahara, Fumiki; Saldivar, Juan-Sebastian; Dharajiya, Nilesh; Sago, Haruhiko; Sekizawa, Akihiko

    2016-07-01

    The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging. PMID:26984559

  8. Translation Start Sequences Affect the Efficiency of Silencing of Agrobacterium tumefaciens T-DNA Oncogenes1

    PubMed Central

    Lee, Hyewon; Humann, Jodi L.; Pitrak, Jennifer S.; Cuperus, Josh T.; Parks, T. Dawn; Whistler, Cheryl A.; Mok, Machteld C.; Ream, L. Walt

    2003-01-01

    Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5′ end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease. PMID:12972655

  9. Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface

    PubMed Central

    Senear, Donald F.; Tretyachenko-Ladokhina, Vira; Opel, Michael L.; Aeling, Kimberly A.; Wesley Hatfield, G.; Franklin, Laurie M.; Darlington, Reuben C.

    2007-01-01

    E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy. PMID:17324943

  10. Integration and bioinformatics analysis of DNA-methylated genes associated with drug resistance in ovarian cancer

    PubMed Central

    YAN, BINGBING; YIN, FUQIANG; WANG, QI; ZHANG, WEI; LI, LI

    2016-01-01

    The main obstacle to the successful treatment of ovarian cancer is the development of drug resistance to combined chemotherapy. Among all the factors associated with drug resistance, DNA methylation apparently plays a critical role. In this study, we performed an integrative analysis of the 26 DNA-methylated genes associated with drug resistance in ovarian cancer, and the genes were further evaluated by comprehensive bioinformatics analysis including gene/protein interaction, biological process enrichment and annotation. The results from the protein interaction analyses revealed that at least 20 of these 26 methylated genes are present in the protein interaction network, indicating that they interact with each other, have a correlation in function, and may participate as a whole in the regulation of ovarian cancer drug resistance. There is a direct interaction between the phosphatase and tensin homolog (PTEN) gene and at least half of the other genes, indicating that PTEN may possess core regulatory functions among these genes. Biological process enrichment and annotation demonstrated that most of these methylated genes were significantly associated with apoptosis, which is possibly an essential way for these genes to be involved in the regulation of multidrug resistance in ovarian cancer. In addition, a comprehensive analysis of clinical factors revealed that the methylation level of genes that are associated with the regulation of drug resistance in ovarian cancer was significantly correlated with the prognosis of ovarian cancer. Overall, this study preliminarily explains the potential correlation between the genes with DNA methylation and drug resistance in ovarian cancer. This finding has significance for our understanding of the regulation of resistant ovarian cancer by methylated genes, the treatment of ovarian cancer, and improvement of the prognosis of ovarian cancer. PMID:27347118

  11. An innovative and integrated approach based on DNA walking to identify unauthorised GMOs.

    PubMed

    Fraiture, Marie-Alice; Herman, Philippe; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

    2014-03-15

    In the coming years, the frequency of unauthorised genetically modified organisms (GMOs) being present in the European food and feed chain will increase significantly. Therefore, we have developed a strategy to identify unauthorised GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. This integrated approach is performed in two successive steps on Bt rice grains. First, the potential presence of unauthorised GMOs is assessed by the qPCR SYBR®Green technology targeting the terminator 35S pCAMBIA element. Second, its presence is confirmed via the characterisation of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. This approach allows to rapidly identify the transgene flanking region and can easily be implemented by the enforcement laboratories. PMID:24206686

  12. Modes of integration of heterologous plasmid DNA into the chromosome of Streptococcus pneumoniae

    SciTech Connect

    Pozzi, G.; Guild, W.R.

    1985-03-01

    The authors compared the efficiencies of two different processes that can direct integration of plasmids into chromosomes of recipient cells during transformation. A donor-recipient system was constructed to allow a single donor plasmid to use either flanking homology, involving an apparent double crossover, or the insertion duplication process that has been described as due to a Campbell-like single crossover between the chromosome and a circular duplex. The latter process gave 600-fold fewer insertions that did the former, confirming expectations from prior work showing that insertion of heterologous DNA by use of flanking homology is highly efficient. It has some advantages for cloning and mapping purposes and can be exploited once it is recognized.

  13. Additional deleterious effects of alcohol consumption on sperm parameters and DNA integrity in diabetic mice.

    PubMed

    Pourentezari, M; Talebi, A R; Mangoli, E; Anvari, M; Rahimipour, M

    2016-06-01

    The aim of this study was to survey the impact of alcohol consumption on sperm parameters and DNA integrity in experimentally induced diabetic mice. A total of 32 adult male mice were divided into four groups: mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg kg(-1) , single dose, intraperitoneal) and basal diet, group 3 received alcohol (10 mg kg(-1) , water soluble) and basal diet, and group 4 received STZ and alcohol for 35 days. The cauda epididymidis of each mouse was dissected and placed in 1 ml of pre-warm Ham's F10 culture medium for 30 min. The swim-out spermatozoa were analysed for count, motility, morphology and viability. Sperm chromatin quality was evaluated with aniline blue, toluidine blue, acridine orange and chromomycin A3 staining. The results showed that all sperm parameters had significant differences (P < 0.05), also when sperm chromatin was assessed with cytochemical tests. There were significant differences (P < 0.001) between the groups. According to our results, alcohol and diabetes can cause abnormalities in sperm parameters and chromatin quality. In addition, alcohol consumption in diabetic mice can intensify sperm chromatin/DNA damage. PMID:26358836

  14. Increasing storage time of extended boar semen reduces sperm DNA integrity.

    PubMed

    Boe-Hansen, Gry B; Ersbøll, Annette K; Greve, Torben; Christensen, Preben

    2005-04-15

    There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA. PMID:15823356

  15. Automatic on-chip RNA-DNA hybridization assay with integrated phase change microvalves

    NASA Astrophysics Data System (ADS)

    Weng, Xuan; Jiang, Hai; Wang, Junsheng; Chen, Shu; Cao, Honghe; Li, Dongqing

    2012-07-01

    An RNA-DNA hybridization assay microfluidic chip integrated with electrothermally actuated phase change microvalves for detecting pathogenic bacteria is presented in this paper. In order to realize the sequential loading and washing processes required in such an assay, gravity-based pressure-driven flow and phase-change microvalves were used in the microfluidic chip. Paraffin wax was used as the phase change material in the valves and thin film heaters were used to electrothermally actuate microvalves. Light absorption measured by a photodetector to determine the concentrations of the samples. The automatic control of the complete assay was implemented by a self-coded LabVIEW program. To examine the performance of this chip, Salmonella was used as a sample pathogen. Significantly, reduction in reagent/sample consumption (up to 20 folds) was achieved by this on-chip assay, compared with using the commercial test kit following the same protocol in conventional labs. The experimental results show that the quantitative detection can be obtained in approximately 26 min, and the detection limit is as low as 103 CFU ml-1. This RNA-DNA hybridization assay microfluidic chip shows an excellent potential in the development of a portable device for point-of-testing applications.

  16. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

    PubMed

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-08-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  17. Pluchea lanceolata protects against Benzo(a) pyrene induced renal toxicity and loss of DNA integrity.

    PubMed

    Jahangir, Tamanna; Safhi, Mohammed M; Sultana, Sarwat; Ahmad, Sayeed

    2013-03-01

    Evidence from epidemiological, experimental and clinical trial data indicates that a plant based diet can reduce the risk of chronic diseases and reduces toxic effects. In the present study, we report the antioxidant and anticlastogenic activity of Pluchea lanceolata (PL), an important medicinal plant, in both in vitro and in vivo model. Benzo(a)pyrene (B(a)P) administration leads to depletion of renal glutathione and its metabolizing enzymes. Pretreatment with PL (100 and 200 mg /kg b.wt) restored renal glutathione content and its dependent enzymes significantly (p<0.001) with simultaneous increase in catalase(CAT), quinone reductase(QR) in mouse kidney. Prophylactic administration of PL prior to B (a) P administration significantly decreased the malondialdehyde(MDA), H2O2 and xanthineoxidase (XO) levels at a significance of p<0.001, at both the doses. PL extract pretreated groups showed marked inhibition in B(a)P induced micronuclei formation in mouse bone marrow cells with simultaneous restoration of DNA integrity, viz. alkaline unwinding assay and DNA damage shown by gel-electrophoresis. HPTLC confirms the presence of quercetin in plant extract which could be responsible for PL protecting efficacy. In conclusion, the present findings strongly support the antioxidant efficacy of PL, possibly by modulation of antioxidant armory. PMID:24170979

  18. An Integrative Multi-scale Analysis of the Dynamic DNA Methylation Landscape in Aging

    PubMed Central

    Yuan, Tian; Jiao, Yinming; de Jong, Simone; Ophoff, Roel A.; Beck, Stephan; Teschendorff, Andrew E.

    2015-01-01

    Recent studies have demonstrated that the DNA methylome changes with age. This epigenetic drift may have deep implications for cellular differentiation and disease development. However, it remains unclear how much of this drift is functional or caused by underlying changes in cell subtype composition. Moreover, no study has yet comprehensively explored epigenetic drift at different genomic length scales and in relation to regulatory elements. Here we conduct an in-depth analysis of epigenetic drift in blood tissue. We demonstrate that most of the age-associated drift is independent of the increase in the granulocyte to lymphocyte ratio that accompanies aging and that enrichment of age-hypermethylated CpG islands increases upon adjustment for cellular composition. We further find that drift has only a minimal impact on in-cis gene expression, acting primarily to stabilize pre-existing baseline expression levels. By studying epigenetic drift at different genomic length scales, we demonstrate the existence of mega-base scale age-associated hypomethylated blocks, covering approximately 14% of the human genome, and which exhibit preferential hypomethylation in age-matched cancer tissue. Importantly, we demonstrate the feasibility of integrating Illumina 450k DNA methylation with ENCODE data to identify transcription factors with key roles in cellular development and aging. Specifically, we identify REST and regulatory factors of the histone methyltransferase MLL complex, whose function may be disrupted in aging. In summary, most of the epigenetic drift seen in blood is independent of changes in blood cell type composition, and exhibits patterns at different genomic length scales reminiscent of those seen in cancer. Integration of Illumina 450k with appropriate ENCODE data may represent a fruitful approach to identify transcription factors with key roles in aging and disease. PMID:25692570

  19. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  20. Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children.

    PubMed Central

    Yaginuma, K; Kobayashi, H; Kobayashi, M; Morishima, T; Matsuyama, K; Koike, K

    1987-01-01

    Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences. Images PMID:3033312

  1. Clinical Integration and How It Affects Student Retention in Undergraduate Athletic Training Programs

    PubMed Central

    Young, Allison; Klossner, Joanne; Docherty, Carrie L; Dodge, Thomas M; Mensch, James M

    2013-01-01

    Context A better understanding of why students leave an undergraduate athletic training education program (ATEP), as well as why they persist, is critical in determining the future membership of our profession. Objective To better understand how clinical experiences affect student retention in undergraduate ATEPs. Design Survey-based research using a quantitative and qualitative mixed-methods approach. Setting Three-year undergraduate ATEPs across District 4 of the National Athletic Trainers' Association. Patients or Other Participants Seventy-one persistent students and 23 students who left the ATEP prematurely. Data Collection and Analysis Data were collected using a modified version of the Athletic Training Education Program Student Retention Questionnaire. Multivariate analysis of variance was performed on the quantitative data, followed by a univariate analysis of variance on any significant findings. The qualitative data were analyzed through inductive content analysis. Results A difference was identified between the persister and dropout groups (Pillai trace = 0.42, F1,92 = 12.95, P = .01). The follow-up analysis of variance revealed that the persister and dropout groups differed on the anticipatory factors (F1,92 = 4.29, P = .04), clinical integration (F1,92 = 6.99, P = .01), and motivation (F1,92 = 43.12, P = .01) scales. Several themes emerged in the qualitative data, including networks of support, authentic experiential learning, role identity, time commitment, and major or career change. Conclusions A perceived difference exists in how athletic training students are integrated into their clinical experiences between those students who leave an ATEP and those who stay. Educators may improve retention by emphasizing authentic experiential learning opportunities rather than hours worked, by allowing students to take on more responsibility, and by facilitating networks of support within clinical education experiences. PMID:23672327

  2. The DnaJ-Like Zinc Finger Domain Protein PSA2 Affects Light Acclimation and Chloroplast Development in Arabidopsis thaliana.

    PubMed

    Wang, Yan-Wen; Chen, Si-Ming; Wang, Wei-Jie; Huang, Xing-Qi; Zhou, Chang-Fang; Zhuang, Zhong; Lu, Shan

    2016-01-01

    The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin, and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development. PMID:27047527

  3. The DnaJ-Like Zinc Finger Domain Protein PSA2 Affects Light Acclimation and Chloroplast Development in Arabidopsis thaliana

    PubMed Central

    Wang, Yan-Wen; Chen, Si-Ming; Wang, Wei-Jie; Huang, Xing-Qi; Zhou, Chang-Fang; Zhuang, Zhong; Lu, Shan

    2016-01-01

    The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin, and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development. PMID:27047527

  4. Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

    PubMed

    Narayanan, Aswathy; Pullepu, Dileep; Reddy, Praveen Kumar; Uddin, Wasim; Kabir, M Anaul

    2016-07-01

    The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors. PMID:26992923

  5. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  6. Identification of Differentially Expressed Genes through Integrated Study of Alzheimer’s Disease Affected Brain Regions

    PubMed Central

    Berretta, Regina; Moscato, Pablo

    2016-01-01

    Background Alzheimer’s disease (AD) is the most common form of dementia in older adults that damages the brain and results in impaired memory, thinking and behaviour. The identification of differentially expressed genes and related pathways among affected brain regions can provide more information on the mechanisms of AD. In the past decade, several studies have reported many genes that are associated with AD. This wealth of information has become difficult to follow and interpret as most of the results are conflicting. In that case, it is worth doing an integrated study of multiple datasets that helps to increase the total number of samples and the statistical power in detecting biomarkers. In this study, we present an integrated analysis of five different brain region datasets and introduce new genes that warrant further investigation. Methods The aim of our study is to apply a novel combinatorial optimisation based meta-analysis approach to identify differentially expressed genes that are associated to AD across brain regions. In this study, microarray gene expression data from 161 samples (74 non-demented controls, 87 AD) from the Entorhinal Cortex (EC), Hippocampus (HIP), Middle temporal gyrus (MTG), Posterior cingulate cortex (PC), Superior frontal gyrus (SFG) and visual cortex (VCX) brain regions were integrated and analysed using our method. The results are then compared to two popular meta-analysis methods, RankProd and GeneMeta, and to what can be obtained by analysing the individual datasets. Results We find genes related with AD that are consistent with existing studies, and new candidate genes not previously related with AD. Our study confirms the up-regualtion of INFAR2 and PTMA along with the down regulation of GPHN, RAB2A, PSMD14 and FGF. Novel genes PSMB2, WNK1, RPL15, SEMA4C, RWDD2A and LARGE are found to be differentially expressed across all brain regions. Further investigation on these genes may provide new insights into the development of AD

  7. DNA integrity assessment in hemocytes of soft-shell clams (Mya arenaria) in the Saguenay Fjord (Québec, Canada).

    PubMed

    Debenest, T; Gagné, F; Burgeot, T; Blaise, C; Pellerin, J

    2013-02-01

    The purpose of this study was to examine the effects of pollution on DNA integrity in the feral soft-shell clam (Mya arenaria) in the Saguenay Fjord. Intertidal clams were collected downstream and upstream of the fjord at sites under anthropogenic pollution. DNA integrity was assessed by following changes in single- and double-stranded breaks, variation in DNA content and micro-nuclei (MN) incidence in hemocytes. The results revealed that clams collected at polluted sites had reduced DNA strand breaks (lower DNA repair activity), increased DNA content variation and MN frequency in hemocytes. The data revealed that DNA content variation was closely related to MN frequency and negatively with DNA strand breaks formation. Water conductivity was also related to reduced MN frequency and DNA content variation, indicating that, in addition to the effects of pollution, the gradual dilution of saltwater could compromise mussel health. PMID:22744162

  8. Campomanesia adamantium extract induces DNA damage, apoptosis, and affects cyclophosphamide metabolism.

    PubMed

    Martello, M D; David, N; Matuo, R; Carvalho, P C; Navarro, S D; Monreal, A C D; Cunha-Laura, A L; Cardoso, C A L; Kassuya, C A L; Oliveira, R J

    2016-01-01

    Campomanesia adamantium (Cambess.) O. Berg. is originally from Brazil. Its leaves and fruits have medicinal properties such as anti-inflammatory, antidiarrheal and antiseptic properties. However, the mutagenic potential of this species has been reported in few studies. This study describes the mutagenic/antimutagenic, splenic phagocytic, and apoptotic activities of C. adamantium hydroethanolic extract with or without cyclophosphamide in Swiss mice. The animals orally received the hydroethanolic extract at doses of 30, 100, or 300 mg/kg with or without 100 mg/kg cyclophosphamide. Mutagenesis was evaluated by performing the micronucleus assay after treatment for 24, 48, and 72 h, while splenic phagocytic and apoptotic effects were investigated after 72 h. Short-term exposure of 30 and 100 mg/kg extract induced mild clastogenic/aneugenic effects and increased splenic phagocytosis and apoptosis in the liver, spleen, and kidneys. When the extract was administered in combination with cyclophosphamide, micronucleus frequency and apoptosis reduced. Extract components might affect cyclophosphamide metabolism, which possibly leads to increased clearance of this chemotherapeutic agent. C. adamantium showed mutagenic activity and it may decrease the effectiveness of drugs with metabolic pathways similar to those associated with cyclophosphamide. Thus, caution should be exercised while consuming these extracts, especially when received in combination with other drugs. PMID:27173259

  9. Emotion malleability beliefs, emotion regulation, and psychopathology: Integrating affective and clinical science.

    PubMed

    Kneeland, Elizabeth T; Dovidio, John F; Joormann, Jutta; Clark, Margaret S

    2016-04-01

    Beliefs that individuals hold about whether emotions are malleable or fixed, also referred to as emotion malleability beliefs, may play a crucial role in individuals' emotional experiences and their engagement in changing their emotions. The current review integrates affective science and clinical science perspectives to provide a comprehensive review of how emotion malleability beliefs relate to emotionality, emotion regulation, and specific clinical disorders and treatment. Specifically, we discuss how holding more malleable views of emotion could be associated with more active emotion regulation efforts, greater motivation to engage in active regulatory efforts, more effort expended regulating emotions, and lower levels of pathological distress. In addition, we explain how extending emotion malleability beliefs into the clinical domain can complement and extend current conceptualizations of major depressive disorder, social anxiety disorder, and generalized anxiety disorder. This may prove important given the increasingly central role emotion dysregulation has been given in conceptualization and intervention for these psychiatric conditions. Additionally, discussion focuses on how emotion beliefs could be more explicitly addressed in existing cognitive therapies. Promising future directions for research are identified throughout the review. PMID:27086086

  10. Deoxynivalenol affects in vitro intestinal epithelial cell barrier integrity through inhibition of protein synthesis

    SciTech Connect

    Van De Walle, Jacqueline; Sergent, Therese; Piront, Neil; Toussaint, Olivier; Schneider, Yves-Jacques; Larondelle, Yvan

    2010-06-15

    Deoxynivalenol (DON), one of the most common mycotoxin contaminants of raw and processed cereal food, adversely affects the gastrointestinal tract. Since DON acts as a protein synthesis inhibitor, the constantly renewing intestinal epithelium could be particularly sensitive to DON. We analyzed the toxicological effects of DON on intestinal epithelial protein synthesis and barrier integrity. Differentiated Caco-2 cells, as a widely used model of the human intestinal barrier, were exposed to realistic intestinal concentrations of DON (50, 500 and 5000 ng/ml) during 24 h. DON caused a concentration-dependent decrease in total protein content associated with a reduction in the incorporation of [{sup 3}H]-leucine, demonstrating its inhibitory effect on protein synthesis. DON simultaneously increased the paracellular permeability of the monolayer as reflected through a decreased transepithelial electrical resistance associated with an increased paracellular flux of the tracer [{sup 3}H]-mannitol. A concentration-dependent reduction in the expression level of the tight junction constituent claudin-4 was demonstrated by Western blot, which was not due to diminished transcription, increased degradation, or NF-{kappa}B, ERK or JNK activation, and was also observed for a tight junction independent protein, i.e. intestinal alkaline phosphatase. These results demonstrate a dual toxicological effect of DON on differentiated Caco-2 cells consisting in an inhibition of protein synthesis as well as an increase in monolayer permeability, and moreover suggest a possible link between them through diminished synthesis of the tight junction constituent claudin-4.

  11. Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

    PubMed Central

    Chen, B; Choi, G H; Nuss, D L

    1993-01-01

    Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents. Images PMID:8344241

  12. Paper electrode integrated lateral flow immunosensor for quantitative analysis of oxidative stress induced DNA damage

    PubMed Central

    Zhu, Xuena; Shah, Pratikkumar; Stoff, Susan; Liu, Hongyun; Li, Chen-zhong

    2014-01-01

    A novel device combining electrochemical and colorimetric detection is developed for the rapid measurement of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a DNA oxidative damage biomarker. The device takes advantage of the speed and low cost of the conventional strip test as well as the high reliability and accuracy of electrochemical assay. Competitive immunoreactions were performed on the lateral flow strip, and the captured 8-OHdG on the control line was determined by chronoamperometric measurement with carbon nanotubes paper as the working electrode. At the same time, the color intensity of the test line was measured by a scanner and analyzed by the ImageJ software. The device was able to detect 8-OHdG concentrations in PBS as low as 2.07 ng mL−1 by the colorimetric method and 3.11 ng mL−1 by the electrochemical method. Furthermore, the device was successfully utilized to detect 8-OHdG in urine with a detection limit of 5.76 ng mL−1 (colorimetric method) and 8.85 ng mL−1 (electrochemical method), respectively. In conclusion, the integrated device with dual detections can provide a rapid, visual, quantitative and feasible detection method for 8-OHdG. The integration of these two methods holds two major advantages over tests based on single method. Firstly, it can provide double confidence on the same assay. Secondly, by involving two methods that differ in principle, the integration could potentially avoid false results coming from one method. In addition, these methods do not require expensive equipment or trained personnel, deeming it suitable for use as a simple, economical, portable field kit for on-site monitoring of 8-OHdG in a variety of clinical settings. PMID:24733353

  13. Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage.

    PubMed

    Bucak, Mustafa Numan; Tuncer, Pürhan Barbaros; Sarıözkan, Serpil; Başpınar, Nuri; Taşpınar, Mehmet; Coyan, Kenan; Bilgili, Ali; Akalın, Pınar Peker; Büyükleblebici, Serhat; Aydos, Sena; Ilgaz, Seda; Sunguroğlu, Asuman; Oztuna, Derya

    2010-12-01

    This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20s in a water bath for the evaluation. The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9±1.3% and 51.3±1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6±2.9% and 54.2±4.9%) and inositol (34.9±2.0% and 47.3±2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance

  14. EVALUATION OF DNA INTEGRITY USING TUNEL AND COMET ASSAY IN HUMAN SEMEN: IMMEDIATE- VERSUS DELAYED-FREEZING

    EPA Science Inventory

    EVALUATION OF DNA INTEGRITY USING TUNEL AND COMET ASSAY IN HUMAN SEMEN: IMMEDIATE- VERSUS DELAYED-FREEZING
    K. Young,* L. Xun,* S. Rothmann,? S. Perreault, ? W. Robbins*
    *University of California, Los Angeles, Los Angeles, California; ?Fertility Solutions Inc., Cleveland, ...

  15. Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction*

    PubMed Central

    Sharadamma, Narayanaswamy; Harshavardhana, Yadumurthy; Ravishankar, Apoorva; Anand, Praveen; Chandra, Nagasuma; Muniyappa, K.

    2014-01-01

    The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins. PMID:25324543

  16. DNA methylome in spleen of avian pathogenic escherichia coli-challenged broilers and integration with mRNA expression

    PubMed Central

    Xu, Haiping; Zhu, Xuenong; Hu, Yongsheng; Li, Zhenhui; Zhang, Xiquan; Nie, Qinghua

    2014-01-01

    Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV. PMID:24599154

  17. DNA methylome in spleen of avian pathogenic Escherichia coli-challenged broilers and integration with mRNA expression.

    PubMed

    Xu, Haiping; Zhu, Xuenong; Hu, Yongsheng; Li, Zhenhui; Zhang, Xiquan; Nie, Qinghua; Nolan, Lisa K; Lamont, Susan J

    2014-01-01

    Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV. PMID:24599154

  18. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

    PubMed

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-01-01

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

  19. Translating theory into practice: integrating the affective and cognitive learning dimensions for effective instruction in engineering education

    NASA Astrophysics Data System (ADS)

    Alias, Maizam; Lashari, Tahira Anwar; Abidin Akasah, Zainal; Jahaya Kesot, Mohd.

    2014-03-01

    Learning in the cognitive domain is highly emphasised and has been widely investigated in engineering education. Lesser emphasis is placed on the affective dimension although the role of affects has been supported by research. The lack of understanding on learning theories and how they may be translated into classroom application of teaching and learning is one factor that contributes to this situation. This paper proposes a working framework for integrating the affective dimension of learning into engineering education that is expected to promote better learning within the cognitive domain. Four major learning theories namely behaviourism, cognitivism, socio-culturalism, and constructivism were analysed and how affects are postulated to influence cognition are identified. The affective domain constructs identified to be important are self-efficacy, attitude and locus of control. Based on the results of the analysis, a framework that integrates methodologies for achieving learning in the cognitive domain with the support of the affective dimension of learning is proposed. It is expected that integrated approach can be used as a guideline to engineering educators in designing effective and sustainable instructional material that would result in the effective engineers for future development.

  20. Factors Affecting the Integration of Computers in Western Sydney Secondary Schools.

    ERIC Educational Resources Information Center

    Morton, Allan

    Integration is based on the assumption that computers should be an integral part of the learning process, both for servicing curriculum needs and as an object for study. The integration of computers into everyday classroom activity has proved to be more slow and difficult than expected, creating the notion that there are incentives enhancing the…

  1. Integrating stakeholder perspectives into the translation of cell-free fetal DNA testing for aneuploidy

    PubMed Central

    2012-01-01

    Background The translation of novel genomic technologies from bench to bedside enjoins the comprehensive consideration of the perspectives of all stakeholders who stand to influence, or be influenced by, the translational course. Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA (cffDNA) circulating in maternal blood is one example of an innovative technology that promises significant benefits for its intended end users; however, it is currently uncertain whether it will achieve widespread clinical implementation. We conducted qualitative interviews with 18 diverse stakeholders in this domain, including prospective users of the technology and healthcare personnel, researchers and developers, and experts in social, legal, and regulatory aspects of genetic technology, and a pilot survey of 62 obstetric healthcare providers. Analysis of interview and survey data was combined with a review of the proceedings of a full-day, multidisciplinary conference on the topic and published scientific and ethics literature surrounding this and other relevant technologies. Discussion We constructed potential pathways for technological implementation, identified broad stakeholder classes party to these translational processes, and performed a preliminary assessment of the viewpoints and interrelations among these diverse stakeholders. Some of the stakeholders whose priorities are critical to understand and integrate into translation include pregnant women and their families; healthcare providers; scientists, their institutions or companies, and the funding agencies that support them; regulatory and judicial bodies; third-party payers; professional societies; educational systems; disability rights communities; and other representatives from civil society. Stakeholder interviews, survey findings, and conference proceedings add complexity to these envisioned pathways and also demonstrate a paramount need to incorporate an iterative stakeholder analysis early and

  2. Host genotype and tumor phenotype of chemokine decoy receptors integrally affect breast cancer relapse

    PubMed Central

    Shao, Zhi-Ming

    2015-01-01

    Purpose Chemokines may play vital roles in breast cancer progression and metastasis. The primary members of chemokine decoy receptors (CDR), DARC and D6, are expressed in breast tumors and lymphatic/hematogenous vessels. CDRs sequestrate the pro-malignant chemokines. We hypothesized that breast cancer patients carrying different levels of CDR expression in tumor and/or in host might have differing clinical outcomes. Methods This prospective observational study measured both expression and germline genotype of DARC and D6 in 463 primary breast cancer patients enrolled between 2004 and 2006. The endpoint was breast cancer relapse-free survival (RFS). Results There was a significant association between the co-expression of CDR (immunohistochemical expression of both DARC and D6) with RFS (hazard ratio [HR] of 0.32, 95% confidence interval [CI] 0.19 to 0.54). Furthermore, the co-genotype of two non-synonymous polymorphisms (with two major alleles of DARC-rs12075 and D6-rs2228468 versus the others) significantly related to relapse. Mechanistically, the variant-alleles of these two polymorphisms significantly decreased by 20–30% of CCL2/CCL5 (CDR ligands) levels relative to their major counterparts. Multivariate analysis highlighted that the co-expression and co-genotype of CDR were independent predictors of RFS, with HR of 0.46 (95% CI 0.27 to 0.80) and 0.56 (95% CI 0.37 to 0.85), respectively. The addition of host CDR genetic information to tumor-based factors (including co-expression of CDR) improved the relapse prediction ability (P = 0.02 of AUC comparison). Conclusion The host genotype and tumor phenotype of CDR integrally affect breast cancer relapse. Host-related factors should be considered for individualized prediction of prognosis. PMID:26314842

  3. Sensory integration dysfunction affects efficacy of speech therapy on children with functional articulation disorders

    PubMed Central

    Tung, Li-Chen; Lin, Chin-Kai; Hsieh, Ching-Lin; Chen, Ching-Chi; Huang, Chin-Tsan; Wang, Chun-Hou

    2013-01-01

    Background Articulation disorders in young children are due to defects occurring at a certain stage in sensory and motor development. Some children with functional articulation disorders may also have sensory integration dysfunction (SID). We hypothesized that speech therapy would be less efficacious in children with SID than in those without SID. Hence, the purpose of this study was to compare the efficacy of speech therapy in two groups of children with functional articulation disorders: those without and those with SID. Method: A total of 30 young children with functional articulation disorders were divided into two groups, the no-SID group (15 children) and the SID group (15 children). The number of pronunciation mistakes was evaluated before and after speech therapy. Results: There were no statistically significant differences in age, sex, sibling order, education of parents, and pretest number of mistakes in pronunciation between the two groups (P > 0.05). The mean and standard deviation in the pre- and post-test number of mistakes in pronunciation were 10.5 ± 3.2 and 3.3 ± 3.3 in the no-SID group, and 10.1 ± 2.9 and 6.9 ± 3.5 in the SID group, respectively. Results showed great changes after speech therapy treatment (F = 70.393; P < 0.001) and interaction between the pre/post speech therapy treatment and groups (F = 11.119; P = 0.002). Conclusions: Speech therapy can improve the articulation performance of children who have functional articulation disorders whether or not they have SID, but it results in significantly greater improvement in children without SID. SID may affect the treatment efficiency of speech therapy in young children with articulation disorders. PMID:23355780

  4. A flowering integrator, SOC1, affects stomatal opening in Arabidopsis thaliana.

    PubMed

    Kimura, Yuriko; Aoki, Saya; Ando, Eigo; Kitatsuji, Ayaka; Watanabe, Aiko; Ohnishi, Masato; Takahashi, Koji; Inoue, Shin-ichiro; Nakamichi, Norihito; Tamada, Yosuke; Kinoshita, Toshinori

    2015-04-01

    Stomatal movements are regulated by multiple environmental signals. Recent investigations indicate that photoperiodic flowering components, such as CRY, GI, CO, FT and TSF, are expressed in guard cells and positively affect stomatal opening in Arabidopsis thaliana. Here we show that SOC1, which encodes a MADS box transcription factor and integrates multiple flowering signals, also exerts a positive effect on stomatal opening. FLC encodes a potent repressor of FT and SOC1, and FRI acts as an activator of FLC. Thus, we examined stomatal phenotypes in FRI-Col, which contains an active FRI allele of accession Sf-2 by introgression. We found higher expression of FLC and lower expression of FT, SOC1 and TSF in guard cells from FRI-Col than in those from Col. Light-induced stomatal opening was significantly suppressed in FRI-Col. Interestingly, vernalization of FRI-Col partially restored light-induced stomatal opening, concomitant with a decrease of FLC and increase of FT, SOC1 and TSF. Furthermore, we observed the constitutive open-stomata phenotype in transgenic plants overexpressing SOC1-GFP (green fluorescent protein) in guard cells (SOC1-GFP overexpressor), and found that light-induced stomatal opening was significantly suppressed in a soc1 knockout mutant. RNA sequencing using epidermis from the SOC1-GFP overexpressor revealed that the expression levels of several genes involved in stomatal opening, such as BLUS1 and the plasma membrane H(+)-ATPases, were higher than those in background plants. From these results, we conclude that SOC1 is involved in the regulation of stomatal opening via transcriptional regulation in guard cells. PMID:25588388

  5. Longer resistance of some DNA traits from BT176 maize to gastric juice from gastrointestinal affected patients.

    PubMed

    Ferrini, A M; Mannoni, V; Pontieri, E; Pourshaban, M

    2007-01-01

    The presence of antibiotic resistance marker genes in genetically engineered plants is one of the most controversial issues related to Genetically Modified Organism (GMO)-containing food, raising concern about the possibility that these markers could increase the pool of antibiotic resistance genes. This study investigates the in vitro survival of genes bla and cryIA(b) of maize Bt176 in human gastric juice samples. Five samples of gastric juice were collected from patients affected by gastro-esophageal reflux or celiac disease and three additional samples were obtained by pH modification with NaHCO3. DNA was extracted from maize Bt176 and incubated with samples of gastric juices at different times. The survival of the target traits (bla gene, whole 1914 bp gene cry1A(b), and its 211 bp fragment) was determined using PCR. The stability of the target genes was an inverse function of their lengths in all the samples. Survival in samples from untreated subjects was below the normal physiological time of gastric digestion. On the contrary, survival time in samples from patients under anti-acid drug treatment or in samples whose pH was modified, resulted strongly increased. Our data indicate the possibility that in particular cases the survival time could be so delayed that, as a consequence, some traits of DNA could reach the intestine. In general, this aspect must be considered for vulnerable consumers (people suffering from gastrointestinal diseases related to altered digestive functionality, physiological problems or drug side-effects) in the risk analysis usually referred to healthy subjects. PMID:17346434

  6. The Effect of Computer-Assisted Learning Integrated with Metacognitive Prompts on Students' Affective Skills

    ERIC Educational Resources Information Center

    Tatar, Nilgün; Akpinar, Ercan; Feyzioglu, Eylem Yildiz

    2013-01-01

    The purpose of this study is to investigate the effect of computer-assisted learning integrated with metacognitive prompts on elementary students' affective skills on the subject of electricity. The researchers developed educational software to enable students to easily and comprehensively learn the concepts in the subject of electricity. A…

  7. MethBank: a database integrating next-generation sequencing single-base-resolution DNA methylation programming data.

    PubMed

    Zou, Dong; Sun, Shixiang; Li, Rujiao; Liu, Jiang; Zhang, Jing; Zhang, Zhang

    2015-01-01

    DNA methylation plays crucial roles during embryonic development. Here we present MethBank (http://dnamethylome.org), a DNA methylome programming database that integrates the genome-wide single-base nucleotide methylomes of gametes and early embryos in different model organisms. Unlike extant relevant databases, MethBank incorporates the whole-genome single-base-resolution methylomes of gametes and early embryos at multiple different developmental stages in zebrafish and mouse. MethBank allows users to retrieve methylation levels, differentially methylated regions, CpG islands, gene expression profiles and genetic polymorphisms for a specific gene or genomic region. Moreover, it offers a methylome browser that is capable of visualizing high-resolution DNA methylation profiles as well as other related data in an interactive manner and thus is of great helpfulness for users to investigate methylation patterns and changes of gametes and early embryos at different developmental stages. Ongoing efforts are focused on incorporation of methylomes and related data from other organisms. Together, MethBank features integration and visualization of high-resolution DNA methylation data as well as other related data, enabling identification of potential DNA methylation signatures in different developmental stages and accordingly providing an important resource for the epigenetic and developmental studies. PMID:25294826

  8. Miniaturized devices towards an integrated lab-on-a-chip platform for DNA diagnostics

    NASA Astrophysics Data System (ADS)

    Kaprou, G.; Papadakis, G.; Kokkoris, G.; Papadopoulos, V.; Kefala, I.; Papageorgiou, D.; Gizeli, E.; Tserepi, A.

    2015-06-01

    Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. LOC systems integrate and scale down one or several laboratory functions on a single chip of a few mm2 to cm2 in size, and account for many advantages on biochemical analyses, such as low sample and reagent consumption, low cost, reduced analysis time, portability and point-of-need compatibility. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. Here, we present a comparison between static chamber and continuous flow miniaturized PCR devices, in terms of energy consumption for devices fabricated on the same material stack, with identical sample volume and channel dimensions. The comparison is implemented by a computational study coupling heat transfer in both solid and fluid, mass conservation of species, and joule heating. Based on the conclusions of this study, we develop low-cost and fast DNA amplification devices for both PCR and isothermal amplification, and we implement them in the detection of mutations related to breast cancer. The devices are fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zones necessary for PCR or isothermal amplification methods.

  9. Study of mitochondrial DNA alteration in the exhaled breath condensate of patients affected by obstructive lung diseases.

    PubMed

    Carpagnano, G E; Lacedonia, D; Carone, M; Soccio, P; Cotugno, G; Palmiotti, G A; Scioscia, G; Foschino Barbaro, M P

    2016-06-01

    Mitochondrial DNA (MtDNA) has been studied as an expression of oxidative stress in asthma, COPD, lung cancer and obstructive sleep apnea, but it has been mainly investigated systemically, although the pathogenetic mechanisms begin in the airways and only later progress to systemic circulation. The aim of this study was to investigate the MtDNA alterations in the exhaled breath condensate (EBC) of patients with asthma, COPD and asthma-COPD overlap syndrome (ACOS). In order to analyze better what happens to mitochondria, both locally and systemically, we compared MtDNA/nDNA in blood and EBC of paired patients. Thirteen (13) COPD patients, 14 asthmatics, 23 ACOS (10 according to Spanish guidelines, 13 in line with GINA guidelines) and 12 healthy subjects were enrolled. Patients underwent clinical and functional diagnostic tests as foreseen by the guidelines. They underwent blood and EBC collection. Content of MtDNA and nuclear DNA (nDNA) was measured in the blood cells and EBC of patients by Real Time PCR. The ratio between MtDNA/nDNA was calculated. For the first time we were able to detect MtDNA/nDNA in the EBC. We found higher exhaled MtDNA/nDNA in COPD, asthmatic and ACOS patients respectively compared to healthy subjects (21.9  ±  4.9 versus 6.51  ±  0.21, p  <  0.05; 7.9  ±  2.5 versus 6.51  ±  0.21, p  =  0.06; 18.3  ±  3.4 versus 6.51  ±  0.21, p  <  0.05). The level of exhaled MtDNA/nDNA was positively correlated with the plasmatic one. The levels of MtDNA/nDNA in the EBC, as expression of oxidative stress, are increased in COPD, asthmatic and ACOS patients compared to healthy subjects. These are preliminary results in a small number of well characterized patients that requires confirmation on a larger population. We support new studies directed toward the analysis of exhaled MtDNA/nDNA as a new exhaled non-invasive marker in other inflammatory/oxidative airways diseases. PMID

  10. Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy.

    PubMed

    Long, Xi; Parks, Joseph W; Bagshaw, Clive R; Stone, Michael D

    2013-02-01

    Single-molecule techniques facilitate analysis of mechanical transitions within nucleic acids and proteins. Here, we describe an integrated fluorescence and magnetic tweezers instrument that permits detection of nanometer-scale DNA structural rearrangements together with the application of a wide range of stretching forces to individual DNA molecules. We have analyzed the force-dependent equilibrium and rate constants for telomere DNA G-quadruplex (GQ) folding and unfolding, and have determined the location of the transition state barrier along the well-defined DNA-stretching reaction coordinate. Our results reveal the mechanical unfolding pathway of the telomere DNA GQ is characterized by a short distance (<1 nm) to the transition state for the unfolding reaction. This mechanical unfolding response reflects a critical contribution of long-range interactions to the global stability of the GQ fold, and suggests that telomere-associated proteins need only disrupt a few base pairs to destabilize GQ structures. Comparison of the GQ unfolded state with a single-stranded polyT DNA revealed the unfolded GQ exhibits a compacted non-native conformation reminiscent of the protein molten globule. We expect the capacity to interrogate macromolecular structural transitions with high spatial resolution under conditions of low forces will have broad application in analyses of nucleic acid and protein folding. PMID:23303789

  11. Does seawater acidification affect survival, growth and shell integrity in bivalve juveniles?

    PubMed

    Bressan, M; Chinellato, A; Munari, M; Matozzo, V; Manci, A; Marčeta, T; Finos, L; Moro, I; Pastore, P; Badocco, D; Marin, M G

    2014-08-01

    Anthropogenic emissions of carbon dioxide are leading to decreases in pH and changes in the carbonate chemistry of seawater. Ocean acidification may negatively affect the ability of marine organisms to produce calcareous structures while also influencing their physiological responses and growth. The aim of this study was to evaluate the effects of reduced pH on the survival, growth and shell integrity of juveniles of two marine bivalves from the Northern Adriatic sea: the Mediterranean mussel Mytilus galloprovincialis and the striped venus clam Chamelea gallina. An outdoor flow-through plant was set up and two pH levels (natural seawater pH as a control, pH 7.4 as the treatment) were tested in long-term experiments. Mortality was low throughout the first experiment for both mussels and clams, but a significant increase, which was sensibly higher in clams, was observed at the end of the experiment (6 months). Significant decreases in the live weight (-26%) and, surprisingly, in the shell length (-5%) were observed in treated clams, but not in mussels. In the controls of both species, no shell damage was ever recorded; in the treated mussels and clams, damage proceeded via different modes and to different extents. The severity of shell injuries was maximal in the mussels after just 3 months of exposure to a reduced pH, whereas it progressively increased in clams until the end of the experiment. In shells of both species, the damaged area increased throughout the experiment, peaking at 35% in mussels and 11% in clams. The shell thickness of the treated and control animals significantly decreased after 3 months in clams and after 6 months in mussels. In the second experiment (3 months), only juvenile mussels were exposed to a reduced pH. After 3 months, the mussels at a natural pH level or pH 7.4 did not differ in their survival, shell length or live weight. Conversely, shell damage was clearly visible in the treated mussels from the 1st month onward. Monitoring the

  12. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  13. Factors That Affect Large Subunit Ribosomal DNA Amplicon Sequencing Studies of Fungal Communities: Classification Method, Primer Choice, and Error

    PubMed Central

    Porter, Teresita M.; Golding, G. Brian

    2012-01-01

    Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve Bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50–100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys. PMID:22558215

  14. Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase.

    PubMed

    De Paepe, Annelies; De Buck, Sylvie; Nolf, Jonah; Van Lerberge, Els; Depicker, Ann

    2013-07-01

    Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants. PMID:23574114

  15. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

    PubMed

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L; Stavans, Joel

    2014-05-20

    The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host. PMID:24799672

  16. Characters and Clues: Factors Affecting Children's Extension of Knowledge through Integration of Separate Episodes

    ERIC Educational Resources Information Center

    Bauer, Patricia J.; King, Jessica E.; Larkina, Marina; Varga, Nicole L.; White, Elizabeth A.

    2012-01-01

    Children build up knowledge about the world and also remember individual episodes. How individual episodes during which children learn new things become integrated with one another to form general knowledge is only beginning to be explored. Integration between separate episodes is called on in educational contexts and in everyday life as a major…

  17. Is the Ability to Integrate Parts into Wholes Affected in Autism Spectrum Disorder?

    ERIC Educational Resources Information Center

    Olu-Lafe, Olufemi; Liederman, Jacqueline; Tager-Flusberg, Helen

    2014-01-01

    There is considerable debate about whether people with autism spectrum disorder (ASD) are biased toward local information and whether this disrupts their ability to integrate two complex shapes elements into a single figure. Moreover, few have examined the relationship between integration ability and ASD symptom severity. Adolescent/adult males…

  18. Curriculum Integration in Context: An Exploration of How Structures and Circumstances Affect Design and Implementation.

    ERIC Educational Resources Information Center

    Johnson, Amy Bell; Charner, Ivan; White, Robin

    In order to obtain firsthand information about different approaches and strategies for curriculum integration, case studies of curriculum integration models were conducted in seven sites across the United States. It was concluded that the presence or lack of certain contextual factors related to structure and operations had implications for the…

  19. Structural diversity of supercoiled DNA

    NASA Astrophysics Data System (ADS)

    Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

    2015-10-01

    By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

  20. Structural diversity of supercoiled DNA

    PubMed Central

    Irobalieva, Rossitza N.; Fogg, Jonathan M.; Catanese, Daniel J.; Sutthibutpong, Thana; Chen, Muyuan; Barker, Anna K.; Ludtke, Steven J.; Harris, Sarah A.; Schmid, Michael F.; Chiu, Wah; Zechiedrich, Lynn

    2015-01-01

    By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function. PMID:26455586

  1. TH-C-18A-09: Exam and Patient Parameters Affecting the DNA Damage Response Following CT Studies

    SciTech Connect

    Elgart, S; Adibi, A; Bostani, M; Ruehm, S; Enzmann, D; McNitt-Gray, M; Iwamoto, K

    2014-06-15

    Purpose: To identify exam and patient parameters affecting the biological response to CT studies using in vivo and ex vivo blood samples. Methods: Blood samples were collected under IRB approval from 16 patients undergoing clinically-indicated CT exams. Blood was procured prior to, immediately after and 30minutes following irradiation. A sample of preexam blood was placed on the patient within the exam region for ex vivo analysis. Whole blood samples were fixed immediately following collection and stained for γH2AX to assess DNA damage response (DDR). Median fluorescence of treated samples was compared to non-irradiated control samples for each patient. Patients were characterized by observed biological kinetic response: (a) fast — phosphorylation increased by 2minutes and fell by 30minutes, (b) slow — phosphorylation continued to increase to 30minutes and (c) none — little change was observed or irradiated samples fell below controls. Total dose values were normalized to exam time for an averaged dose-rate in dose/sec for each exam. Relationships between patient biological responses and patient and exam parameters were investigated. Results: A clearer dose response at 30minutes is observed for young patients (<61yoa; R2>0.5) compared to old patients (>61yoa; R{sup 2}<0.11). Fast responding patients were significantly younger than slow responding patients (p<0.05). Unlike in vivo samples, age did not significantly affect the patient response ex vivo. Additionally, fast responding patients received exams with significantly smaller dose-rate than slow responding patients (p<0.05). Conclusion: Age is a significant factor in the biological response suggesting that DDR may be more rapid in a younger population and slower as the population ages. Lack of an agerelated response ex vivo suggests a systemic response to radiation not present when irradiated outside the body. Dose-rate affects the biological response suggesting that patient response may be related to

  2. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

    PubMed Central

    Doolittle-Hall, Janet M.; Cunningham Glasspoole, Danielle L.; Seaman, William T.; Webster-Cyriaque, Jennifer

    2015-01-01

    Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. PMID:26569308

  3. HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

    Cancer.gov

    Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

  4. HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

    Cancer.gov

    Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

  5. Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

    NASA Technical Reports Server (NTRS)

    Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan; Lu, Thomas; Chao, Tien-Hsin

    2011-01-01

    In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5 FPGA to an embedded system consisting of a PowerPC and a Xilinx Virtex 5 FPGA. The FTS instrument features a novel liquid crystal waveguide, which consequently eliminates all moving parts from the instrument. The addition of the eDNA architecture to do the control and data processing has resulted in a highly fault-tolerant FTS instrument. The case study has shown that the early stage prototype of the autonomous self-healing eDNA architecture is expensive in terms of execution time.

  6. From sample to PCR product in under 45 minutes: a polymeric integrated microdevice for clinical and forensic DNA analysis.

    PubMed

    Lounsbury, Jenny A; Karlsson, Anne; Miranian, Daniel C; Cronk, Stephen M; Nelson, Daniel A; Li, Jingyi; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2013-04-01

    The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the β-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the β-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice

  7. Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

    NASA Astrophysics Data System (ADS)

    Vivas, Paula; Velmurugu, Yogambigai; Kuznetsov, Serguei V.; Rice, Phoebe A.; Ansari, Anjum

    2013-09-01

    Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SKbi ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SKuni < 0.5. This result is counterintuitive because there

  8. Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins.

    PubMed

    Park, So-Yon; Vaghchhipawala, Zarir; Vasudevan, Balaji; Lee, Lan-Ying; Shen, Yunjia; Singer, Kamy; Waterworth, Wanda M; Zhang, Zhanyuan J; West, Christopher E; Mysore, Kirankumar S; Gelvin, Stanton B

    2015-03-01

    Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR), play an important role in 'classical' (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a 'backup' (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA Ligase VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration. PMID:25641249

  9. Local chromatin structure of heterochromatin regulates repeatedDNA stability, nucleolus structure, and genome integrity

    SciTech Connect

    Peng, Jamy C.

    2007-05-05

    Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

  10. Does Ataxia Telangiectasia Mutated (ATM) protect testicular and germ cell DNA integrity by regulating the redox status?

    PubMed

    Godschalk, Roger W L; Vanhees, Kimberly; Maas, Lou; Drittij, Marie-Jose; Pachen, Daniëlle; van Doorn-Khosrovani, Sahar van Waalwijk; van Schooten, Frederik J; Haenen, Guido R M M

    2016-08-01

    A balanced redox homeostasis in the testis is essential for genetic integrity of sperm. Reactive oxygen species can disturb this balance by oxidation of glutathione, which is regenerated using NADPH, formed by glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is regulated by the Ataxia Telangiectasia Mutated (Atm) protein. Therefore, we studied the redox status and DNA damage in testes and sperm of mice that carried a deletion in Atm. The redox status in heterozygote mice, reflected by glutathione levels and antioxidant capacity, was lower than in wild type mice, and in homozygotes the redox status was even lower. The redox status correlated with oxidative DNA damage that was highest in mice that carried Atm deletions. Surprisingly, G6PDH activity was highest in homozygotes carrying the deletion. These data indicate that defective Atm reduces the redox homeostasis of the testis and genetic integrity of sperm by regulating glutathione levels independently from G6PDH activity. PMID:27318254

  11. Characters and clues: Factors affecting children’s extension of knowledge through integration of separate episodes

    PubMed Central

    Bauer, Patricia J.; King, Jessica E.; Larkina, Marina; Varga, Nicole L.; White, Elizabeth A.

    2012-01-01

    Children build up knowledge about the world and also remember individual episodes. How individual episodes during which children learn new things become integrated with one another to form general knowledge is only beginning to be explored. Integration between separate episodes is called on in educational contexts and in everyday life as a major means of extending knowledge and organizing information. Bauer and San Souci (2010) provided an initial demonstration that 6-year-olds extend their knowledge by integrating between separate but related episodes; the episodes shared a high level of surface similarity. Experiments 1A and 1B of the current research were tests of integration under low and high levels of surface similarity, respectively. In Experiment 1A, when surface similarity of the episodes was low, 6-year-olds integrated between passages of text, yet their performance was not as robust as observed previously. In Experiment 1B, when surface similarity of the episodes was high, a replication of Bauer and San Souci’s results was observed. In Experiment 2, we tested whether a “hint” to consult the information learned in the passages improved performance even when surface level similarity was low. The hint had a strong facilitating effect. Possible mechanisms of integration between separate yet related episodes are discussed. PMID:22153911

  12. RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity.

    PubMed

    Yard, Brian D; Reilly, Nicole M; Bedenbaugh, Michael K; Pittman, Douglas L

    2016-06-01

    The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex. PMID:27161866

  13. Effect of vitamin E on sperm parameters and DNA integrity in sodium arsenite-treated rats

    PubMed Central

    Momeni, Hamid Reza; Eskandari, Najmeh

    2012-01-01

    Background: Arsenic as an environmental toxicant is able to exert malformations in male reproductive system by inducing oxidative stress. Vitamin E (Vit.E) is known as antioxidant vitamin. Objective: The aim of this study was to investigate the harmful effects of sodium arsenite on sperm parameters and the antioxidant effects of Vit.E on sperm anomalies in sodium arsenite treated rats. Materials and Methods: Adult male rats were divided into 4 groups: control, sodium arsenite (8 mg/kg/day), Vit.E (100 mg/kg/day) and sodium arsenite+Vit.E. Oral treatments were performed till 8 weeks. Body and left testis weight were recorded and then left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze number, motility, viability and abnormalities of the sperm. Sperm chromatin quality was assessed by nuclear staining using acridine orange and aniline blue. Results: Body and testis weight showed no significant change in 4 groups (p>0.05). A significant decrease in the number, motility, viability and normal sperm morphology was found in sodium arsenite-treated rats compared to the control (p<0.001). Sodium arsenite had no effect on sperm DNA integrity and histon-protamine replacement (p>0.05). In sodium arsenite+Vit.E group, Vit.E could significantly compensate the harmful effects of sodium arsenite on sperm number, motility, viability and morphology compared to sodium arsenite group. In addition, sperm viability and motility was significantly increased in rats treated with Vit.E alone compared to the control and sodium arsenite+Vit.E group. Conclusion: Vitamin E could compensate the adverse effects of sodium arsenite on sperm parameters in adult rats. PMID:25243001

  14. A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element.

    PubMed

    Miyazaki, Ryo; van der Meer, Jan Roelof

    2013-07-01

    During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria. PMID:23686268

  15. Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions.

    PubMed

    Hollox, E J; Poulter, M; Wang, Y; Krause, A; Swallow, D M

    1999-01-01

    In most mammals lactase activity declines after weaning when lactose is no longer part of the diet, but in many humans lactase activity persists into adult life. The difference responsible for this phenotypic polymorphism has been shown to be cis-acting to the lactase gene. The causal sequence difference has not been found so far, but a number of polymorphic sites have been found within and near to the lactase gene. We have shown previously that in Europeans there are two polymorphic sites in a small region between 974 bp and 852 bp upstream from the start of transcription, which are detectable by denaturing gradient gel electrophoresis (DGGE). In this study, analysis of individuals from five other population groups by the same DGGE method reveals four new alleles resulting from three additional nucleotide changes within this very small region. Analysis of sequence in four primate species and comparison with the published pig sequence shows that the overall sequence of this highly variable human region is conserved in pigs as well as primates, and that it lies within a 1kb region which has been shown to control lactase downregulation in pigs. Electrophoretic mobility shift assay (EMSA) studies were carried out to determine whether common variation affected protein-DNA binding and several binding activities were found using this technique. A novel two base-pair deletion that is common in most populations tested, but is not present in Europeans, caused no change in binding activity. However, a previously published C to T transition at -958bp dramatically reduced binding activity, although the functional significance of this is not clear. PMID:10573012

  16. Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

    PubMed Central

    Koukolíková-Nicola, Z; Raineri, D; Stephens, K; Ramos, C; Tinland, B; Nester, E W; Hohn, B

    1993-01-01

    The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer. Images PMID:8380800

  17. How Distance Affects Semantic Integration in Discourse: Evidence from Event-Related Potentials

    PubMed Central

    Yang, Xiaohong; Chen, Shuang; Chen, Xuhai; Yang, Yufang

    2015-01-01

    Event-related potentials were used to investigate whether semantic integration in discourse is influenced by the number of intervening sentences between the endpoints of integration. Readers read discourses in which the last sentence contained a critical word that was either congruent or incongruent with the information introduced in the first sentence. Furthermore, for the short discourses, the first and last sentence were intervened by only one sentence while for the long discourses, they were intervened by three sentences. We found that the incongruent words elicited an N400 effect for both the short and long discourses. However, a P600 effect was only observed for the long discourses, but not for the short ones. These results suggest that although readers can successfully integrate upcoming words into the existing discourse representation, the effort required for this integration process is modulated by the number of intervening sentences. Thus, discourse distance as measured by the number of intervening sentences should be taken as an important factor for semantic integration in discourse. PMID:26569606

  18. Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The extensive use of DNA barcoding technology in a large inventory of Macrolepidoptera and their parasitoids is documented. The methodology used and its practical applications are summarized, and numerous examples of how DNA barcoding has untangled complexes of cryptic species of butterflies, moths...

  19. Integration Strategy Is a Key Step in Network-Based Analysis and Dramatically Affects Network Topological Properties and Inferring Outcomes

    PubMed Central

    Jin, Nana; Wu, Deng; Gong, Yonghui; Bi, Xiaoman; Jiang, Hong; Li, Kongning; Wang, Qianghu

    2014-01-01

    An increasing number of experiments have been designed to detect intracellular and intercellular molecular interactions. Based on these molecular interactions (especially protein interactions), molecular networks have been built for using in several typical applications, such as the discovery of new disease genes and the identification of drug targets and molecular complexes. Because the data are incomplete and a considerable number of false-positive interactions exist, protein interactions from different sources are commonly integrated in network analyses to build a stable molecular network. Although various types of integration strategies are being applied in current studies, the topological properties of the networks from these different integration strategies, especially typical applications based on these network integration strategies, have not been rigorously evaluated. In this paper, systematic analyses were performed to evaluate 11 frequently used methods using two types of integration strategies: empirical and machine learning methods. The topological properties of the networks of these different integration strategies were found to significantly differ. Moreover, these networks were found to dramatically affect the outcomes of typical applications, such as disease gene predictions, drug target detections, and molecular complex identifications. The analysis presented in this paper could provide an important basis for future network-based biological researches. PMID:25243127

  20. Effect of dilution rate on feline urethral sperm motility, viability, and DNA integrity.

    PubMed

    Prochowska, Sylwia; Niżański, Wojciech; Ochota, Małgorzata; Partyka, Agnieszka

    2014-12-01

    This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10(6)/mL, 5 × 10(6)/mL, and 1 × 10(6)/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10(6)/mL dilution rate, spermatozoa diluted to 1 × 10(6) sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates. PMID:25262548

  1. Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

    PubMed Central

    He, Yuhui; Tsutsui, Makusu; Scheicher, Ralph H.; Fan, Chun; Taniguchi, Masateru; Kawai, Tomoji

    2013-01-01

    Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account. PMID:23931325

  2. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics

    PubMed Central

    Rose, Sasha J.; Babrak, Lmar M.; Bermudez, Luiz E.

    2015-01-01

    Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections. PMID:26010725

  3. Perceptual and affective mechanisms in facial expression recognition: An integrative review.

    PubMed

    Calvo, Manuel G; Nummenmaa, Lauri

    2016-09-01

    Facial expressions of emotion involve a physical component of morphological changes in a face and an affective component conveying information about the expresser's internal feelings. It remains unresolved how much recognition and discrimination of expressions rely on the perception of morphological patterns or the processing of affective content. This review of research on the role of visual and emotional factors in expression recognition reached three major conclusions. First, behavioral, neurophysiological, and computational measures indicate that basic expressions are reliably recognized and discriminated from one another, albeit the effect may be inflated by the use of prototypical expression stimuli and forced-choice responses. Second, affective content along the dimensions of valence and arousal is extracted early from facial expressions, although this coarse affective representation contributes minimally to categorical recognition of specific expressions. Third, the physical configuration and visual saliency of facial features contribute significantly to expression recognition, with "emotionless" computational models being able to reproduce some of the basic phenomena demonstrated in human observers. We conclude that facial expression recognition, as it has been investigated in conventional laboratory tasks, depends to a greater extent on perceptual than affective information and mechanisms. PMID:26212348

  4. The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology.

    PubMed

    Sohrabi, Mohsen; Nair, Raj G; Samaranayake, Lakshman P; Zhang, Li; Zulfiker, Abu Hasanat Md; Ahmetagic, Adnan; Good, David; Wei, Ming Q

    2016-03-01

    Recent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling. Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozyme-achromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others. PMID:26812577

  5. The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model.

    PubMed

    Xiao, Sha; Cui, Su; Lu, Xiaobo; Guan, Yangyang; Li, Dandan; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; van der Straaten, Tahar

    2016-08-01

    Nucleotide excision repair (NER) is an important defense mechanism of the body to exogenous carcinogens and mutagens, such as benzo[a]pyrene (B[a]P). Genetic polymorphisms in ERCC2/XPD, a critical element in NER, are thought to be associated with individual's cancer susceptibility. Although ERCC2/XPD Lys751Gln (rs13181) is the most studied polymorphism, the impact of this polymorphism on DNA repair capacity to carcinogen remains unclear. In the present study, cDNA clones carrying different genotypes of ERCC2/XPD (Lys751Gln) were introduced into an ERCC2/XPD deficient cell line (UV5) in a well-controlled biological system. After B[a]P treatment, cell growth inhibition rates and DNA damage levels in all cells were detected respectively. As expected, we found that the DNA repair capacity in UV5 cells was restored to levels similar to wildtype parent AA8 cells upon introduction of the cDNA clone of ERCC2/XPD (Lys751). Interestingly, after B[a]P treatment, transfected cells expressing variant ERCC2/XPD (751Gln) showed an enhanced cellular sensitivity and a diminished DNA repair capacity. The wildtype genotype AA (Lys) was found to be associated with a higher DNA repair capacity as compared to its polymorphic genotype CC (Gln). These data indicate that ERCC2/XPD Lys751Gln polymorphism affects DNA repair capacity after exposure to environmental carcinogens such as B[a]P in this well-controlled in vitro system and could act as a biomarker to increase the predictive value to develop cancer. PMID:27139774

  6. Variables that Affect Math Teacher Candidates' Intentions to Integrate Computer-Assisted Mathematics Education (CAME)

    ERIC Educational Resources Information Center

    Erdogan, Ahmet

    2010-01-01

    Based on Social Cognitive Carier Theory (SCCT) (Lent, Brown, & Hackett, 1994, 2002), this study tested the effects of mathematics teacher candidates' self-efficacy in, outcome expectations from, and interest in CAME on their intentions to integrate Computer-Assisted Mathematics Education (CAME). While mathematics teacher candidates' outcome…

  7. Factors Affecting Technology Integration in K-12 Classrooms: A Path Model

    ERIC Educational Resources Information Center

    Inan, Fethi A.; Lowther, Deborah L.

    2010-01-01

    The purpose of this study was to examine the direct and indirect effects of teachers' individual characteristics and perceptions of environmental factors that influence their technology integration in the classroom. A research-based path model was developed to explain causal relationships between these factors and was tested based on data gathered…

  8. Unserved, Unseen, and Unheard: Integrating Programs for HIV-Infected and HIV-Affected Older Adults

    ERIC Educational Resources Information Center

    Emlet, Charles A.; Poindexter, Cynthia Cannon

    2004-01-01

    This article explores the parallel structures and service delivery systems of the Older Americans' Act and the Ryan White CARE Act, argues that social workers should have a working knowledge of both pieces of public policy, and suggests integration or coordination of aging and HIV services. Two vignettes illuminate the issues and implications for…

  9. Sperm DNA integrity in frozen-thawed semen from Italian Mediterranean Buffalo bulls and its relationship to in vivo fertility.

    PubMed

    Serafini, Rosanna; Love, Charles C; Coletta, Angelo; Mari, Gaetano; Mislei, Beatrice; Caso, Chiara; Di Palo, Rossella

    2016-09-01

    The relationship among sperm attributes of DNA integrity, sperm motility, morphology, viability, acrosome integrity and in vivo fertility of frozen-thawed Italian Mediterranean Buffalo (IMB) sperm has not been reported. Straws of frozen-thawed semen samples from three bulls were examined. Sperm DNA assays (i.e., neutral Comet assay, Sperm Bos Halomax-SBH and Sperm Chromatin Structure Assay-SCSA) were not correlated to each other (P>0.05). Many neutral Comet assay measures were correlated to total sperm motility-TMOT (% head-H-DNA, r=0.74; Olive moment, r=-0.76; P<0.05) and coiled tails (r-values ranged from% H-DNA, r=-0.80 to tail length, r=-0.71; P<0.05). The COMP-αt was negatively correlated to viable acrosome intact (VAI) sperm, and distal droplets (r=-0.60 and -0.61; P<0.05), whereas Mean-αt and Mode-αt were positively correlated to bent midpieces (r=0.63 and 0.61; P<0.05). The SBH assay was positively correlated to non-viable acrosome damaged (NVAD) sperm (r=0.60; P<0.05) and negatively correlated to viable acrosome damaged (VAD) sperm (r=-0.63; P<0.05). The overall pregnancy rate (PR-at 30 and 45d post artificial insemination-AI) and the calving rate were 57%, 55% and 45%, respectively. Among sperm features analyzed the area under the Receiver Operating Characteristic (ROC) Curve was significant (P<0.05) for TMOT, NVAD, Standard Deviation-αt (SD-αt) and neutral comet measures (Olive tail moment and tail moment, % H- DNA and tail area) in estimating pregnancy. PMID:27421229

  10. Integrated analysis of DNA methylation profiles and gene expression profiles to identify genes associated with pilocytic astrocytomas.

    PubMed

    Zhou, Ruigang; Man, Yigang

    2016-04-01

    The present study performed an integral analysis of the gene expression and DNA methylation profile of pilocytic astrocytomas (PAs). Weighted gene co-expression network analysis (WGCNA) was also performed to examine and identify the genes correlated to PAs, to identify candidate therapeutic targets for the treatment of PAs. The DNA methylation profile and gene expression profile were downloaded from the Gene Expression Omnibus database. Following screening of the differentially expressed genes (DEGs) and differentially methylated regions (DMRs), respectively, integrated analysis of the DEGs and DMRs was performed to detect their correlation. Subsequently, the WGCNA algorithm was applied to identify the significant modules and construct the co‑expression network associated with PAs. Furthermore, Gene Ontology enrichment analysis of the associated genes was performed using the Database for Annotation, Visualization and Integrated Discovery. A total number of 2,259 DEGs and 235 DMRs were screened out. Integrated analysis revealed that 30 DEGs were DMRs with prominent negative correlation (cor=‑0.82; P=0.02). Based on the DEGs, the gene co‑expression network was constructed, and nine network modules associated with PAs were identified. The functional analysis results showed that genes relevant to PAs were closely associated with cell differentiation modulation. The screened PA-associated genes were significantly different at the expression and methylation levels. These genes may be used as reliable candidate target genes for the treatment of PAs. PMID:26934913

  11. Integrated analysis of DNA methylation profiles and gene expression profiles to identify genes associated with pilocytic astrocytomas

    PubMed Central

    ZHOU, RUIGANG; MAN, YIGANG

    2016-01-01

    The present study performed an integral analysis of the gene expression and DNA methylation profile of pilocytic astrocytomas (PAs). Weighted gene co-expression network analysis (WGCNA) was also performed to examine and identify the genes correlated to PAs, to identify candidate therapeutic targets for the treatment of PAs. The DNA methylation profile and gene expression profile were downloaded from the Gene Expression Omnibus database. Following screening of the differentially expressed genes (DEGs) and differentially methylated regions (DMRs), respectively, integrated analysis of the DEGs and DMRs was performed to detect their correlation. Subsequently, the WGCNA algorithm was applied to identify the significant modules and construct the co-expression network associated with PAs. Furthermore, Gene Ontology enrichment analysis of the associated genes was performed using the Database for Annotation, Visualization and Integrated Discovery. A total number of 2,259 DEGs and 235 DMRs were screened out. Integrated analysis revealed that 30 DEGs were DMRs with prominent negative correlation (cor=−0.82; P=0.02). Based on the DEGs, the gene co-expression network was constructed, and nine network modules associated with PAs were identified. The functional analysis results showed that genes relevant to PAs were closely associated with cell differentiation modulation. The screened PA-associated genes were significantly different at the expression and methylation levels. These genes may be used as reliable candidate target genes for the treatment of PAs. PMID:26934913

  12. Integrated Hatchery Operations : Existing Policy Affecting Hatcheries in the Columbia River Basin, 1992 Annual Report.

    SciTech Connect

    Shelldrake, Tom

    1993-05-01

    Collected together in this document is relevant laws and policy of the US Fish and Wildlife Service, Washington State Department of Wildlife, Oregon State, Washington Department of Fisheries, and Idaho Department of Fish and Game as they affect hatcheries in the Columbia River Basin.

  13. Technology Integration before Student Outcomes: Factors Affecting Teacher Adoption of Technology in India

    ERIC Educational Resources Information Center

    Bandyopadhyay, Alankar

    2013-01-01

    Since the 1920s, ICTs have been endorsed as solutions to challenges of access and quality in education. Proponents have also supported technology use in education on grounds that it could potentially impact cognitive, affective, and pedagogical outcomes. Based on these perceived benefits, many developed and developing countries have been…

  14. Affective Aspects on Mathematics Conceptualization: From Dichotomies to an Integrated Approach

    ERIC Educational Resources Information Center

    Araujo, Claudia Roberta; Andrade, Fernanda; Hazin, Izabel; Falcao, Jorge Tarcisio da Rocha; do Nascimento, Jorge Costa; Lessa, Monica Maria Lins

    2003-01-01

    The present paper aims to propose a theoretical reflection in order to overcome a strong tradition in psychology concerning the analysis of cognition and affectivity as dichotomic processes explaining human behaviours. A general theory of the human subject is presented to discussion, followed by the proposition of a new unit of analysis for the…

  15. Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

    PubMed

    Norgard, M V; Keem, K; Monahan, J J

    1978-07-01

    The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA. PMID:365684

  16. Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars.

    PubMed

    Geering, A D; Olszewski, N E; Dahal, G; Thomas, J E; Lockhart, B E

    2001-07-01

    Summary Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l'Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l'Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the 'dead' virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants. PMID:20573008

  17. Bias and other limitations affect measures of journals in integrative and complementary medicine

    PubMed Central

    Fan, Ka-wai

    2015-01-01

    Publishing articles in a prestigious journal is a golden rule for university professors and researchers nowadays. Impact factor, journal rank, and citation count, included in Science Citation Index managed by Thomson Reuters Web of Science, are the most important indicators for evaluating the quality of academic journals. By listing the journals encompassed in the “Integrative and Complementary Medicine” category of Science Citation Index from 2003 to 2013, this paper examines the publication trends of journals in the category. The examination includes number, country of origin, ranking, and languages of journals. Moreover, newly listed or removed journals in the category, journal publishers, and open access strategies are examined. It is concluded that the role of journal publisher should not be undermined in the “Integrative and Complementary Medicine” category. PMID:26213508

  18. Crossmodal deficit in dyslexic children: practice affects the neural timing of letter-speech sound integration

    PubMed Central

    Žarić, Gojko; Fraga González, Gorka; Tijms, Jurgen; van der Molen, Maurits W.; Blomert, Leo; Bonte, Milene

    2015-01-01

    A failure to build solid letter-speech sound associations may contribute to reading impairments in developmental dyslexia. Whether this reduced neural integration of letters and speech sounds changes over time within individual children and how this relates to behavioral gains in reading skills remains unknown. In this research, we examined changes in event-related potential (ERP) measures of letter-speech sound integration over a 6-month period during which 9-year-old dyslexic readers (n = 17) followed a training in letter-speech sound coupling next to their regular reading curriculum. We presented the Dutch spoken vowels /a/ and /o/ as standard and deviant stimuli in one auditory and two audiovisual oddball conditions. In one audiovisual condition (AV0), the letter “a” was presented simultaneously with the vowels, while in the other (AV200) it was preceding vowel onset for 200 ms. Prior to the training (T1), dyslexic readers showed the expected pattern of typical auditory mismatch responses, together with the absence of letter-speech sound effects in a late negativity (LN) window. After the training (T2), our results showed earlier (and enhanced) crossmodal effects in the LN window. Most interestingly, earlier LN latency at T2 was significantly related to higher behavioral accuracy in letter-speech sound coupling. On a more general level, the timing of the earlier mismatch negativity (MMN) in the simultaneous condition (AV0) measured at T1, significantly related to reading fluency at both T1 and T2 as well as with reading gains. Our findings suggest that the reduced neural integration of letters and speech sounds in dyslexic children may show moderate improvement with reading instruction and training and that behavioral improvements relate especially to individual differences in the timing of this neural integration. PMID:26157382

  19. EVALUATION OF CHROMOSOME BREAKAGE AND DNA INTEGRITY IN SPERM: AN INVESTIGATION OF REMOTE SEMEN COLLECTION CONDITIONS

    EPA Science Inventory

    Home collection of ejaculated semen would facilitate participation rates and geographic diversity in reproductive epidemiology studies. Our study addressed concerns that home collection and overnight mail return might induce chromosome/DNA damage. We collected semen from 10 hea...

  20. An integrated microfluidic device for rapid cell lysis and DNA purification of epithelial cell samples.

    PubMed

    Ha, Seung-Mo; Cho, Woong; Ahn, Yoomin; Hwang, Seung Yong

    2011-05-01

    In this paper, we describe the design and fabrication of a microfluidic device for cell lysis and DNA purification, and the results of device tests using a real sample of buccal cells. Cell lysis was thermally executed for two minutes at 80 degrees C in a serpentine type microreactor (20 microL) using an Au microheater with a microsensor. The DNA was then mixed with other residual products and purified by a new filtration process involving micropillars and 50-80 microm microbeads. The entire process of sample loading, cell lysis, DNA purification, and sample extraction was successfully completed in the microchip within five minutes. Sample preparation within the microchip was verified by performing a SY158 gene PCR analysis and gel electrophoresis on the products obtained from the chip. The new purification method enhanced DNA purity from 0.93 to 1.62 after purification. PMID:21780436

  1. DNA Integrity and Shock Wave Transformation Efficiency of Bacteria and Fungi

    NASA Astrophysics Data System (ADS)

    Loske, Achim M.; Campos-Guillén, Juan; Fernández, Francisco; Pastrana, Xóchitl; Magaña-Ortíz, Denis; Coconi-Linares, Nancy; Ortíz-Vázquez, Elizabeth; Gómez-Lim, Miguel

    Delivery of DNA into bacteria and fungi is essential in medicine and biotechnology to produce metabolites, enzymes, antibiotics and proteins. So far, protocols to genetically transform bacteria and fungi are inefficient and have low reproducibility.

  2. Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

    PubMed Central

    Nelson, Glyn; Hall, Philip; Miwa, Satomi; Kirkwood, Thomas B. L.; Shanley, Daryl P.

    2015-01-01

    Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level. PMID:26020242

  3. Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

    PubMed Central

    Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

    2008-01-01

    Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

  4. Repetitive, marker-free, site-specific integration as a novel tool for multiple chromosomal integration of DNA.

    PubMed

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

    2013-06-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB(min)), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB(min) site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps. PMID:23542630

  5. Valproic Acid Affects Membrane Trafficking and Cell-Wall Integrity in Fission Yeast

    PubMed Central

    Miyatake, Makoto; Kuno, Takayoshi; Kita, Ayako; Katsura, Kosaku; Takegawa, Kaoru; Uno, Satoshi; Nabata, Toshiya; Sugiura, Reiko

    2007-01-01

    Valproic acid (VPA) is widely used to treat epilepsy and manic-depressive illness. Although VPA has been reported to exert a variety of biochemical effects, the exact mechanisms underlying its therapeutic effects remain elusive. To gain further insights into the molecular mechanisms of VPA action, a genetic screen for fission yeast mutants that show hypersensitivity to VPA was performed. One of the genes that we identified was vps45+, which encodes a member of the Sec1/Munc18 family that is implicated in membrane trafficking. Notably, several mutations affecting membrane trafficking also resulted in hypersensitivity to VPA. These include ypt3+ and ryh1+, both encoding a Rab family protein, and apm1+, encoding the μ1 subunit of the adaptor protein complex AP-1. More importantly, VPA caused vacuolar fragmentation and inhibited the glycosylation and the secretion of acid phosphatase in wild-type cells, suggesting that VPA affects membrane trafficking. Interestingly, the cell-wall-damaging agents such as micafungin or the inhibition of calcineurin dramatically enhanced the sensitivity of wild-type cells to VPA. Consistently, VPA treatment of wild-type cells enhanced their sensitivity to the cell-wall-digesting enzymes. Altogether, our results suggest that VPA affects membrane trafficking, which leads to the enhanced sensitivity to cell-wall damage in fission yeast. PMID:17287531

  6. Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

    PubMed Central

    Lancini, Cesare; van den Berk, Paul C.M.; Vissers, Joseph H.A.; Gargiulo, Gaetano; Song, Ji-Ying; Hulsman, Danielle; Serresi, Michela; Tanger, Ellen; Blom, Marleen; Vens, Conchita; van Lohuizen, Maarten; Jacobs, Heinz

    2014-01-01

    Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling. Notably, USP3 deletion increased the levels of histone ubiquitination in adult tissues, reduced the hematopoietic stem cell (HSC) reserves over time, and shortened animal life span. Mechanistically, our data show that USP3 is important in HSC homeostasis, preserving HSC self-renewal, and repopulation potential in vivo and proliferation in vitro. A defective DDR and unresolved spontaneous DNA damage contribute to cell cycle restriction of Usp3Δ/Δ HSCs. Beyond the hematopoietic system, Usp3Δ/Δ animals spontaneously developed tumors, and primary Usp3Δ/Δ cells failed to preserve chromosomal integrity. These findings broadly support the regulation of chromatin ubiquitination as a key pathway in preserving tissue function through modulation of the response to genotoxic stress. PMID:25113974

  7. Misconduct Policies, Academic Culture and Career Stage, Not Gender or Pressures to Publish, Affect Scientific Integrity

    PubMed Central

    Fanelli, Daniele; Costas, Rodrigo; Larivière, Vincent

    2015-01-01

    The honesty and integrity of scientists is widely believed to be threatened by pressures to publish, unsupportive research environments, and other structural, sociological and psychological factors. Belief in the importance of these factors has inspired major policy initiatives, but evidence to support them is either non-existent or derived from self-reports and other sources that have known limitations. We used a retrospective study design to verify whether risk factors for scientific misconduct could predict the occurrence of retractions, which are usually the consequence of research misconduct, or corrections, which are honest rectifications of minor mistakes. Bibliographic and personal information were collected on all co-authors of papers that have been retracted or corrected in 2010-2011 (N=611 and N=2226 papers, respectively) and authors of control papers matched by journal and issue (N=1181 and N=4285 papers, respectively), and were analysed with conditional logistic regression. Results, which avoided several limitations of past studies and are robust to different sampling strategies, support the notion that scientific misconduct is more likely in countries that lack research integrity policies, in countries where individual publication performance is rewarded with cash, in cultures and situations were mutual criticism is hampered, and in the earliest phases of a researcher’s career. The hypothesis that males might be prone to scientific misconduct was not supported, and the widespread belief that pressures to publish are a major driver of misconduct was largely contradicted: high-impact and productive researchers, and those working in countries in which pressures to publish are believed to be higher, are less-likely to produce retracted papers, and more likely to correct them. Efforts to reduce and prevent misconduct, therefore, might be most effective if focused on promoting research integrity policies, improving mentoring and training, and encouraging

  8. Misconduct Policies, Academic Culture and Career Stage, Not Gender or Pressures to Publish, Affect Scientific Integrity.

    PubMed

    Fanelli, Daniele; Costas, Rodrigo; Larivière, Vincent

    2015-01-01

    The honesty and integrity of scientists is widely believed to be threatened by pressures to publish, unsupportive research environments, and other structural, sociological and psychological factors. Belief in the importance of these factors has inspired major policy initiatives, but evidence to support them is either non-existent or derived from self-reports and other sources that have known limitations. We used a retrospective study design to verify whether risk factors for scientific misconduct could predict the occurrence of retractions, which are usually the consequence of research misconduct, or corrections, which are honest rectifications of minor mistakes. Bibliographic and personal information were collected on all co-authors of papers that have been retracted or corrected in 2010-2011 (N=611 and N=2226 papers, respectively) and authors of control papers matched by journal and issue (N=1181 and N=4285 papers, respectively), and were analysed with conditional logistic regression. Results, which avoided several limitations of past studies and are robust to different sampling strategies, support the notion that scientific misconduct is more likely in countries that lack research integrity policies, in countries where individual publication performance is rewarded with cash, in cultures and situations were mutual criticism is hampered, and in the earliest phases of a researcher's career. The hypothesis that males might be prone to scientific misconduct was not supported, and the widespread belief that pressures to publish are a major driver of misconduct was largely contradicted: high-impact and productive researchers, and those working in countries in which pressures to publish are believed to be higher, are less-likely to produce retracted papers, and more likely to correct them. Efforts to reduce and prevent misconduct, therefore, might be most effective if focused on promoting research integrity policies, improving mentoring and training, and encouraging

  9. Trans-activation function of a 3 prime truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

    SciTech Connect

    Takada, Shinako; Koike, Katsuro )

    1990-08-01

    To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3{prime} end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.

  10. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

    PubMed Central

    Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

    2014-01-01

    Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy. PMID:24583643

  11. Connecting the dots: how local structure affects global integration in infants

    PubMed Central

    Palomares, Melanie; Pettet, Mark; Vildavski, Vladimir; Hou, Chuan; Norcia, Anthony

    2009-01-01

    Glass patterns are moirés created from a sparse random dot field paired with its spatially-shifted copy. Because discrimination of these patterns is not based on local features, they have been used extensively to study global integration processes. Here, we investigated whether 4–5.5 month old infants are sensitive to the global structure of Glass patterns by measuring Visual Evoked Potentials (VEPs). Although we found strong responses to the appearance of the constituent dots, we found sensitivity to the global structure of the Glass patterns in the infants only over a very limited range of spatial separation. In contrast, we observed robust responses in the infants when we connected the dot pairs of the Glass pattern with lines. Moreover, both infants and adults showed differential responses to exchanges between line patterns portraying different global structures. A control study varying luminance contrast in adults suggests that infant sensitivity to global structure is not primarily limited by reduced element visibility. Together our results suggest that the insensitivity to structure in conventional Glass patterns is due to inefficiencies in extracting the local orientation cues generated by the dot pairs. Once the local orientations are made unambiguous or when the interpolation span is small, infants can integrate these signals over the image. PMID:19642888

  12. Institutional issues affecting the integration and use of remotely sensed data and geographic information systems

    USGS Publications Warehouse

    Lauer, D.T.; Estes, J.E.; Jensen, J.R.; Greenlee, D.D.

    1991-01-01

    The developers as well as the users of remotely sensed data and geographic information system (GIS) techniques are associated with nearly all types of institutions in government, industry, and academia. Individuals in these various institutions often find the barriers to accepting remote sensing and GIS are not necessarily technical in nature, but can be attributed to the institutions themselves. Several major institutional issues that affect the technologies of remote sensing and GIS are data availability, data marketing and costs, equipment availability and costs, standards and practices, education and training, and organizational infrastructures. Not only are problems associated with these issues identified, but needs and opportunities also are discussed. -from Authors

  13. Gene copy number variations in the leukocyte genome of hepatocellular carcinoma patients with integrated hepatitis B virus DNA

    PubMed Central

    Xu, Guixia; Cheng, Kai; Cao, Guangwen; Wu, Mengchao; Cheng, Shuqun; Liu, Shanrong

    2016-01-01

    Integration of hepatitis B virus (HBV) DNA into the human liver cell genome is believed to promote HBV-related carcinogenesis. This study aimed to quantify the integration of HBV DNA into the leukocyte genome in hepatocellular carcinoma (HCC) patients in order to identify potential biomarkers for HBV-related diseases. Whole-genome comparative genomic hybridization (CGH) chip array analyses were performed to screen gene copy number variations (CNV) in the leukocyte genome, and the results were confirmed by quantitative polymerase chain reaction (qPCR). The commonly detected regions included chromosome arms 19p, 5q, 1q and 15p, where 200 copy number gain events and 270 copy number loss events were noted. In particular, gains were observed in 5q35.3 (OR4F3) and 19p13.3 (OR4F17) in 90% of the samples. Successful homologous recombination of OR4F3 and the HBV P gene was demonstrated, and the amplification at 5q35.3 is potentially associated with the integration of HBV P gene into natural killer cells isolated from peripheral blood mononuclear cells (PBMCs). Receiver operating characteristic (ROC) curve analysis indicated that the combination of OR4F3 and OR4F17 a novel potential biomarker of HBV-related diseases. PMID:26769853

  14. Integrating prior knowledge in multiple testing under dependence with applications to detecting differential DNA methylation.

    PubMed

    Kuan, Pei Fen; Chiang, Derek Y

    2012-09-01

    DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This article aims to introduce a robust testing and probe ranking procedure based on a nonhomogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, Journal of the Royal Statistical Society: Series B (Statistical Methodology)71, 393-424) and propose modeling the nonnull using a nonparametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites. PMID:22260651

  15. PRMT5 Protects Genomic Integrity during Global DNA Demethylation in Primordial Germ Cells and Preimplantation Embryos

    PubMed Central

    Kim, Shinseog; Günesdogan, Ufuk; Zylicz, Jan J.; Hackett, Jamie A.; Cougot, Delphine; Bao, Siqin; Lee, Caroline; Dietmann, Sabine; Allen, George E.; Sengupta, Roopsha; Surani, M. Azim

    2014-01-01

    Summary Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation. PMID:25457166

  16. An integrated multiple capillary array electrophoresis system for high-throughput DNA sequencing

    SciTech Connect

    Lu, X.

    1998-03-27

    A capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies. There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk, and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 {micro}m o.d., 75 {micro}m i.d and the other format is square capillary with 300 {micro}m out edge and 75 {micro}m inner edge. This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.

  17. Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

    PubMed

    Ohyama, Tatsuya; Hayakawa, Masato; Nishikawa, Shin; Kurita, Noriyuki

    2011-06-01

    Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti-inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti-inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA. PMID:21328406

  18. 17β-Hydroxysteroid dehydrogenase type 10 predicts survival of patients with colorectal cancer and affects mitochondrial DNA content.

    PubMed

    Amberger, Albert; Deutschmann, Andrea J; Traunfellner, Pia; Moser, Patrizia; Feichtinger, René G; Kofler, Barbara; Zschocke, Johannes

    2016-04-28

    Mitochondrial energy production is reduced in tumor cells, and altered mitochondrial respiration contributes to tumor progression. Synthesis of proteins coded by mitochondrial DNA (mtDNA) requires the correct processing of long polycistronic precursor RNA molecules. Mitochondrial RNase P, composed of three different proteins (MRPP1, HSD10, and MRPP3), is necessary for correct RNA processing. Here we analyzed the role of RNase P proteins in colorectal cancer. High HSD10 expression was found in 28%; high MRPP1 expression in 40% of colorectal cancers, respectively. Expression of both proteins was not significantly associated with clinicopathological parameters. Survival analysis revealed that loss of HSD10 expression is associated with poor prognosis. Cox regression demonstrated that patients with high HSD10 tumors are at lower risk. High HSD10 expression was significantly associated with high mtDNA content in tumor tissue. A causal effect of HSD10 overexpression or knock down with increased or reduced mtDNA levels, respectively, was confirmed in tumor cell lines. Our data suggest that HSD10 plays a role in alterations of energy metabolism by regulating mtDNA content in colorectal carcinomas, and HSD10 protein analysis may be of prognostic value. PMID:26884257

  19. BRIT1 regulates early DNA damage response, chromosomal integrity,and cancer

    SciTech Connect

    Rai, Rekha; Dai, Hui; Multani, Asha S.; Li, Kaiyi; Chin, Koei; Gray, Joe; Lahad, John P.; Liang, Jiyong; Mills, Gordon B.; Meric-Bernstam, Funda; Lin, Shiaw-Yih

    2006-08-24

    BRIT1, initially identified as an hTERT repressor, hasadditional functions at DNA damage checkpoints. Here, we demonstratedthat BRIT1 formed nuclear foci minutes after irradiation. The foci ofBRIT1 co-localized with 53BP1, MDC1, NBS1, ATM, RPA, and ATR. BRIT1 wasrequired for activation of these elements, indicating that BRIT1 is aproximal factor in the DNA damage response pathway. Depletion of BRIT1increased the accumulation of chromosomal aberrations. In addition,decreased levels of BRIT1 were detected in several types of human cancerwith BRIT1 expression being inversely correlated with genomic instabilityand metastasis. These results identify BRIT1 as a crucial DNA damageregulator in the ATM/ATR pathways and suggest that it functions as atumor suppressor gene.

  20. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.

    PubMed

    van den Brink-van der Laan, Els; Killian, J Antoinette; de Kruijff, Ben

    2004-11-01

    Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure. PMID:15519321

  1. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

    PubMed

    Adams, Andrea J; LaBonte, John P; Ball, Morgan L; Richards-Hrdlicka, Kathryn L; Toothman, Mary H; Briggs, Cheryl J

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum

  2. DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR

    PubMed Central

    Adams, Andrea J.; LaBonte, John P.; Ball, Morgan L.; Richards-Hrdlicka, Kathryn L.; Toothman, Mary H.; Briggs, Cheryl J.

    2015-01-01

    Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80–90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum

  3. Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III.

    PubMed

    Bonora, Elena; Porcelli, Anna Maria; Gasparre, Giuseppe; Biondi, Annalisa; Ghelli, Anna; Carelli, Valerio; Baracca, Alessandra; Tallini, Giovanni; Martinuzzi, Andrea; Lenaz, Giorgio; Rugolo, Michela; Romeo, Giovanni

    2006-06-15

    Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids. PMID:16778181

  4. Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.

    PubMed

    Inaba, Yasushi; Miyashita, Satoshi; Somfai, Tamás; Geshi, Masaya; Matoba, Satoko; Dochi, Osamu; Nagai, Takashi

    2016-04-01

    This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells. PMID:26996887

  5. Effect of bile salts on the DNA and membrane integrity of enteric bacteria.

    PubMed

    Merritt, Megan E; Donaldson, Janet R

    2009-12-01

    Enteric bacteria are able to resist the high concentrations of bile encountered throughout the gastrointestinal tract. Here we review the current mechanisms identified in the enteric bacteria Salmonella, Escherichia coli, Bacillus cereus and Listeria monocytogenes to resist the dangerous effects of bile. We describe the role of membrane transport systems, and their connection with DNA repair pathways, in conferring bile resistance to these enterics. We discuss the findings from recent investigations that indicate bile tolerance is dependent upon being able to resist the detergent properties of bile at both the membrane and DNA level. PMID:19762477

  6. Parameters affecting image quality with Time-Resolved Optical Integrative Neutron (TRION) detector

    NASA Astrophysics Data System (ADS)

    Mor, I.; Vartsky, D.; Feldman, G.; Dangendorf, V.; Bar, D.; Goldberg, M. B.; Tittelmeier, K.; Bromberger, B.; Weierganz, M.; Brandis, M.

    2011-06-01

    We have investigated by simulations and experimentally the parameters that affect image quality (contrast and spatial-resolution) of the fast neutron TRION detector. A scintillating fiber screen with 0.5×0.5 mm 2 square fibers, few centimeters thick, provides superior spatial-resolution to that of a slab scintillator of the same thickness. A detailed calculation of the neutron interaction processes that influence the point-spread function (PSF) in the scintillating screen has been performed using the GEANT 3.21 code. The calculations showed that neutron scattering within the screen accounts for a significant loss of image contrast. The factors that limit the spatial-resolution of the image are the cross-sectional scintillating-fiber dimensions within the screen and the spatial response of the image-intensifier. A deconvolution method has been applied for restoring the contrast and the spatial-resolution of the fast neutron image.

  7. TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

    PubMed

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R; Kantarjian, Hagop M; Liang, Shoudan; Estecio, Marcos R; Godley, Lucy A; Issa, Jean-Pierre J

    2015-07-15

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites. PMID:25972343

  8. Psychophysiology of neural, cognitive and affective integration: fMRI and autonomic indicants.

    PubMed

    Critchley, Hugo D

    2009-08-01

    Behaviour is shaped by environmental challenge in the context of homoeostatic need. Emotional and cognitive processes evoke patterned changes in bodily state that may signal emotional state to others. This dynamic modulation of visceral state is neurally mediated by sympathetic and parasympathetic divisions of the autonomic nervous system. Moreover neural afferents convey representations of the internal state of the body back to the brain to further influence emotion and cognition. Neuroimaging and lesion studies implicate specific regions of limbic forebrain in the behavioural generation of autonomic arousal states. Activity within these regions may predict emotion-specific autonomic response patterns within and between bodily organs, with implications for psychosomatic medicine. Feedback from the viscera is mapped hierarchically in the brain to influence efferent signals, and ultimately at the cortical level to engender and reinforce affective responses and subjective feeling states. Again neuroimaging and patient studies suggest discrete neural substrates for these representations, notably regions of insula and orbitofrontal cortex. Individual differences in conscious access to these interoceptive representations predict differences in emotional experience, but equally the misperception of heightened arousal level may evoke changes in emotional behaviour through engagement of the same neural centres. Perturbation of feedback may impair emotional reactivity and, in the context of inflammatory states give rise to cognitive, affective and psychomotor expressions of illness. Changes in visceral state during emotion may be mirrored in the responses of others, permitting a corresponding representation in the observer. The degree to which individuals are susceptible to this 'contagion' predicts individual differences in questionnaire ratings of empathy. Together these neuroimaging and clinical studies highlight the dynamic relationship between mind and body and help

  9. Abnormal pattern of post-gamma-ray DNA replication in radioresistant fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome.

    PubMed Central

    Mirzayans, R.; Aubin, R. A.; Bosnich, W.; Blattner, W. A.; Paterson, M. C.

    1995-01-01

    Non-malignant dermal fibroblast strains, cultured from affected members of a Li-Fraumeni syndrome (LFS) family with diverse neoplasms associated with radiation exposure, display a unique increased resistance to the lethal effects of gamma-radiation. In the studies reported here, this radioresistance (RR) trait has been found to correlate strongly with an abnormal pattern of post-gamma-ray DNA replicative synthesis, as monitored by radiolabelled thymidine incorporation and S-phase cell autoradiography. In particular, the time interval between the gamma-ray-induced shutdown of DNA synthesis and its subsequent recovery was greater in all four RR strains examined and the post-recovery replication rate was much higher and was maintained longer than in normal and spousal controls. Alkaline sucrose sedimentation profiles of pulse-labelled cellular DNA indicated that the unusual pattern of DNA replication in irradiated RR strains may be ascribed to anomalies in both replicon initiation and DNA chain elongation processes. Moreover, the RR strain which had previously displayed the highest post-gamma-ray clonogenic survival was found to harbour a somatic (codon 234) mutation (presumably acquired during culture in vitro) in the same conserved region of the p53 tumour-suppressor gene as the germline (codon 245) mutation in the remaining three RR strains from other family members, thus coupling the RR phenotype and abnormal post-gamma-ray DNA synthesis pattern with faulty p53 expression. Significantly, these two aberrant radioresponse end points, along with documented anomalies in c-myc and c-raf-1 proto-oncogenes, are unprecedented among other LFS families carrying p53 germline mutations. We thus speculate that this peculiar cancer-prone family may possess in its germ line a second, as yet unidentified, genetic defect in addition to the p53 mutation. Images Figure 8 PMID:7779715

  10. USF-1 Is Critical for Maintaining Genome Integrity in Response to UV-Induced DNA Photolesions

    PubMed Central

    Mouchet, Nicolas; Vaulont, Sophie; Prince, Sharon; Galibert, Marie-Dominique

    2012-01-01

    An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect against this, cells have evolved effective mechanisms to recognize DNA damage, signal its presence, and mediate its repair. While these responses are expected to be highly regulated because they are critical to avoid human diseases, very little is known about the regulation of the expression of genes involved in mediating their effects. The Nucleotide Excision Repair (NER) is the major DNA–repair process involved in the recognition and removal of UV-mediated DNA damage. Here we use a combination of in vitro and in vivo assays with an intermittent UV-irradiation protocol to investigate the regulation of key players in the DNA–damage recognition step of NER sub-pathways (TCR and GGR). We show an up-regulation in gene expression of CSA and HR23A, which are involved in TCR and GGR, respectively. Importantly, we show that this occurs through a p53 independent mechanism and that it is coordinated by the stress-responsive transcription factor USF-1. Furthermore, using a mouse model we show that the loss of USF-1 compromises DNA repair, which suggests that USF-1 plays an important role in maintaining genomic stability. PMID:22291606

  11. A Protocol to Preserve the Integrity of Stable Fly (Diptera: Muscidae) DNA for Long Distance Shipment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Population genetic studies on a global scale may be hampered by the ability to acquire quality samples from distant countries. Preservation methods must be adequate to prevent the samples from decay during shipping, so an adequate quantity of quality DNA can be extracted for analysis, and materials...

  12. Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

    SciTech Connect

    Qingbro, Li; Liu, Zhaowei; Monroe, Heidi M; Culiat, Cymbeline T

    2002-08-01

    We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.

  13. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    PubMed Central

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  14. Integrated assessment of oxidative stress and DNA damage in earthworms (Eisenia fetida) exposed to azoxystrobin.

    PubMed

    Han, Yingnan; Zhu, Lusheng; Wang, Jinhua; Wang, Jun; Xie, Hui; Zhang, Shumin

    2014-09-01

    Azoxystrobin has been widely used in recent years. The present study investigated the oxidative stress and DNA damage effects of azoxystrobin on earthworms (Eisenia fetida). Earthworms were exposed to different azoxystrobin concentrations in an artificial soil (0, 0.1, 1, and 10mg/kg) and sampled on days 7, 14, 21, and 28. Superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione-S-transferase (GST), reactive oxygen species (ROS), and malondialdehyde (MDA) content were measured by an ultraviolet spectrophotometer to determine the antioxidant responses and lipid peroxidation. Single cell gel electrophoresis (SCGE) was used to detect DNA damage in the coelomocytes. Compared with these in the controls, earthworms exposed to azoxystrobin had excess ROS accumulation and greater SOD, POD, and GST activity while the opposite trend occurred for CAT activity. MDA content increased after 14-day exposure, and DNA damage was enhanced with an increase in the concentration of azoxystrobin. In conclusion, azoxystrobin caused oxidative stress leading to lipid peroxidation and DNA damage in earthworms. PMID:25011117

  15. Metabolic dynamics analysis by massive data integration: application to tsunami-affected field soils in Japan.

    PubMed

    Ogura, Tatsuki; Date, Yasuhiro; Tsuboi, Yuuri; Kikuchi, Jun

    2015-08-21

    A new metabolic dynamics analysis approach has been developed in which massive data sets from time-series of (1)H and (13)C NMR spectra are integrated in combination with microbial variability to characterize the biomass degradation process using field soil microbial communities. On the basis of correlation analyses that revealed relationships between various metabolites and bacteria, we efficiently monitored the metabolic dynamics of saccharides, amino acids, and organic acids, by assessing time-course changes in the microbial and metabolic profiles during biomass degradation. Specific bacteria were found to support specific steps of metabolic pathways in the degradation process of biomass to short chain fatty acids. We evaluated samples from agricultural and abandoned fields contaminated by the tsunami that followed the Great East earthquake in Japan. Metabolic dynamics and activities in the biomass degradation process differed considerably between soil from agricultural and abandoned fields. In particular, production levels of short chain fatty acids, such as acetate and propionate, which were considered to be produced by soil bacteria such as Sedimentibacter sp. and Coprococcus sp., were higher in the soil from agricultural fields than from abandoned fields. Our approach could characterize soil activity based on the metabolic dynamics of microbial communities in the biomass degradation process and should therefore be useful in future investigations of the environmental effects of natural disasters on soils. PMID:25997449

  16. Integrating Epigenomic Elements and GWASs Identifies BDNF Gene Affecting Bone Mineral Density and Osteoporotic Fracture Risk

    PubMed Central

    Guo, Yan; Dong, Shan-Shan; Chen, Xiao-Feng; Jing, Ying-Aisha; Yang, Man; Yan, Han; Shen, Hui; Chen, Xiang-Ding; Tan, Li-Jun; Tian, Qing; Deng, Hong-Wen; Yang, Tie-Lin

    2016-01-01

    To identify susceptibility genes for osteoporosis, we conducted an integrative analysis that combined epigenomic elements and previous genome-wide association studies (GWASs) data, followed by validation at population and functional levels, which could identify common regulatory elements and predict new susceptibility genes that are biologically meaningful to osteoporosis. By this approach, we found a set of distinct epigenomic elements significantly enriched or depleted in the promoters of osteoporosis-associated genes, including 4 transcription factor binding sites, 27 histone marks, and 21 chromatin states segmentation types. Using these epigenomic marks, we performed reverse prediction analysis to prioritize the discovery of new candidate genes. Functional enrichment analysis of all the prioritized genes revealed several key osteoporosis related pathways, including Wnt signaling. Genes with high priority were further subjected to validation using available GWASs datasets. Three genes were significantly associated with spine bone mineral density, including BDNF, PDE4D, and SATB2, which all closely related to bone metabolism. The most significant gene BDNF was also associated with osteoporotic fractures. RNA interference revealed that BDNF knockdown can suppress osteoblast differentiation. Our results demonstrated that epigenomic data could be used to indicate common epigenomic marks to discover additional loci with biological functions for osteoporosis. PMID:27465306

  17. Study of the impacts of regulations affecting the acceptance of integrated community energy systems. Final report

    SciTech Connect

    Feurer, Duane A.; Weaver, Clifford L.; Rielley, Kevin J.; Gallagher, Kevin C.; Harmon, Susan B.; Hejna, David T.; Kitch, Edmund W.

    1981-01-01

    A detailed description is presented of the laws and programs of the State of North Carolina governing the regulation of public energy utilities, the siting of energy generating and transmission facilities, the municipal franchising of public energy utilities, and the prescription of rates to be charged by utilities including attendant problems of cost allocations, rate base and operating expense determinations, and rate of return allowances. These laws and programs are analyzed to identify impediments which they may present to the implementation of Integrated Community Energy Systems (ICES). This report is one of fifty-one separate volumes which describe such regulatory programs at the Federal level and in each state as background to the report entitled Community Energy Systems and the Law of Public Utilities - Volume One: An Overview. This report also contains a summary of a strategy described in Volume One - An Overview for overcoming these impediments by working within the existing regulatory framework and by making changes in the regulatory programs to enhance the likelihood of ICES implementation.

  18. A review of anatomical and mechanical factors affecting vertebral body integrity

    PubMed Central

    2004-01-01

    Background: The aetiology of osteoporotic vertebral fracture is multifactorial and may be conceptualised using a systems framework. Previous studies have established several correlates of vertebral fracture including reduced vertebral cross-sectional area, weakness in back extensor muscles, reduced bone mineral density, increasing age, worsening kyphosis and recent vertebral fracture. Alterations in these physical characteristics may influence biomechanical loads and neuromuscular control of the trunk and contribute to changes in subregional bone mineral density of the vertebral bodies. Methods: This review discusses factors that have received less attention in the literature, which may contribute to the development of vertebral fracture. A literature review was conducted using electronic databases including Medline, Cinahl and ISI Web of Science to examine the potential contribution of trabecular architecture, subregional bone mineral density, vertebral geometry, muscle force, muscle strength, neuromuscular control and intervertebral disc integrity to the aetiology of osteoporotic vertebral fracture. Interpretation: A better understanding of factors such as biomechanical loading and neuromuscular control of the trunk may help to explain the high incidence of subsequent vertebral fracture after sustaining an initial vertebral fracture. Consideration of these issues may be important in the development of prevention and management strategies. PMID:15912196

  19. Early maternal loss affects social integration of chimpanzees throughout their lifetime.

    PubMed

    Kalcher-Sommersguter, Elfriede; Preuschoft, Signe; Franz-Schaider, Cornelia; Hemelrijk, Charlotte K; Crailsheim, Karl; Massen, Jorg J M

    2015-01-01

    The long-term effects of early adverse experiences on later psychosocial functioning are well described in humans, but sparsely documented for chimpanzees. In our earlier studies, we investigated the effects of maternal and social deprivation on three groups of ex-laboratory chimpanzees who experienced either an early or later onset of long-term deprivation. Here we expand our research by adding data on subjects that came from two stable zoo groups. The groups comprised of early maternally deprived wild-caught chimpanzees and non-deprived zoo-born chimpanzees. We found that compared to zoo chimpanzees, ex-laboratory chimpanzees were more restricted regarding their association partners in the newly formed groups, but not during their second year of group-life, indicating that social stability has an important influence on the toleration of association partners close-by. Social grooming activity, however, was impaired in early long-term deprived ex-laboratory chimpanzees as well as in early maternally deprived zoo chimpanzees compared to non-deprived zoo chimpanzees. Thus, we conclude that early maternal loss has lifelong effects on the social integration of chimpanzees which becomes evident in their grooming networks. Although the retrospective nature of our study prevents a clear causal explanation, our results are of importance for understanding the development of social competence in chimpanzees. PMID:26552576

  20. Early maternal loss affects social integration of chimpanzees throughout their lifetime

    PubMed Central

    Kalcher-Sommersguter, Elfriede; Preuschoft, Signe; Franz-Schaider, Cornelia; Hemelrijk, Charlotte K.; Crailsheim, Karl; Massen, Jorg J. M.

    2015-01-01

    The long-term effects of early adverse experiences on later psychosocial functioning are well described in humans, but sparsely documented for chimpanzees. In our earlier studies, we investigated the effects of maternal and social deprivation on three groups of ex-laboratory chimpanzees who experienced either an early or later onset of long-term deprivation. Here we expand our research by adding data on subjects that came from two stable zoo groups. The groups comprised of early maternally deprived wild-caught chimpanzees and non-deprived zoo-born chimpanzees. We found that compared to zoo chimpanzees, ex-laboratory chimpanzees were more restricted regarding their association partners in the newly formed groups, but not during their second year of group-life, indicating that social stability has an important influence on the toleration of association partners close-by. Social grooming activity, however, was impaired in early long-term deprived ex-laboratory chimpanzees as well as in early maternally deprived zoo chimpanzees compared to non-deprived zoo chimpanzees. Thus, we conclude that early maternal loss has lifelong effects on the social integration of chimpanzees which becomes evident in their grooming networks. Although the retrospective nature of our study prevents a clear causal explanation, our results are of importance for understanding the development of social competence in chimpanzees. PMID:26552576

  1. [Temperament traits associated with bipolar affective disorder: an integrative literature review].

    PubMed

    Vasconcelos, Alina Gomide; Malloy-Diniz, Leandro Fernandes; Nascimento, Elizabeth do; Neves, Fernando; Corrêa, Humberto

    2011-01-01

    Studies have suggested an association between temperament characteristics and adjustment and psychiatric disorders, describing them as different manifestations of vulnerability to psychopathology. The objective of this study was to conduct an integrative review of the literature on temperament traits typical of bipolar patients in relation to the general population. A systematic search was conducted on the MEDLINE, PsycINFO and LILACS databases, using the headings bipolar disorder, temperament and/or personality, between January 2000 and December 2010. The search was performed in January 2011. A total of 199 articles were identified for potential inclusion in the review. After application of the exclusion criteria, a total of 15 articles were selected and their full texts analyzed. Review of the selected studies revealed heterogeneity in terms of sample profile and specific temperament traits assessed with the appropriate instruments. Temperament traits in bipolar patients are identified based on different theoretical models. The results of five studies consistently showed that neuroticism is a distinct personality trait in the temperament profile of bipolar patients. Future reviews should use more specific keywords and limit the search to studies with a longitudinal design. PMID:25924090

  2. Integrating Epigenomic Elements and GWASs Identifies BDNF Gene Affecting Bone Mineral Density and Osteoporotic Fracture Risk.

    PubMed

    Guo, Yan; Dong, Shan-Shan; Chen, Xiao-Feng; Jing, Ying-Aisha; Yang, Man; Yan, Han; Shen, Hui; Chen, Xiang-Ding; Tan, Li-Jun; Tian, Qing; Deng, Hong-Wen; Yang, Tie-Lin

    2016-01-01

    To identify susceptibility genes for osteoporosis, we conducted an integrative analysis that combined epigenomic elements and previous genome-wide association studies (GWASs) data, followed by validation at population and functional levels, which could identify common regulatory elements and predict new susceptibility genes that are biologically meaningful to osteoporosis. By this approach, we found a set of distinct epigenomic elements significantly enriched or depleted in the promoters of osteoporosis-associated genes, including 4 transcription factor binding sites, 27 histone marks, and 21 chromatin states segmentation types. Using these epigenomic marks, we performed reverse prediction analysis to prioritize the discovery of new candidate genes. Functional enrichment analysis of all the prioritized genes revealed several key osteoporosis related pathways, including Wnt signaling. Genes with high priority were further subjected to validation using available GWASs datasets. Three genes were significantly associated with spine bone mineral density, including BDNF, PDE4D, and SATB2, which all closely related to bone metabolism. The most significant gene BDNF was also associated with osteoporotic fractures. RNA interference revealed that BDNF knockdown can suppress osteoblast differentiation. Our results demonstrated that epigenomic data could be used to indicate common epigenomic marks to discover additional loci with biological functions for osteoporosis. PMID:27465306

  3. Improved Pulsed-Field Gel Electrophoresis Procedure for the Analysis of F. columnare Isolates Previously Affected by DNA Degradation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strain...

  4. Ischemic preconditioning affects long-term cell fate through DNA damage-related molecular signaling and altered proliferation.

    PubMed

    Kapoor, Sorabh; Berishvili, Ekaterine; Bandi, Sriram; Gupta, Sanjeev

    2014-10-01

    Despite the potential of ischemic preconditioning for organ protection, long-term effects in terms of molecular processes and cell fates are ill defined. We determined consequences of hepatic ischemic preconditioning in rats, including cell transplantation assays. Ischemic preconditioning induced persistent alterations; for example, after 5 days liver histology was normal, but γ-glutamyl transpeptidase expression was observed, with altered antioxidant enzyme content, lipid peroxidation, and oxidative DNA adducts. Nonetheless, ischemic preconditioning partially protected from toxic liver injury. Similarly, primary hepatocytes from donor livers preconditioned with ischemia exhibited undesirably altered antioxidant enzyme content and lipid peroxidation, but better withstood insults. However, donor hepatocytes from livers preconditioned with ischemia did not engraft better than hepatocytes from control livers. Moreover, proliferation of hepatocytes from donor livers preconditioned with ischemia decreased under liver repopulation conditions. Hepatocytes from donor livers preconditioned with ischemia showed oxidative DNA damage with expression of genes involved in MAPK signaling that impose G1/S and G2/M checkpoint restrictions, including p38 MAPK-regulated or ERK-1/2-regulated cell-cycle genes such as FOS, MAPK8, MYC, various cyclins, CDKN2A, CDKN2B, TP53, and RB1. Thus, although ischemic preconditioning allowed hepatocytes to better withstand secondary insults, accompanying DNA damage and molecular events simultaneously impaired their proliferation capacity over the long term. Mitigation of ischemic preconditioning-induced DNA damage and deleterious molecular perturbations holds promise for advancing clinical applications. PMID:25128377

  5. Decontamination by Persteril 36 may affect the reliability of DNA-based detection of biological warfare agents-short communication.

    PubMed

    Josefiova, Jirina; Pospisek, Martin; Vanek, Daniel

    2016-09-01

    Persteril 36 is a disinfectant with a broad spectrum of antimicrobial activity. Because of its bactericidal, virucidal, fungicidal, and sporicidal effectiveness, it is used as a disinfectant against biological warfare agents in the emergency and army services. In case of an attack with potentially harmful biological agents, a person's gear or afflicted skin is sprayed with a diluted solution of Persteril 36 as a precaution. Subsequently, the remains of the biological agents are analyzed. However, the question remains concerning whether DNA can be successfully analyzed from Persteril 36-treated dead bacterial cells. Spore-forming Bacillus subtilis and Gram-negative Pseudomonas aeruginosa and Xanthomonas campestris were splattered on a camouflage suit and treated with 2 or 0.2 % Persteril 36. After the disinfectant vaporized, the bacterial DNA was extracted and quantified by real-time PCR. A sufficient amount of DNA was recovered for downstream analysis only in the case of spore-forming B. subtilis treated with a 0.2 % solution of Persteril 36. The bacterial DNA was almost completely destroyed in Gram-negative bacteria or after treatment with the more concentrated solution in B. subtilis. This phenomenon can lead to false-negative results during the identification of harmful microorganisms. PMID:26910525

  6. Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA.

    PubMed

    Hirabayashi, Masumi; Kato, Megumi; Ishikawa, Ayako; Kaneko, Ryosuke; Yagi, Takeshi; Hochi, Shinichi

    2005-04-01

    Factors affecting the efficiency of producing transgenic rats by intracytoplasmic sperm injection (ICSI)-mediated DNA transfer were investigated. Epididymal spermatozoa from Sprague-Dawley (SD) rats were sonicated and/or frozen-thawed for cutting the tail and membrane disruption. The sperm heads were exposed for 1 min to different concentrations (0.02-2.5 microg/ml) of 3.0 kb enhanced green fluorescent protein (EGFP) DNA solution, and then microinjected into the denuded F1 hybrid (Donryu x LEW) rat oocytes. The optimal concentration of EGFP DNA solution was 0.1 microg/ml, as determined by the in vitro developmental competence into morulae/blastocysts of the ICSI oocytes and the EGFP expression of the resultant embryos. The efficiency of producing transgenic rat offspring (per transferred zygote) was 2.8%, 1.6%, and 3.3% in the oocytes injected with sonicated, frozen-thawed, and sonicated + frozen-thawed sperm heads, respectively. The founder transgenic rats carrying the EGFP gene transmitted their transgenes to their progeny according to the Mendelian fashion, suggesting the stable incorporation of the transgenes into the rat genomes. Four rat strains (F344, LEW, Donryu, and SD) were compared for their suitability as sperm/oocyte donors for the production of transgenic rats by ICSI with sonicated, frozen-thawed and solution of EGFP DNA-exposed sperm heads. The efficiency of producing transgenic rats in the SD strain (8.2%) was higher than that in the LEW strain (0.9%), while those in the F344 and Donryu strains (4.3%-4.4%) were intermediate. One plasmid DNA (Fyn, 5.0 kb) and two BAC DNA (BAC/Fyn, 208 kb; Svet1/IRES-Cre, 186 kb) were successfully introduced into the SD rat genomes via ICSI, with the producing efficiencies of 2.8%, 0.9%, and 2.4%, respectively. PMID:15685640

  7. Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates.

    PubMed

    Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S; Schatz, David G

    2007-08-31

    The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. PMID:17613522

  8. An integrative affect regulation process model of internalized weight bias and intuitive eating in college women.

    PubMed

    Webb, Jennifer B; Hardin, Abigail S

    2016-07-01

    The present study extended the weight stigma and well-being process model (Tylka et al., 2014) by examining three affect regulation pathways that may help simultaneously explain the predicted inverse association between internalized weight bias and intuitive eating. A weight-diverse sample of 333 college women completed an online survey assessing internalized weight stigma, intuitive eating, body shame, body image flexibility, and self-compassion. Self-reported height and weight were used to calculate body mass index (BMI). Non-parametric bootstrap resampling procedures were computed to ascertain the presence of the indirect effects of internalized weight bias on intuitive eating via the three hypothesized mediators controlling for BMI in a combined model. Results demonstrated that body image flexibility significantly and self-compassion marginally contributed unique variance in accounting for this relationship. Our preliminary cross-sectional findings contribute to a nascent body of scholarship seeking to provide a theoretically-driven understanding of how negative and positive forms of experiencing and relating to the body may co-occur within individuals. Results also point to potential target variables to consider incorporating in later-stage efforts to promote more adaptive ways of eating amidst internalized weight stigma. PMID:26893074

  9. Food contaminant zearalenone and its metabolites affect cytokine synthesis and intestinal epithelial integrity of porcine cells.

    PubMed

    Marin, Daniela E; Motiu, Monica; Taranu, Ionelia

    2015-06-01

    The intestinal epithelium is the first barrier against food contaminants. Zearalenone (ZEN) is an estrogenic mycotoxin that was identified as a common contaminant of cereal grains and food and feedstuffs. In the present study, we have investigated the in vitro effects of ZEN and some of its metabolites (α-ZOL, β-ZOL) in concentrations of 10-100 µM on a swine epithelial cell line: Intestinal porcine epithelial cells (IPEC-1). We demonstrated that both ZEN metabolites were more toxic for IPEC cells as resulted from the XTT test, while for doses lower than 10 µM, only β-ZOL showed a more pronounced cytotoxicity versus epithelial cells as resulted from neutral red assay. ZEN has no effect on TER values, while α-ZOL significantly decreased the TER values, starting with day 4 of treatment. β-ZOL had a dual effect, firstly it induced a significant increase of TER, and then, starting on day 6, it induced a dramatic decrease of TER values as compared with on day 0. Concerning the cytokine synthesis, our results showed that ZEN has a tendency to increase the synthesis of IL-8 and IL-10. By contrast, α- and β-ZOL decreased the expression of both IL-8 and IL-10, in a dose dependent manner. In conclusion, our results showed that ZEN and its metabolites differently affected porcine intestinal cell viability, transepithelial resistance and cytokine synthesis with important implication for gut health. PMID:26035492

  10. Food Contaminant Zearalenone and Its Metabolites Affect Cytokine Synthesis and Intestinal Epithelial Integrity of Porcine Cells

    PubMed Central

    Marin, Daniela E.; Motiu, Monica; Taranu, Ionelia

    2015-01-01

    The intestinal epithelium is the first barrier against food contaminants. Zearalenone (ZEN) is an estrogenic mycotoxin that was identified as a common contaminant of cereal grains and food and feedstuffs. In the present study, we have investigated the in vitro effects of ZEN and some of its metabolites (α-ZOL, β-ZOL) in concentrations of 10–100 µM on a swine epithelial cell line: Intestinal porcine epithelial cells (IPEC-1). We demonstrated that both ZEN metabolites were more toxic for IPEC cells as resulted from the XTT test, while for doses lower than 10 µM, only β-ZOL showed a more pronounced cytotoxicity versus epithelial cells as resulted from neutral red assay. ZEN has no effect on TER values, while α-ZOL significantly decreased the TER values, starting with day 4 of treatment. β-ZOL had a dual effect, firstly it induced a significant increase of TER, and then, starting on day 6, it induced a dramatic decrease of TER values as compared with on day 0. Concerning the cytokine synthesis, our results showed that ZEN has a tendency to increase the synthesis of IL-8 and IL-10. By contrast, α- and β-ZOL decreased the expression of both IL-8 and IL-10, in a dose dependent manner. In conclusion, our results showed that ZEN and its metabolites differently affected porcine intestinal cell viability, transepithelial resistance and cytokine synthesis with important implication for gut health. PMID:26035492

  11. Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant.

    PubMed

    Durand, Adeline; Sinha, Anurag Kumar; Dard-Dascot, Cloelia; Michel, Bénédicte

    2016-06-01

    Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo. PMID:27280472

  12. Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant

    PubMed Central

    Durand, Adeline

    2016-01-01

    Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo. PMID:27280472

  13. In Vitro HIV-1 LTR Integration into T-Cell Activation Gene CD27 Segment and the Decoy Effect of Modified-Sequence DNA

    PubMed Central

    Ohmori, Rei; Tsuruyama, Tatsuaki

    2012-01-01

    Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors. PMID:23209625

  14. Disruption of Amyloid Plaques Integrity Affects the Soluble Oligomers Content from Alzheimer Disease Brains

    PubMed Central

    Moyano, Javier; Sanchez-Mico, María; Torres, Manuel; Davila, Jose Carlos; Vizuete, Marisa; Gutierrez, Antonia; Vitorica, Javier

    2014-01-01

    The implication of soluble Abeta in the Alzheimer’s disease (AD) pathology is currently accepted. In fact, the content of soluble extracellular Abeta species, such as monomeric and/or oligomeric Abeta, seems to correlate with the clinico-pathological dysfunction observed in AD patients. However, the nature (monomeric, dimeric or other oligomers), the relative abundance, and the origin (extra-/intraneuronal or plaque-associated), of these soluble species are actually under debate. In this work we have characterized the soluble (defined as soluble in Tris-buffered saline after ultracentrifugation) Abeta, obtained from hippocampal samples of Braak II, Braak III–IV and Braak V–VI patients. Although the content of both Abeta40 and Abeta42 peptides displayed significant increase with pathology progression, our results demonstrated the presence of low, pg/µg protein, amount of both peptides. This low content could explain the absence (or below detection limits) of soluble Abeta peptides detected by western blots or by immunoprecipitation-western blot analysis. These data were in clear contrast to those published recently by different groups. Aiming to explain the reasons that determine these substantial differences, we also investigated whether the initial homogenization could mobilize Abeta from plaques, using 12-month-old PS1xAPP cortical samples. Our data demonstrated that manual homogenization (using Dounce) preserved the integrity of Abeta plaques whereas strong homogenization procedures (such as sonication) produced a vast redistribution of the Abeta species in all soluble and insoluble fractions. This artifact could explain the dissimilar and somehow controversial data between different groups analyzing human AD samples. PMID:25485545

  15. The ability of Hepatitis B surface antigen DNA vaccine to elicit cell-mediated immune responses, but not antibody responses, was affected by the deglysosylation of S antigen.

    PubMed

    Xing, Yiping; Huang, Zuhu; Lin, Yan; Li, Jun; Chou, Te-Hui; Lu, Shan; Wang, Shixia

    2008-09-19

    Hepatitis B Virus (HBV) infection remains a major worldwide infectious disease with serious long-term morbidity and mortality. The limited selections of drug treatment are not able to control the progress of disease in people with active and persistent HBV infection. Immunotherapy to control the degree of viral infection is one possible alternative solution to this challenge. HBV DNA vaccines, with their strong ability to induce cell-mediated immune responses, offer an attractive option. HBV surface protein is important in viral immunity. Re-establishing anti-S immunity in chronic HBV infected patients will bring significant benefit to the patients. Previous studies have shown that HBV S DNA vaccines are immunogenic in a number of animal studies. In the current study, we further investigated the effect of glycosylation to the expression and immunogenicity of S DNA vaccines. Our results demonstrate that deglycosylation at the two potential N-linked glycosylation sites in S protein resulted in a significant decrease of S-specific cell-mediated immune responses, but did not affect anti-S antibody responses. This finding provides important direction to the development of S DNA vaccines to elicit the optimal and balanced antibody and cell-mediated immune responses to treat people with HBV chronic infections. PMID:18462847

  16. Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination.

    PubMed

    Slon Campos, J L; Poggianella, M; Marchese, S; Bestagno, M; Burrone, O R

    2015-11-01

    Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV. PMID:26358704

  17. Retroposons do jump: a B2 element recently integrated in an 18S rDNA gene.

    PubMed Central

    Oberbäumer, I

    1992-01-01

    Several cDNA clones were isolated from cDNA libraries constructed with mRNA longer than 28S RNA from the murine cell line PYS-2/12. The plasmids have inserts containing 1-1.2 kb of the ribosomal 5' external transcribed spacer followed by nearly 700 nt of sequence for 18S rRNA and ending with a B2 element (retroposon). The cloned sequence differed in a few positions from published ribosomal sequences. The 3' adjacent genomic sequence was obtained by polymerase chain reaction (PCR) and showed that the B2 element has a poly(A) tail of about 50 nt and is surrounded by perfect direct repeats of 15 nt. Analysis of genomic DNA from several murine cell lines revealed that PYS cells contain at least one copy of 18S RNA with the B2 element which is not present in the genome of other murine cell lines derived from the same teratocarcinoma. Similarly, rRNA transcripts containing the B2 element were only detected in PYS cells. According to the publication dates of the different cell lines, the B2 element must have been integrated into an rRNA transcription unit during the years 1970 through 1974 thus proving that retroposons (SINEs) can still be inserted into the genome in our times. Images PMID:1311830

  18. Integrating affective and cognitive correlates of heart rate variability: A structural equation modeling approach.

    PubMed

    Mann, Sarah L; Selby, Edward A; Bates, Marsha E; Contrada, Richard J

    2015-10-01

    High frequency heart rate variability (HRV) is a measure of neurocardiac communication thought to reflect predominantly parasympathetic cardiac regulation. Low HRV has been associated empirically with clinical and subclinical levels of anxiety and depression and, more recently, high levels of HRV have been associated with better performance on some measures of executive functioning (EF). These findings have offered support for theories proposing HRV as an index measure of a broad, self-regulatory capacity underlying aspects of emotion regulation and executive control. This study sought to test that proposition by using a structural equation modeling approach to examine the relationships of HRV to negative affect (NA) and EF in a large sample of U.S. adults ages 30s-80s. HRV was modeled as a predictor of an NA factor (self-reported trait anxiety and depression symptoms) and an EF factor (performance on three neuropsychological tests tapping facets of executive abilities). Alternative models also were tested to determine the utility of HRV for predicting NA and EF, with and without statistical control of demographic and health-related covariates. In the initial structural model, HRV showed a significant positive relationship to EF and a nonsignificant relationship to NA. In a covariate-adjusted model, HRV's associations with both constructs were nonsignificant. Age emerged as the only significant predictor of NA and EF in the final model, showing inverse relationships to both. Findings may reflect population and methodological differences from prior research; they also suggest refinements to the interpretations of earlier findings and theoretical claims regarding HRV. PMID:26168884

  19. Extracellular S100A4 affects endothelial cell integrity and stimulates transmigration of A375 melanoma cells.

    PubMed

    Herwig, Nadine; Belter, Birgit; Pietzsch, Jens

    2016-09-01

    High extracellular S100A4 level proves a specific characteristic of some cancer cases, including malignant melanoma. Concerning the latter, extracellular S100A4 in an autocrine manner was shown to promote prometastatic activation of A375 cells by interaction with the receptor for advanced glycation endproducts (RAGE). We hypothesized that interaction of extracellular S100A4 with RAGE in a paracrine manner will affect endothelial cell (EC) integrity thus further promoting melanoma metastasis. We investigated the influence of recombinant and cell (A375)-derived S100A4 on junction protein expression and EC (hCMEC/D3) integrity by measuring transendothelial electrical resistance (TEER). Decrease of TEER and diminished expression of both occludin and VE-cadherin revealed the loss of EC integrity. Transmigration of transgenic A375 cells (A375-hS100A4/A375-hRAGE) through the EC monolayer was significantly higher compared to wild-type A375 cells, and was substantially decreased by sRAGE. A pilot study in mice, intracardially injected with A375-hS100A4 or A375-hRAGE cells, showed lower survival rates and a higher incidence of metastases compared to wild-type A375 cells. Tumor development was mostly located in the brain, bones, and ovaries. These findings provide further evidence on extracellular S100A4 as paracrine mediator of prometastatic endothelial dysfunction involving its interaction with RAGE. PMID:27387233

  20. An affected core drives network integration deficits of the structural connectome in 22q11.2 deletion syndrome.

    PubMed

    Váša, František; Griffa, Alessandra; Scariati, Elisa; Schaer, Marie; Urben, Sébastien; Eliez, Stephan; Hagmann, Patric

    2016-01-01

    Chromosome 22q11.2 deletion syndrome (22q11DS) is a genetic disease known to lead to cerebral structural alterations, which we study using the framework of the macroscopic white-matter connectome. We create weighted connectomes of 44 patients with 22q11DS and 44 healthy controls using diffusion tensor magnetic resonance imaging, and perform a weighted graph theoretical analysis. After confirming global network integration deficits in 22q11DS (previously identified using binary connectomes), we identify the spatial distribution of regions responsible for global deficits. Next, we further characterize the dysconnectivity of the deficient regions in terms of sub-network properties, and investigate their relevance with respect to clinical profiles. We define the subset of regions with decreased nodal integration (evaluated using the closeness centrality measure) as the affected core (A-core) of the 22q11DS structural connectome. A-core regions are broadly bilaterally symmetric and consist of numerous network hubs - chiefly parietal and frontal cortical, as well as subcortical regions. Using a simulated lesion approach, we demonstrate that these core regions and their connections are particularly important to efficient network communication. Moreover, these regions are generally densely connected, but less so in 22q11DS. These specific disturbances are associated to a rerouting of shortest network paths that circumvent the A-core in 22q11DS, "de-centralizing" the network. Finally, the efficiency and mean connectivity strength of an orbito-frontal/cingulate circuit, included in the affected regions, correlate negatively with the extent of negative symptoms in 22q11DS patients, revealing the clinical relevance of present findings. The identified A-core overlaps numerous regions previously identified as affected in 22q11DS as well as in schizophrenia, which approximately 30-40% of 22q11DS patients develop. PMID:26870660

  1. An affected core drives network integration deficits of the structural connectome in 22q11.2 deletion syndrome

    PubMed Central

    Váša, František; Griffa, Alessandra; Scariati, Elisa; Schaer, Marie; Urben, Sébastien; Eliez, Stephan; Hagmann, Patric

    2015-01-01

    Chromosome 22q11.2 deletion syndrome (22q11DS) is a genetic disease known to lead to cerebral structural alterations, which we study using the framework of the macroscopic white-matter connectome. We create weighted connectomes of 44 patients with 22q11DS and 44 healthy controls using diffusion tensor magnetic resonance imaging, and perform a weighted graph theoretical analysis. After confirming global network integration deficits in 22q11DS (previously identified using binary connectomes), we identify the spatial distribution of regions responsible for global deficits. Next, we further characterize the dysconnectivity of the deficient regions in terms of sub-network properties, and investigate their relevance with respect to clinical profiles. We define the subset of regions with decreased nodal integration (evaluated using the closeness centrality measure) as the affected core (A-core) of the 22q11DS structural connectome. A-core regions are broadly bilaterally symmetric and consist of numerous network hubs — chiefly parietal and frontal cortical, as well as subcortical regions. Using a simulated lesion approach, we demonstrate that these core regions and their connections are particularly important to efficient network communication. Moreover, these regions are generally densely connected, but less so in 22q11DS. These specific disturbances are associated to a rerouting of shortest network paths that circumvent the A-core in 22q11DS, “de-centralizing” the network. Finally, the efficiency and mean connectivity strength of an orbito-frontal/cingulate circuit, included in the affected regions, correlate negatively with the extent of negative symptoms in 22q11DS patients, revealing the clinical relevance of present findings. The identified A-core overlaps numerous regions previously identified as affected in 22q11DS as well as in schizophrenia, which approximately 30–40% of 22q11DS patients develop. PMID:26870660

  2. Integration host factor and LuxR synergistically bind DNA to coactivate quorum-sensing genes in Vibrio harveyi.

    PubMed

    Chaparian, Ryan R; Olney, Stephen G; Hustmyer, Christine M; Rowe-Magnus, Dean A; van Kessel, Julia C

    2016-09-01

    The cell-cell signaling process called quorum sensing allows bacteria to control behaviors in response to changes in population density. In Vibrio harveyi, the master quorum-sensing transcription factor LuxR is a member of the TetR family of transcription factors that both activates and represses genes to coordinate group behaviors, including bioluminescence. Here, we show that integration host factor (IHF) is a key coactivator of the luxCDABE bioluminescence genes that is required together with LuxR for precise timing and expression levels of bioluminescence during quorum sensing. IHF binds to multiple sites in the luxCDABE promoter and bends the DNA in vitro. IHF and LuxR synergistically bind luxCDABE promoter DNA at overlapping, essential binding sites that are required for maximal gene expression in vivo. RNA-seq analysis demonstrated that IHF regulates 300 genes in V. harveyi, and among these are a core set of 19 genes that are also directly bound and regulated by LuxR. We validated these global analyses by demonstrating that both IHF and LuxR are required for transcriptional activation of the osmotic stress response genes betIBA-proXWV. These data suggest that IHF plays an integral role in one mechanism of transcriptional activation by the LuxR-type family of quorum-sensing regulators in vibrios. PMID:27191515

  3. DIETARY ARSENITE AFFECTS DIMETHYLHYDRAZINE (DMH)-INDUCED ABERRANT CRYPT FORMATION IN COLON AND GLOBAL DNA METHYLATION IN LIVER OF RATS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work has shown that arsenic (As) affects methionine metabolism. Alterations in methionine metabolism can affect cancer processes. To determine the effect of dietary As on DMH-induced aberrant crypt formation in colon Fisher-344 male, weanling rats (N=20/group) were fed diets containing 0, 0...

  4. LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair

    PubMed Central

    Liu, Xue-Song; Chandramouly, Gurushankar; Rass, Emilie; Guan, Yinghua; Wang, Guocan; Hobbs, Robin M.; Rajendran, Anbazhagan; Xie, Anyong; Shah, Jagesh V.; Davis, Anthony J.; Scully, Ralph; Lunardi, Andrea; Pandolfi, Pier Paolo

    2015-01-01

    Leukemia/lymphoma-related factor (LRF) is a POZ/BTB and Krüppel (POK) transcriptional repressor characterized by context-dependent key roles in cell fate decision and tumorigenesis. Here we demonstrate an unexpected transcription-independent function for LRF in the classical non-homologous end joining (cNHEJ) pathway of double-strand break (DSB) repair. We find that LRF loss in cell lines and mouse tissues results in defective cNHEJ, genomic instability and hypersensitivity to ionizing radiation. Mechanistically, we show that LRF binds and stabilizes DNA-PKcs on DSBs, in turn favouring DNA-PK activity. Importantly, LRF loss restores ionizing radiation sensitivity to p53 null cells, making LRF an attractive biomarker to direct p53-null LRF-deficient tumours towards therapeutic treatments based on genotoxic agents or PARP inhibitors following a synthetic lethal strategy. PMID:26446488

  5. LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair.

    PubMed

    Liu, Xue-Song; Chandramouly, Gurushankar; Rass, Emilie; Guan, Yinghua; Wang, Guocan; Hobbs, Robin M; Rajendran, Anbazhagan; Xie, Anyong; Shah, Jagesh V; Davis, Anthony J; Scully, Ralph; Lunardi, Andrea; Pandolfi, Pier Paolo

    2015-01-01

    Leukemia/lymphoma-related factor (LRF) is a POZ/BTB and Krüppel (POK) transcriptional repressor characterized by context-dependent key roles in cell fate decision and tumorigenesis. Here we demonstrate an unexpected transcription-independent function for LRF in the classical non-homologous end joining (cNHEJ) pathway of double-strand break (DSB) repair. We find that LRF loss in cell lines and mouse tissues results in defective cNHEJ, genomic instability and hypersensitivity to ionizing radiation. Mechanistically, we show that LRF binds and stabilizes DNA-PKcs on DSBs, in turn favouring DNA-PK activity. Importantly, LRF loss restores ionizing radiation sensitivity to p53 null cells, making LRF an attractive biomarker to direct p53-null LRF-deficient tumours towards therapeutic treatments based on genotoxic agents or PARP inhibitors following a synthetic lethal strategy. PMID:26446488

  6. Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA.

    PubMed Central

    Seiki, M; Hattori, S; Hirayama, Y; Yoshida, M

    1983-01-01

    Human retrovirus adult T-cell leukemia virus (ATLV) has been shown to be closely associated with human adult T-cell leukemia (ATL) [Yoshida, M., Miyoshi, I. & Hinuma, Y. (1982) Proc. Natl. Acad. Sci. USA 79, 2031-2035]. The provirus of ATLV integrated in DNA of leukemia T cells from a patient with ATL was molecularly cloned and the complete nucleotide sequence of 9,032 bases of the proviral genome was determined. The provirus DNA contains two long terminal repeats (LTRs) consisting of 755 bases, one at each end, which are flanked by a 6-base direct repeat of the cellular DNA sequence. The nucleotides in the LTR could be arranged into a unique secondary structure, which could explain transcriptional termination within the 3' LTR but not in the 5' LTR. The nucleotide sequence of the provirus contains three large open reading frames, which are capable of coding for proteins of 48,000, 99,000, and 54,000 daltons. The three open frames are in this order from the 5' end of the viral genome and the predicted 48,000-dalton polypeptide is a precursor of gag proteins, because it has an identical amino acid sequence to that of the NH2 terminus of human T-cell leukemia virus (HTLV) p24. The open frames coding for 99,000- and 54,000-dalton polypeptides are thought to be the pol and env genes, respectively. On the 3' side of these three open frames, the ATLV sequence has four smaller open frames in various phases; these frames may code for 10,000-, 11,000-, 12,000-, and 27,000-dalton polypeptides. Although one or some of these open frames could be the transforming gene of this virus, in preliminary analysis, DNA of this region has no homology with the normal human genome. PMID:6304725

  7. STAT3 regulated ATR via microRNA-383 to control DNA damage to affect apoptosis in A431 cells.

    PubMed

    Liao, Xing-Hua; Zheng, Li; He, Hong-Peng; Zheng, De-Liang; Wei, Zhao-Qiang; Wang, Nan; Dong, Jian; Ma, Wen-Jian; Zhang, Tong-Cun

    2015-11-01

    Skin cancer is a major cause of morbidity and mortality worldwide. Mounting evidence shows that exposure of the skin to solar UV radiation results in inflammation, oxidative stress, DNA damage, dysregulation of cellular signaling pathways and immunosuppression thereby resulting in skin cancer. Signal transducer and activator of transcription 3 (STAT3) is well known to function as an anti-apoptotic factor, especially in numerous malignancies, but the relationship between STAT3 activation and DNA damage response in skin cancer is still not fully understood. We now report that STAT3 inhibited DNA damage induced by UV and STAT3 mediated upregulation of GADD45γ and MDC-1 and the phosphorylation of H2AX in UV induced DNA damage. Notably, STAT3 can increase the expression of ATR in A431 cells. Luciferase assay shows that STAT3 activates the transcription of ATR promoter. More importantly, microRNA-383 suppressed ATR expression by targeting 3' (untranslated regions)UTR of ATR in A431 cells, and STAT3 down-regulates the transcription of miR-383 promoter. Thus, these results reveal the new insight that ATR is down-regulated by STAT3-regulated microRNA-383 in A431 cells. Moreover, overexpression of STAT3 enhanced expression of antiapoptosis genes BCL-1 and MCL-1, and depletion of STAT3 sensitized A431 cells to apoptotic cell death following UV. Collectively, these studies suggest that STAT3 may be a potential target for both the prevention and treatment of human skin cancer. PMID:26261078

  8. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    SciTech Connect

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  9. Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1*

    PubMed Central

    Boesler, Benjamin; Meier, Doreen; Förstner, Konrad U.; Friedrich, Michael; Hammann, Christian; Sharma, Cynthia M.; Nellen, Wolfgang

    2014-01-01

    The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in D. discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi-related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1-encoded proteins, and the loss of most DIRS-1-derived secondary siRNAs. Simultaneously, extrachromosomal single-stranded DIRS-1 DNA accumulated in the cytoplasm of agnA− strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA− strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon regulation. However, in agnA−/agnB− double mutant strains strongly reduced accumulation of extrachromosomal DNA compared with the single agnA− strains was observed. PMID:25352599

  10. Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

    PubMed Central

    Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

    2015-01-01

    Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

  11. Phenolic composition and inhibitory effect against oxidative DNA damage of cooked cowpeas as affected by simulated in vitro gastrointestinal digestion.

    PubMed

    Nderitu, Alice M; Dykes, Linda; Awika, Joseph M; Minnaar, Amanda; Duodu, Kwaku G

    2013-12-01

    Cowpeas contain phenolic compounds with potential health benefits. The effect of simulated gastrointestinal digestion on phenolic composition of cooked cowpeas and the ability of the digests to inhibit radical-induced DNA damage was determined. A red and a cream-coloured cowpea type were used. The phenolic composition of acetone extracts and enzyme digests of cooked cowpeas was determined using UPLC-MS. Compounds such as p-hydroxybenzoic acid, p-coumaric acid, coumaroylaldaric acid and feruloylaldaric acid were present in the acetone extracts of the cooked cowpeas but were not detected in the enzyme digests. Glycosides of quercetin and myricetin decreased upon in vitro gastrointestinal digestion of cooked cowpeas whereas flavan-3-ols were hardly present except catechin glucoside. The enzyme digest of the red cowpea type was about thrice as effective as that of the cream cowpea type in protecting DNA from oxidative damage. The observation that enzyme digests of cooked cowpeas inhibited radical-induced DNA damage suggests that cowpea phenolics retain some radical scavenging activity after gastrointestinal digestion. PMID:23870889

  12. Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

    PubMed Central

    Puchta, H; Kocher, S; Hohn, B

    1992-01-01

    Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared. Images PMID:1630452

  13. DNA barcodes for assessment of the biological integrity of aquatic ecosystems

    EPA Science Inventory

    Water quality regulations and aquatic ecosystem monitoring increasingly rely on direct assessments of biological integrity. Because these aquatic “bioassessments” evaluate the incidence and abundance of sensitive aquatic species, they are able to measure cumulative ecosystem eff...

  14. Factors affecting efficiency of introducing foreign DNA and RNA into parthenogenetic or in vitro-fertilized porcine eggs by cytoplasmic microinjection.

    PubMed

    Liu, Shuai; Liu, XiaoQun; Huang, HaiYan; Liu, QingYou; Su, XiaoPing; Zhu, Peng; Li, HongLi; Cui, KuiQing; Xie, BingKun; Shi, DeShun

    2016-08-01

    Cytoplasmic microinjection (CI) of foreign gene into in vivo fertilized zygotes has emerged as a useful tool for transgenic pig production. In the current study, we investigated factors affecting transgenic efficiency and developmental potential of parthenogenetic (PA) and in vitro-fertilized (IVF) porcine embryos produced by CI. These factors included adding of RNase inhibitor, DNA or RNA concentration, injection time, and different structures of plasmids. Our results showed that adding of 1-4 U/μL of RNase inhibitor did not have negative effect on development potential of CI-PA embryos, and RNase inhibitor injection significantly increased EGFP expressing rate of CI-PA embryos. High injection DNA concentration and long injection interval after PA significantly reduced blastocyst formation. Different molecular structures such as DNA or RNA affected CI-PA embryos development, and RNA had little harmful effect on pig's early embryonic development. EGFP expression rate of CI-IVF embryos was improved following the increase of foreign DNA concentration, but blastocyst formation rate was decreased. Injection time after IVF did not show any significant difference on embryonic development, but longer interval resulted in a significantly lower EGFP expressing rate. Cas9 mRNA and myostatin (GDF-8) sgRNA co-injection indicated that the mutation rate of CI-IVF group was significantly higher than that of CI-PA. The CI-IVF-generated embryos were then transferred to six recipient pigs, but no live piglets were obtained. The following pronuclear formation assessment showed more than 76.1% IVF zygotes were polyspermy. These results demonstrate that CI-PA and CI-IVF were effective methods for production of transgenic pig embryos. However, polyspermic fertilization and poor quality of porcine IVF blastocysts are still the main problem of resulting in pregnancy failure. PMID:27130683

  15. Dme-miR-314-3p modulation in Cr(VI) exposed Drosophila affects DNA damage repair by targeting mus309.

    PubMed

    Chandra, Swati; Khatoon, Rehana; Pandey, Ashutosh; Saini, Sanjay; Vimal, Divya; Singh, Pallavi; Chowdhuri, D Kar

    2016-03-01

    microRNAs (miRNAs) as one of the major epigenetic modulators negatively regulate mRNAs at post transcriptional level. It was therefore hypothesized that modulation of miRNAs by hexavalent Chromium [Cr(VI)], a priority environmental chemical, can affect DNA damage. In a genetically tractable model, Drosophila melanogaster, role of maximally up-regulated miRNA, dme-miR-314-3p, on DNA damage was examined by exposing the third instar larvae to 5.0-20.0 μg/ml Cr(VI) for 24 and 48 h. mus309, a Drosophila homologue of human Bloom's syndrome and predicted as one of the potential targets of this miRNA, was confirmed as its target by 5'RLM-RACE assay. A significant down-regulation of mus309 was observed in dme-miR-314-3p overexpression strain (myo-gal4>UAS-miR-314-3p) as compared with that in parental strains (myo-gal4 and UAS-miR-314-3p) and in w(1118). A significant increase in DNA damage including double strand breaks generation was observed in exposed myo-gal4>UAS-miR-314 and mus309 mutants as compared with that in parental strain and in unexposed control. A significant down-regulation of cell cycle regulation genes (CycA, CycB and cdc2) was observed in these exposed genotypes. Collectively, the study demonstrates that dme-miR-314-3p can mediate the downregulation of repair deficient gene mus309 leading to increased DNA damage and cell cycle arrest in exposed organism which may affect Cr(VI) mediated carcinogenesis. PMID:26590872

  16. Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa.

    PubMed

    Gloor, Kayleen T; Winget, Doug; Swanson, William F

    2006-09-01

    In response to growing terrorism concerns, the Transportation Security Administration now requires that all checked baggage at U.S. airports be scanned through a cabinet x-ray system, which may increase risk of radiation damage to transported biologic samples and other sensitive genetic material. The objective of this study was to investigate the effect of these new airport security regulations on the viability and DNA integrity of frozen felid spermatozoa. Semen was collected from two domestic cats (Felis silvestris catus) and one fishing cat (Prionailurus viverrinus), cryopreserved in plastic freezing straws, and transferred into liquid nitrogen dry shippers for security screening. Treatment groups included frozen samples from each male scanned once or three times using a Transportation Security Administration-operated cabinet x-ray system, in addition to non-scanned samples (i.e., negative control) and samples previously scanned three times and exposed to five additional high-intensity x-ray bursts (i.e., positive control). Dosimeters placed in empty dry shippers were used to quantify radiation exposure. Following treatment, straws were thawed and spermatozoa analyzed for post-thaw motility (percentage motile and rate of progressive movement), acrosome status, and DNA integrity using single-cell gel electrophoresis (i.e., the comet assay). Dosimeter measurements determined that each airport screening procedure produced approximately 16 mrem of radiation exposure. Our results indicated that all levels of radiation exposure adversely affected (P < 0.05) post-thaw sperm motility, but the percentage of acrosome-intact spermatozoa did not differ (P > 0.05) among treatment groups. Results also showed that the amount of double-stranded DNA damage was greater (P < 0.05) in sperm samples from both cat species scanned three times compared to samples scanned once or negative controls. Findings suggest that new airport security measures may cause radiation-induced damage to

  17. DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

    PubMed Central

    Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

    2016-01-01

    Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network

  18. Integrating the Regulation of Affect, Behavior, and Cognition into Self-Regulated Learning Paradigms among Secondary and Post-Secondary Students

    ERIC Educational Resources Information Center

    Ben-Eliyahu, Adar; Linnenbrink-Garcia, Lisa

    2015-01-01

    An integrative framework for investigating self-regulated learning situated in students' favorite and least favorite courses was empirically tested in a sample of 178 high school and 280 college students. Building on cognitive, clinical, social, and educational conceptions of self-regulation, the current paper integrated affective (e.g.,…

  19. Ion and solvent density distributions around canonical B-DNA from integral equations

    PubMed Central

    Howard, Jesse J.; Lynch, Gillian C.; Pettitt, B. Montgomery

    2011-01-01

    We calculate the water and ion spatial distributions around charged oligonucleotides using a renormalized three-dimensional reference interaction site theory coupled with the HNC closure. Our goal is to understand the balance between inter-DNA strand forces and solvation forces as a function of oligonucleotide length in the short strand limit. The DNA is considered in aqueous electrolyte solutions of 1 M KCl, 0.1 M KCl or 0.1 M NaCl. The current theoretical results are compared to MD simulations and experiments. It is found that the IE theory replicates the MD and the experimental results for the base-specific hydration patterns in both the major and minor grooves. We are also able to discern characteristic structural pattern differences between Na+ and K+ ions. When compared to Poisson-Boltzmann methods the IE theory, like simulation, predicts a richly structured ion environment which is better described as multi-layer rather than double-layer. PMID:21190358

  20. Integrating microsatellite DNA markers and otolith geochemistry to assess population structure of European hake (Merluccius merluccius)

    NASA Astrophysics Data System (ADS)

    Tanner, Susanne E.; Pérez, Montse; Presa, Pablo; Thorrold, Simon R.; Cabral, Henrique N.

    2014-04-01

    Population structure and natal origins of European hake were investigated using microsatellite DNA markers and otolith geochemistry data. Five microsatellites were sequenced and otolith core geochemical composition was determined from age-1 hake collected in the northeast Atlantic Ocean and the Mediterranean Sea. Microsatellites provided evidence of a major genetic split in the vicinity of the Strait of Gibraltar, separating the Atlantic and the Mediterranean populations, with the exception of the Gulf of Cádiz. Based on classification models using otolith core geochemical values, individual natal origins were identified, although with an increased error rate. Coupling genotype and otolith data increased the classification accuracy of individuals to their potential natal origins while providing evidence of movement between the northern and southern stock units in the Atlantic Ocean. Information obtained by the two natural markers on population structure of European hake was complementary as the two markers act at different spatio-temporal scales. Otolith geochemistry provides information over an ecological time frame and on a fine spatial scale, while microsatellite DNA markers report on gene flow over evolutionary time scales and therefore act on a broader spatio-temporal resolution. Thus, this study confirmed the value of otolith geochemistry to complement the assessment of early life stage dispersal in populations with high gene flow and low genetic divergence.

  1. Green reconstruction of the tsunami-affected areas in India using the integrated coastal zone management concept.

    PubMed

    Sonak, Sangeeta; Pangam, Prajwala; Giriyan, Asha

    2008-10-01

    A tsunami, triggered by a massive undersea earthquake off Sumatra in Indonesia, greatly devastated the lives, property and infrastructure of coastal communities in the coastal states of India, Andaman and Nicobar Islands, Indonesia, Sri Lanka, Malaysia and Thailand. This event attracted the attention of environmental managers at all levels, local, national, regional and global. It also shifted the focus from the impact of human activities on the environment to the impacts of natural hazards. Recovery/reconstruction of these areas is highly challenging. A clear understanding of the complex dynamics of the coast and the types of challenges faced by the several stakeholders of the coast is required. Issues such as sustainability, equity and community participation assume importance. The concept of ICZM (integrated coastal zone management) has been effectively used in most parts of the world. This concept emphasizes the holistic assessment of the coast and a multidisciplinary analysis using participatory processes. It integrates anthropocentric and eco-centric approaches. This paper documents several issues involved in the recovery of tsunami-affected areas and recommends the application of the ICZM concept to the reconstruction efforts. PMID:17544565

  2. Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

    PubMed

    Kellogg, G E; Scarsdale, J N; Fornari, F A

    1998-10-15

    The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents. PMID:9753742

  3. Identification and hydropathic characterization of structural features affecting sequence specificity for doxorubicin intercalation into DNA double-stranded polynucleotides.

    PubMed Central

    Kellogg, G E; Scarsdale, J N; Fornari, F A

    1998-01-01

    The computer molecular modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field function that includes hydrogen bonding, coulombic and hydrophobic terms, was used to study sequence-selective doxorubicin binding/intercalation in the 64 unique CAxy, CGxy, TAxy, TGxy base pair quartet combinations. The CAAT quartet sequence is shown to have the highest binding score of the 64 combinations. Of the two regularly alternating polynucleotides, d(CGCGCG)2and d(TATATA)2, the HINT calculated binding scores reveal doxorubicin binds preferentially to d(TATATA)2. Although interactions of the chromophore with the DNA base pairs defining the intercalation site [I-1] [I+1] and the neighboring [I+2] base pair are predominant, the results obtained with HINT indicate that the base pair [I+3] contributes significantly to the sequence selectivity of doxorubicin by providing an additional hydrogen bonding opportunity for the N3' ammonium of the daunosamine sugar moiety in approximately 25% of the sequences. This observation, that interactions involving a base pair [I+3] distal to the intercalation site play a significant role in stabilizing/destabilizing the intercalation of doxorubicin into the various DNA sequences, has not been previously reported. In general terms, this work shows that molecular modeling and careful analysis of molecular interactions can have a significant role in designing and evaluating nucleotides and antineoplastic agents. PMID:9753742

  4. DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro.

    PubMed Central

    Scarlato, V; Aricò, B; Rappuoli, R

    1993-01-01

    The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the PTOX promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription. Images PMID:8393006

  5. Diverse pathways to positive and negative affect in adulthood and later life: an integrative approach using recursive partitioning.