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Sample records for affect dna integrity

  1. 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

  2. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa

    PubMed Central

    Anbari, Fatemeh; Halvaei, Iman; Nabi, Ali; Ghazali, Shahin; Khalili, Mohammad Ali; Johansson, Lars

    2016-01-01

    AIM: The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. METHODS: A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. RESULTS: There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ (P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ (P = 0.02). CONCLUSIONS: The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs. PMID:28216914

  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. Retroviral DNA Integration

    PubMed Central

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  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. 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.

  7. Assessment of affect integration: validation of the affect consciousness construct.

    PubMed

    Solbakken, Ole André; Hansen, Roger Sandvik; Havik, Odd E; Monsen, Jon T

    2011-05-01

    Affect integration, or the capacity to utilize the motivational and signal properties of affect for personal adjustment, is assumed to be an important aspect of psychological health and functioning. Affect integration has been operationalized through the affect consciousness (AC) construct as degrees of awareness, tolerance, nonverbal expression, and conceptual expression of nine discrete affects. A semistructured Affect Consciousness Interview (ACI) and separate Affect Consciousness Scales (ACSs) have been developed to specifically assess these aspects of affect integration. This study explored the construct validity of AC in a Norwegian clinical sample including estimates of reliability and assessment of structure by factor analyses. External validity issues were addressed by examining the relationships between scores on the ACSs and self-rated symptom- and interpersonal problem measures as well as independent, observer-based ratings of personality disorder criteria and the Global Assessment of Functioning (GAF) scale from the Diagnostic and Statistical Manual of Mental Disorders (4th ed. [DSM-IV]; American Psychiatric Association, 1994).

  8. Integrated Sensing Using DNA Nanoarchitectures

    DTIC Science & Technology

    2014-05-20

    characterization of vibrational modes in artificially designed DNA monocrystal, Chemical Physics , (11 2013): 121. doi: 10.1016/j.chemphys.2013.08.015 Xiaoning...M. Rahman, M. L. Norton. Observation of terahertz absorption signatures in microliter DNA solutions, Applied Physics Letters, (01 2013): 23701...Modification of surface bound DNA nanoarchitectures with biomolecules and conjugates," ACS, Indianapolis, IN. (September 8, 2013). Rahman, M. (Author

  9. DNA Topoisomerase Iα Affects the Floral Transition1[OPEN

    PubMed Central

    Gong, Ximing; Shen, Lisha; Peng, Ya Zhi; Gan, Yinbo

    2017-01-01

    DNA topoisomerases modulate DNA topology to maintain chromosome superstructure and genome integrity, which is indispensable for DNA replication and RNA transcription. Their function in plant development still remains largely unknown. Here, we report a hitherto unidentified role of Topoisomerase Iα (TOP1α) in controlling flowering time in Arabidopsis (Arabidopsis thaliana). Loss of function of TOP1α results in early flowering under both long and short days. This is attributed mainly to a decrease in the expression of a central flowering repressor, FLOWERING LOCUS C (FLC), and its close homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, during the floral transition. TOP1α physically binds to the genomic regions of FLC, MAF4, and MAF5 and promotes the association of RNA polymerase II complexes to their transcriptional start sites. These correlate with the changes in histone modifications but do not directly affect nucleosome occupancy at these loci. Our results suggest that TOP1α mediates DNA topology to facilitate the recruitment of RNA polymerase II at FLC, MAF4, and MAF5 in conjunction with histone modifications, thus facilitating the expression of these key flowering repressors to prevent precocious flowering in Arabidopsis. PMID:27837087

  10. 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.

  11. 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

  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. Topological friction strongly affects viral DNA ejection

    PubMed Central

    Marenduzzo, Davide; Micheletti, Cristian; Orlandini, Enzo; Sumners, De Witt

    2013-01-01

    Bacteriophages initiate infection by releasing their double-stranded DNA into the cytosol of their bacterial host. However, what controls and sets the timescales of DNA ejection? Here we provide evidence from stochastic simulations which shows that the topology and organization of DNA packed inside the capsid plays a key role in determining these properties. Even with similar osmotic pressure pushing out the DNA, we find that spatially ordered DNA spools have a much lower effective friction than disordered entangled states. Such spools are only found when the tendency of nearby DNA strands to align locally is accounted for. This topological or conformational friction also depends on DNA knot type in the packing geometry and slows down or arrests the ejection of twist knots and very complex knots. We also find that the family of (2, 2k+1) torus knots unravel gradually by simplifying their topology in a stepwise fashion. Finally, an analysis of DNA trajectories inside the capsid shows that the knots formed throughout the ejection process mirror those found in gel electrophoresis experiments for viral DNA molecules extracted from the capsids. PMID:24272939

  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.

  15. Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment

    SciTech Connect

    Tsuchiya, Hiroyuki; Harashima, Hideyoshi; Kamiya, Hiroyuki . E-mail: hirokam@pharm.hokudai.ac.jp

    2005-11-04

    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment ({approx}600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

  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. 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

  18. The human specialized DNA polymerases and non-B DNA: vital relationships to preserve genome integrity.

    PubMed

    Boyer, Anne-Sophie; Grgurevic, Srdana; Cazaux, Christophe; Hoffmann, Jean-Sébastien

    2013-11-29

    In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.

  19. Developing Hierarchical Structures Integrating Cognition and Affect.

    ERIC Educational Resources Information Center

    Hurst, Barbara Martin

    Several categories of the affective domain are important to the schooling process. Schools are delegated the responsibility of helping students to clarify their esthetic, instrumental, and moral values. Three areas of affect are related to student achievement: subject-related affect, school-related affect, and academic self concept. In addition,…

  20. T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

    PubMed

    van Kregten, Maartje; de Pater, Sylvia; Romeijn, Ron; van Schendel, Robin; Hooykaas, Paul J J; Tijsterman, Marcel

    2016-10-31

    Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown(1). Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

  1. Thematic Teaching: Integrating Cognitive and Affective Outcomes in Elementary Classrooms.

    ERIC Educational Resources Information Center

    Brodeur, Doris R.

    1998-01-01

    Defines thematic teaching, also known as interdisciplinary or authentic instruction, as representing cross-disciplinary programs which integrate cognitive, affective, and psychomotor outcomes. Highlights include integrating thematic teaching into elementary school classrooms, cognitive and social learning theories, motivation, cooperative…

  2. Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

    PubMed Central

    Adams, A K; Holm, C

    1996-01-01

    To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. PMID:8756617

  3. RECQL4 LOCALIZES TO MITOCHONDRIA AND PRESERVES MITOCHONDRIAL DNA INTEGRITY

    PubMed Central

    Croteau, Deborah L.; Rossi, Marie L.; Canugovi, Chandrika; Tian, Jane; Sykora, Peter; Ramamoorthy, Mahesh; Wang, ZhengMing; Singh, Dharmendra Kumar; Akbari, Mansour; Kasiviswanathan, Rajesh; Copeland, William C.; Bohr, Vilhelm A.

    2012-01-01

    SUMMARY RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability and cancer predisposition. RECQL4 is a member of the RecQ-helicase family, and has many similarities to WRN protein, which is also implicated in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4’s helicase activity. RECQL4 is the first 3′ to 5′ RecQ helicase to be found in both human and mouse mitochondria and the loss of RECQL4 alters mitochondrial integrity. PMID:22296597

  4. DNA minicircles clarify the specific role of DNA structure on retroviral integration.

    PubMed

    Pasi, Marco; Mornico, Damien; Volant, Stevenn; Juchet, Anna; Batisse, Julien; Bouchier, Christiane; Parissi, Vincent; Ruff, Marc; Lavery, Richard; Lavigne, Marc

    2016-09-19

    Chromatin regulates the selectivity of retroviral integration into the genome of infected cells. At the nucleosome level, both histones and DNA structure are involved in this regulation. We propose a strategy that allows to specifically study a single factor: the DNA distortion induced by the nucleosome. This strategy relies on mimicking this distortion using DNA minicircles (MCs) having a fixed rotational orientation of DNA curvature, coupled with atomic-resolution modeling. Contrasting MCs with linear DNA fragments having identical sequences enabled us to analyze the impact of DNA distortion on the efficiency and selectivity of integration. We observed a global enhancement of HIV-1 integration in MCs and an enrichment of integration sites in the outward-facing DNA major grooves. Both of these changes are favored by LEDGF/p75, revealing a new, histone-independent role of this integration cofactor. PFV integration is also enhanced in MCs, but is not associated with a periodic redistribution of integration sites, thus highlighting its distinct catalytic properties. MCs help to separate the roles of target DNA structure, histone modifications and integrase (IN) cofactors during retroviral integration and to reveal IN-specific regulation mechanisms.

  5. Experimental factors affecting the robustness of DNA methylation analysis

    PubMed Central

    Pharo, Heidi D.; Honne, Hilde; Vedeld, Hege M.; Dahl, Christina; Andresen, Kim; Liestøl, Knut; Jeanmougin, Marine; Guldberg, Per; Lind, Guro E.

    2016-01-01

    Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data. PMID:27671843

  6. Intranuclear DNA density affects chromosome condensation in metazoans.

    PubMed

    Hara, Yuki; Iwabuchi, Mari; Ohsumi, Keita; Kimura, Akatsuki

    2013-08-01

    Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or "intranuclear DNA density." Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.

  7. Retroviral Integrase Proteins and HIV-1 DNA Integration*

    PubMed Central

    Krishnan, Lavanya; Engelman, Alan

    2012-01-01

    Retroviral integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions. PMID:23043109

  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.

  10. Preservation of DNA integrity and neuronal degeneration.

    PubMed

    Francisconi, Simona; Codenotti, Mara; Ferrari-Toninelli, Giulia; Uberti, Daniela; Memo, Maurizio

    2005-04-01

    The mismatch repair system (MMR) is an important member of the DNA checkpoint, that includes a number of protein deputed to control genomic stability through cell cycle arrest, DNA repair, and apoptosis. Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of MSH2, perhaps the most relevant component of the MMR system. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes.

  11. Genomic landscape of human, bat, and ex vivo DNA transposon integrations.

    PubMed

    Campos-Sánchez, Rebeca; Kapusta, Aurélie; Feschotte, Cédric; Chiaromonte, Francesca; Makova, Kateryna D

    2014-07-01

    The integration and fixation preferences of DNA transposons, one of the major classes of eukaryotic transposable elements, have never been evaluated comprehensively on a genome-wide scale. Here, we present a detailed study of the distribution of DNA transposons in the human and bat genomes. We studied three groups of DNA transposons that integrated at different evolutionary times: 1) ancient (>40 My) and currently inactive human elements, 2) younger (<40 My) bat elements, and 3) ex vivo integrations of piggyBat and Sleeping Beauty elements in HeLa cells. Although the distribution of ex vivo elements reflected integration preferences, the distribution of human and (to a lesser extent) bat elements was also affected by selection. We used regression techniques (linear, negative binomial, and logistic regression models with multiple predictors) applied to 20-kb and 1-Mb windows to investigate how the genomic landscape in the vicinity of DNA transposons contributes to their integration and fixation. Our models indicate that genomic landscape explains 16-79% of variability in DNA transposon genome-wide distribution. Importantly, we not only confirmed previously identified predictors (e.g., DNA conformation and recombination hotspots) but also identified several novel predictors (e.g., signatures of double-strand breaks and telomere hexamer). Ex vivo integrations showed a bias toward actively transcribed regions. Older DNA transposons were located in genomic regions scarce in most conserved elements-likely reflecting purifying selection. Our study highlights how DNA transposons are integral to the evolution of bat and human genomes, and has implications for the development of DNA transposon assays for gene therapy and mutagenesis applications.

  12. 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

  13. The Zeamine Antibiotics Affect the Integrity of Bacterial Membranes

    PubMed Central

    Masschelein, Joleen; Clauwers, Charlien; Stalmans, Karen; Nuyts, Koen; De Borggraeve, Wim; Briers, Yves; Aertsen, Abram; Michiels, Chris W.

    2014-01-01

    The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBDKZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of the OM rather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components. PMID:25452285

  14. Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host.

    PubMed Central

    Fleming, J G; Summers, M D

    1991-01-01

    The polydnavirus Campoletis sonorensis virus (CsV) is present in the oviducts of all adult C. sonorensis female wasps and appears to be required for these wasps to parasitize hosts successfully. Physical mapping, Southern blot analysis, and nucleotide sequence analysis demonstrate that the viral DNA B-specific sequences in cloned wasp DNA are colinear with viral genomic segment DNA B from nucleocapsids and are covalently linked to nonviral wasp sequences. Integrated DNA B terminates in 59-nucleotide imperfect direct repeats, but a single repeat exists in the extrachromosomal superhelical viral DNA B. Sequences near each junction form imperfect inverted repeats with sequences near the ends of an internal viral 540-base-pair repeat element gene. CsV appears to be the first documented integrated, nonretroviral DNA virus of insects and probably is vertically transmitted as a provirus. Images PMID:1946402

  15. 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

  16. Seasonal Liza aurata tissue-specific DNA integrity in a multi-contaminated coastal lagoon (Ria de Aveiro, Portugal).

    PubMed

    Oliveira, M; Maria, V L; Ahmad, I; Pacheco, M; Santos, M A

    2010-10-01

    In this study, the DNA integrity of golden grey mullet (Liza aurata) collected in differently contaminated sites of a coastal lagoon, Ria de Aveiro (Portugal), was assessed, over the period of 1 year, using the DNA alkaline unwinding assay, in four different tissues (gill, kidney, liver and blood) and compared to a reference site. The four tissues displayed different DNA integrity basal levels, clearly affected by seasonal factors. Gill and kidney were, respectively, the most and least sensitive tissues. All sites demonstrated the capacity to interfere with DNA integrity. The sites displaying the highest and lowest DNA damage capability were, respectively, Barra (subject to naval traffic) and Vagos (contaminated with polycyclic aromatic hydrocarbons). In terms of seasonal variability, autumn seems to be the more critical season (more DNA damage) unlike summer when no DNA damage was found in any tissue. Data recommend the continued monitoring of this aquatic system.

  17. DNA Ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair

    PubMed Central

    Gao, Yankun; Katyal, Sachin; Lee, Youngsoo; Zhao, Jingfeng; Rehg, Jerold E.; Russell, Helen R.; McKinnon, Peter J.

    2011-01-01

    DNA replication and repair in mammalian cells involves three distinct DNA ligases; ligase I (Lig1), ligase III (Lig3) and ligase IV (Lig4)1. Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway2,3. Lig3 is also present in the mitochondria where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc14. However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality5. Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss6, further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair. PMID:21390131

  18. 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

  19. On the question of the integration of exogenous bacterial DNA into plant DNA.

    PubMed Central

    Kleinhofs, A; Eden, F C; Chilton, M D; Bendich, A J

    1975-01-01

    Extensive studies with pea, tomato, and barley failed to confirm the evidence presented by previous investigators for integration or replication of exogenously applied bacterial DNA in these plants. Labeled DNA of buoyant density in CsCl intermediate between that of high density donor bacterial DNA and of plant DNA was never observed with axenic plants. Intermediate peaks, similar to those used as evidence for recombination by earlier investigators, were observed only when the plants were contaminated with bacteria. Plant DNA prepared by a published procedure [Ledoux, L. & Huart, R. (1969) J. Mol. Biol. 43, 243-262] was found to be contaminated with unidentified impurities. Such DNA was partially protected from the action of DNase and produced aberrant banding patterns in CsCl after shearing. Much of the published evidence for integration of foreign DNA in plants is based upon experiments with plant DNA prepared by this procedure. We conclude that contamination is the likely explanation for what has been interpreted as evidence for integration. PMID:809769

  20. 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

  1. 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…

  2. 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.

  3. Integrating DNA-based data into bioassessments improves ...

    EPA Pesticide Factsheets

    The integration of DNA-based identification methods into bioassessments could result in more accurate representations of species distributions and species-habitat relationships. DNA-based approaches may be particularly informative for tracking the distributions of rare and/or invasive species that can comprise a small proportion of samples or are difficult to identify morphologically. In 2012 and 2013, we used a combination of morphological and DNA-based methods (meta-barcoding) to identify fish eggs and larvae collected in the St. Louis River estuary area, Minnesota. We found a large proportion of cases where a lack of agreement occurred between species identified at a site using morphological versus DNA identification, prompting a discussion of how to best reconcile these differences. Choices made during sampling collection, DNA amplification/extraction, and bioinformatics processing influence the DNA-morphology match. The distribution of some species (including several invasives) and their relationships to habitat changed after DNA-data was incorporated. Results highlight how incorporating of DNA-data may get us closer to the “truth”, which has large ramifications in the search for rare species and when understanding the environmental drivers of species distributions is important for management. not applicable

  4. Affective state and community integration after traumatic brain injury.

    PubMed

    Juengst, Shannon B; Arenth, Patricia M; Raina, Ketki D; McCue, Michael; Skidmore, Elizabeth R

    2014-12-01

    Previous studies investigating the relationship between affective state and community integration have focused primarily on the influence of depression and anxiety. In addition, they have focused on frequency of participation in various activities, failing to address an individual's subjective satisfaction with participation. The purpose of this study was to examine how affective state contributes to frequency of participation and satisfaction with participation after traumatic brain injury among participants with and without a current major depressive episode. Sixty-four community-dwelling participants with a history of complicated mild-to-severe traumatic brain injury participated in this cross-sectional cohort study. High positive affect contributed significantly to frequency of participation (β = 0.401, P = 0.001), and both high positive affect and low negative affect significantly contributed to better satisfaction with participation (F2,61 = 13.63, P < 0.001). Further investigation to assess the direction of these relationships may better inform effective targets for intervention. These findings highlight the importance of assessing affective state after traumatic brain injury and incorporating a subjective measure of participation when considering community integration outcomes.

  5. An integrated web medicinal materials DNA database: MMDBD (Medicinal Materials DNA Barcode Database)

    PubMed Central

    2010-01-01

    Background Thousands of plants and animals possess pharmacological properties and there is an increased interest in using these materials for therapy and health maintenance. Efficacies of the application is critically dependent on the use of genuine materials. For time to time, life-threatening poisoning is found because toxic adulterant or substitute is administered. DNA barcoding provides a definitive means of authentication and for conducting molecular systematics studies. Owing to the reduced cost in DNA authentication, the volume of the DNA barcodes produced for medicinal materials is on the rise and necessitates the development of an integrated DNA database. Description We have developed an integrated DNA barcode multimedia information platform- Medicinal Materials DNA Barcode Database (MMDBD) for data retrieval and similarity search. MMDBD contains over 1000 species of medicinal materials listed in the Chinese Pharmacopoeia and American Herbal Pharmacopoeia. MMDBD also contains useful information of the medicinal material, including resources, adulterant information, medical parts, photographs, primers used for obtaining the barcodes and key references. MMDBD can be accessed at http://www.cuhk.edu.hk/icm/mmdbd.htm. Conclusions This work provides a centralized medicinal materials DNA barcode database and bioinformatics tools for data storage, analysis and exchange for promoting the identification of medicinal materials. MMDBD has the largest collection of DNA barcodes of medicinal materials and is a useful resource for researchers in conservation, systematic study, forensic and herbal industry. PMID:20576098

  6. Integration of amplified differential gene expression (ADGE) and DNA microarray.

    PubMed

    Chen, Zhijian J; Gaté, Laurent; Davis, Warren; Ile, Kristina E; Tew, Kenneth D

    2002-12-01

    Amplified Differential Gene Expression (ADGE) provides a new concept that the ratios of differentially expressed genes are magnified before detection in order to improve both sensitivity and accuracy. This technology is now implemented with integration of DNA reassociation and PCR. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. These two techniques are mutually synergistic: the quadratic magnification of ratios of differential gene expression achieved by ADGE improves the detection sensitivity and accuracy; the PCR amplification of templates enhances the signal intensity and reduces the requirement for large amounts of starting material; the high throughput for DNA microarray is maintained.

  7. 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…

  8. Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response.

    PubMed

    Stephenson, Anthony A; Taggart, David J; Suo, Zucai

    2017-04-13

    Specialized DNA polymerases, such as DNA polymerase lambda (Polλ), are important players in DNA damage tolerance and repair pathways. Knowing how DNA polymerases are regulated and recruited to sites of DNA damage is imperative to understanding these pathways. Recent work has suggested that Polλ plays a role in several distinct DNA damage tolerance and repair pathways. In this paper, we report previously unknown roles of the N-terminal domains of human Polλ for modulating its involvement in DNA damage tolerance and repair. By using Western blot analysis, fluorescence microscopy, and cell survival assays, we found that the BRCA1 C-terminal (BRCT) and proline/serine-rich (PSR) domains of Polλ affect its cellular localization and DNA damage responses. The nuclear localization signal (NLS) of Polλ was necessary to overcome the impediment of its nuclear localization caused by its BRCT and PSR domains. Induction of DNA damage resulted in recruitment of Polλ to chromatin, which was controlled by its BRCT and PSR domains. In addition, the presence of both domains was required for Polλ-mediated tolerance of oxidative DNA damage but not DNA methylation damage. These findings suggest that the N-terminal domains of Polλ are important for regulating its responses to DNA damage.

  9. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair.

    PubMed

    Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-01

    Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80-95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.

  10. Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2016-11-24

    The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

  11. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse

    PubMed Central

    Krzeminski, Patryk; Corchete, Luis A.; García, Juan L.; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M.; Martín, Ana A.; Hernández-Rivas, Jesús M.; García-Sanz, Ramón; Miguel, Jesús F. San; Gutiérrez, Norma C.

    2016-01-01

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse. PMID:27811368

  12. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

    PubMed

    Krzeminski, Patryk; Corchete, Luis A; García, Juan L; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M; Martín, Ana A; Hernández-Rivas, Jesús M; García-Sanz, Ramón; San Miguel, Jesús F; Gutiérrez, Norma C

    2016-12-06

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

  13. T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites

    PubMed Central

    Brunaud, Véronique; Balzergue, Sandrine; Dubreucq, Bertrand; Aubourg, Sébastien; Samson, Franck; Chauvin, Stéphanie; Bechtold, Nicole; Cruaud, Corinne; DeRose, Richard; Pelletier, Georges; Lepiniec, Loïc; Caboche, Michel; Lecharny, Alain

    2002-01-01

    A statistical analysis of 9000 flanking sequence tags characterizing transferred DNA (T-DNA) transformants in Arabidopsis sheds new light on T-DNA insertion by illegitimate recombination. T-DNA integration is favoured in plant DNA regions with an A-T-rich content. The formation of a short DNA duplex between the host DNA and the left end of the T-DNA sets the frame for the recombination. The sequence immediately downstream of the plant A-T-rich region is the master element for setting up the DNA duplex, and deletions into the left end of the integrated T-DNA depend on the location of a complementary sequence on the T-DNA. Recombination at the right end of the T-DNA with the host DNA involves another DNA duplex, 2–3 base pairs long, that preferentially includes a G close to the right end of the T-DNA. PMID:12446565

  14. META2: Intercellular DNA Methylation Pairwise Annotation and Integrative Analysis

    PubMed Central

    2016-01-01

    Genome-wide deciphering intercellular differential DNA methylation as well as its roles in transcriptional regulation remains elusive in cancer epigenetics. Here we developed a toolkit META2 for DNA methylation annotation and analysis, which aims to perform integrative analysis on differentially methylated loci and regions through deep mining and statistical comparison methods. META2 contains multiple versatile functions for investigating and annotating DNA methylation profiles. Benchmarked with T-47D cell, we interrogated the association within differentially methylated CpG (DMC) and region (DMR) candidate count and region length and identified major transition zones as clues for inferring statistically significant DMRs; together we validated those DMRs with the functional annotation. Thus META2 can provide a comprehensive analysis approach for epigenetic research and clinical study. PMID:28116291

  15. 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.

  16. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing.

    PubMed

    Blazej, Robert G; Kumaresan, Palani; Mathies, Richard A

    2006-05-09

    An efficient, nanoliter-scale microfabricated bioprocessor integrating all three Sanger sequencing steps, thermal cycling, sample purification, and capillary electrophoresis, has been developed and evaluated. Hybrid glass-polydimethylsiloxane (PDMS) wafer-scale construction is used to combine 250-nl reactors, affinity-capture purification chambers, high-performance capillary electrophoresis channels, and pneumatic valves and pumps onto a single microfabricated device. Lab-on-a-chip-level integration enables complete Sanger sequencing from only 1 fmol of DNA template. Up to 556 continuous bases were sequenced with 99% accuracy, demonstrating read lengths required for de novo sequencing of human and other complex genomes. The performance of this miniaturized DNA sequencer provides a benchmark for predicting the ultimate cost and efficiency limits of Sanger sequencing.

  17. The FACT complex promotes avian leukosis virus DNA integration.

    PubMed

    Winans, Shelby; Larue, Ross C; Abraham, Carly M; Shkriabai, Nikolozi; Skopp, Amelie; Winkler, Duane; Kvaratskhelia, Mamuka; Beemon, Karen L

    2017-01-25

    All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. Here, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-LTR circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.

  18. 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.

  19. 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.

  20. 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

  1. Affect integration as a predictor of change: affect consciousness and treatment response in open-ended psychotherapy.

    PubMed

    Solbakken, Ole André; Hansen, Roger Sandvik; Havik, Odd E; Monsen, Jon Trygve

    2012-01-01

    The present study investigated the relationship between baseline levels of affect integration and the magnitude of change during and after open-ended psychotherapy. Affect integration reflects the capacity for accessing and utilizing the adaptive properties of affects for personal adjustment, along with the more general capability of tolerating and regulating affective activation. It is thus a capacity with relevance for the postulated mechanisms of change in various treatment modalities. Overall, the results indicated that patients with more severe problems in affect integration had larger improvements in symptoms, interpersonal and personality problems in open-ended treatment than those with less severe problems. This was also the case when examining the predictive effects of the integration of specific affects on changes in interpersonal relatedness. It was indicated that increasing problems with the integration of discrete affects were associated with distinct patterns of change in different interpersonal problem domains.

  2. A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

    PubMed Central

    Gallagher, Renata C.; Blackburn, Elizabeth H.

    1998-01-01

    In the ciliated protozoan Tetrahymena thermophila the ribosomal DNA (rDNA) minichromosome replicates partially under cell cycle control and is also subject to a copy number control mechanism. The relationship between rDNA replication and rRNA gene transcription was investigated by the analysis of replication, transcription, and DNA-protein interactions in a mutant rDNA, the rmm3 rDNA. The rmm3 (for rDNA maturation or maintenance mutant 3) rDNA contains a single-base deletion in the rRNA promoter region, in a phylogenetically conserved sequence element that is repeated in the replication origin region of the rDNA minichromosome. The multicopy rmm3 rDNA minichromosome has a maintenance defect in the presence of a competing rDNA allele in heterozygous cells. No difference in the level of rRNA transcription was found between wild-type and rmm3 strains. However, rmm3 rDNA replicating intermediates exhibited an enhanced pause in the region of the replication origin, roughly 750 bp upstream from the rmm3 mutation. In footprinting of isolated nuclei, the rmm3 rDNA lacked the wild-type dimethyl sulfate (DMS) footprint in the promoter region adjacent to the base change. In addition, a DMS footprint in the origin region was lost in the rmm3 rDNA minichromosome. This is the first reported correlation in this system between an rDNA minichromosome maintenance defect and an altered footprint in the origin region. Our results suggest that a promoter region mutation can affect replication without detectably affecting transcription. We propose a model in which interactions between promoter and origin region complexes facilitate replication and maintenance of the Tetrahymena rDNA minichromosome. PMID:9566921

  3. Retroviruses integrate into a shared, non-palindromic DNA motif.

    PubMed

    Kirk, Paul D W; Huvet, Maxime; Melamed, Anat; Maertens, Goedele N; Bangham, Charles R M

    2016-11-14

    Many DNA-binding factors, such as transcription factors, form oligomeric complexes with structural symmetry that bind to palindromic DNA sequences(1). Palindromic consensus nucleotide sequences are also found at the genomic integration sites of retroviruses(2-6) and other transposable elements(7-9), and it has been suggested that this palindromic consensus arises as a consequence of the structural symmetry in the integrase complex(2,3). However, we show here that the palindromic consensus sequence is not present in individual integration sites of human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1), but arises in the population average as a consequence of the existence of a non-palindromic nucleotide motif that occurs in approximately equal proportions on the plus strand and the minus strand of the host genome. We develop a generally applicable algorithm to sort the individual integration site sequences into plus-strand and minus-strand subpopulations, and use this to identify the integration site nucleotide motifs of five retroviruses of different genera: HTLV-1, HIV-1, murine leukaemia virus (MLV), avian sarcoma leucosis virus (ASLV) and prototype foamy virus (PFV). The results reveal a non-palindromic motif that is shared between these retroviruses.

  4. 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

  5. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-strand break repair model for T-DNA integration.

    PubMed

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-02-28

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-strand break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-strand break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosome 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. This article is protected by copyright. All rights reserved.

  6. Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.

    PubMed

    Chen, Huang; Zhu, Zaifang; Lu, Joann Juan; Liu, Shaorong

    2015-02-03

    Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary-hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

  7. Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

    PubMed

    Fleischhacker, Michael; Schmidt, Bernd; Weickmann, Sabine; Fersching, Debora M I; Leszinski, Gloria S; Siegele, Barbara; Stötzer, Oliver J; Nagel, Dorothea; Holdenrieder, Stefan

    2011-11-20

    Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.

  8. Exploration mode affects visuohaptic integration of surface orientation.

    PubMed

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

    2014-11-20

    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.

  9. 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.

  10. 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

  11. How hormone receptor-DNA binding affects nucleosomal DNA: the role of symmetry.

    PubMed Central

    Bishop, T C; Kosztin, D; Schulten, K

    1997-01-01

    Molecular dynamics simulations have been employed to determine the optimal conformation of an estrogen receptor DNA binding domain dimer bound to a consensus response element, ds(AGGTCACAGTGACCT), and to a nonconsensus response element, ds(AGAACACAGTGACCT). The structures simulated were derived from a crystallographic structure and solvated by a sphere (45-A radius) of explicit water and counterions. Long-range electrostatic interactions were accounted for during 100-ps simulations by means of a fast multipole expansion algorithm combined with a multiple time-step scheme in the molecular dynamics package NAMD. The simulations demonstrate that the dimer induces a bent and underwound (10.7 bp/turn) conformation in the DNA. The bending reflects the dyad symmetry of the receptor dimer and can be described as an S-shaped curve in the helical axis of DNA when projected onto a plane. A similar bent and underwound conformation is observed for nucleosomal DNA near the nucleosome's dyad axis that reflects the symmetry of the histone octamer. We propose that when a receptor dimer binds to a nucleosome, the most favorable dimer-DNA and histone-DNA interactions are achieved if the respective symmetry axes are aligned. Such positioning of a receptor dimer over the dyad of nucleosome B in the mouse mammary tumor virus promoter is in agreement with experiment. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 9 FIGURE 11 PMID:9129808

  12. 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

  13. AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS(1).

    PubMed

    Kato, Syou; Misawa, Kazuharu; Takahashi, Fumio; Sakayama, Hidetoshi; Sano, Satomi; Kosuge, Keiko; Kasai, Fumie; Watanabe, Makoto M; Tanaka, Jiro; Nozaki, Hisayoshi

    2011-10-01

    Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat-based dimorphism of chloroplast DNA (cpDNA). Here, we examined the "shallow" and "deep" subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima's D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both "shallow" and "deep" haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat-based dimorphism of cpDNA from field-collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes.

  14. Factors affecting rural volunteering in palliative care - an integrated review.

    PubMed

    Whittall, Dawn; Lee, Susan; O'Connor, Margaret

    2016-12-01

    To review factors shaping volunteering in palliative care in Australian rural communities using Australian and International literature. Identify gaps in the palliative care literature and make recommendations for future research. A comprehensive literature search was conducted using Proquest, Scopus, Sage Premier, Wiley online, Ovid, Cochran, Google Scholar, CINAHL and Informit Health Collection. The literature was synthesised and presented in an integrated thematic narrative. Australian Rural communities. While Australia, Canada, the United States (US) and the United Kingdom (UK) are leaders in palliative care volunteer research, limited research specifically focuses on volunteers in rural communities with the least occurring in Australia. Several interrelated factors influence rural palliative care provision, in particular an increasingly ageing population which includes an ageing volunteer and health professional workforce. Also current and models of palliative care practice fail to recognise the innumerable variables between and within rural communities such as distance, isolation, lack of privacy, limited health care services and infrastructure, and workforce shortages. These issues impact palliative care provision and are significant for health professionals, volunteers, patients and caregivers. The three key themes of this integrated review include: (i) Geography, ageing rural populations in palliative care practice, (ii) Psychosocial impact of end-end-of life care in rural communities and (iii) Palliative care models of practice and volunteering in rural communities. The invisibility of volunteers in rural palliative care research is a concern in understanding the issues affecting the sustainability of quality palliative care provision in rural communities. Recommendations for future Australian research includes examination of the suitability of current models of palliative care practice in addressing the needs of rural communities; the recruitment

  15. 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

  16. 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

  17. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.

    PubMed

    Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

    2016-09-09

    Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

  18. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    PubMed Central

    Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

    2016-01-01

    Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA. DOI: http://dx.doi.org/10.7554/eLife.18574.001 PMID:27612385

  19. Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions.

    PubMed

    Schneeberger, Richard G; Zhang, Ke; Tatarinova, Tatiana; Troukhan, Max; Kwok, Shing F; Drais, Josh; Klinger, Kevin; Orejudos, Francis; Macy, Kimberly; Bhakta, Amit; Burns, James; Subramanian, Gopal; Donson, Jonathan; Flavell, Richard; Feldmann, Kenneth A

    2005-10-01

    Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5' and 3' flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5' upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.

  20. DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

    PubMed Central

    Glover, T W; Coyle-Morris, J; Pearce-Birge, L; Berger, C; Gemmill, R M

    1986-01-01

    Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression. Images Fig. 1 PMID:2420174

  1. TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells

    PubMed Central

    Altilia, Serena; Santoro, Aurelia; Malagoli, Davide; Lanzarini, Catia; Álvarez, Josué Adolfo Ballesteros; Galazzo, Gianluca; Porter, Donald Carl; Crocco, Paolina; Rose, Giuseppina; Passarino, Giuseppe; Roninson, Igor Boris; Franceschi, Claudio; Salvioli, Stefano

    2012-01-01

    Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations. PMID:22289634

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Impact of radio frequency electromagnetic radiation on DNA integrity in the male germline.

    PubMed

    Aitken, R J; Bennetts, L E; Sawyer, D; Wiklendt, A M; King, B V

    2005-06-01

    Concern has arisen over human exposures to radio frequency electromagnetic radiation (RFEMR), including a recent report indicating that regular mobile phone use can negatively impact upon human semen quality. These effects would be particularly serious if the biological effects of RFEMR included the induction of DNA damage in male germ cells. In this study, mice were exposed to 900 MHz RFEMR at a specific absorption rate of approximately 90 mW/kg inside a waveguide for 7 days at 12 h per day. Following exposure, DNA damage to caudal epididymal spermatozoa was assessed by quantitative PCR (QPCR) as well as alkaline and pulsed-field gel electrophoresis. The treated mice were overtly normal and all assessment criteria, including sperm number, morphology and vitality were not significantly affected. Gel electrophoresis revealed no gross evidence of increased single- or double-DNA strand breakage in spermatozoa taken from treated animals. However, a detailed analysis of DNA integrity using QPCR revealed statistically significant damage to both the mitochondrial genome (p < 0.05) and the nuclear beta-globin locus (p < 0.01). This study suggests that while RFEMR does not have a dramatic impact on male germ cell development, a significant genotoxic effect on epididymal spermatozoa is evident and deserves further investigation.

  9. 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.

  10. 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.

  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. 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...

  13. The circumplex model of affect: An integrative approach to affective neuroscience, cognitive development, and psychopathology

    PubMed Central

    Posner, Jonathan; Russell, James A.; Peterson, Bradley S.

    2008-01-01

    The circumplex model of affect proposes that all affective states arise from cognitive interpretations of core neural sensations that are the product of two independent neurophysiological systems. This model stands in contrast to theories of basic emotions, which posit that a discrete and independent neural system subserves every emotion. We propose that basic emotion theories no longer explain adequately the vast number of empirical observations from studies in affective neuroscience, and we suggest that a conceptual shift is needed in the empirical approaches taken to the study of emotion and affective psychopathologies. The circumplex model of affect is more consistent with many recent findings from behavioral, cognitive neuroscience, neuroimaging, and developmental studies of affect. Moreover, the model offers new theoretical and empirical approaches to studying the development of affective disorders as well as the genetic and cognitive underpinnings of affective processing within the central nervous system. PMID:16262989

  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. Post-translational modifications of proliferating cell nuclear antigen: A key signal integrator for DNA damage response (Review).

    PubMed

    Zhu, Qiong; Chang, Yuxiao; Yang, Jin; Wei, Quanfang

    2014-05-01

    Previous studies have shown that the post-translational modifications of proliferating cell nuclear antigen (PCNA) may be crucial in influencing the cellular choice between different pathways, such as the cell cycle checkpoint, DNA repair or apoptosis pathways, in order to maintain genomic stability. DNA damage leads to replication stress and the subsequent induction of PCNA modification by small ubiquitin (Ub)-related modifiers and Ub, which has been identified to affect multiple biological processes of genomic DNA. Thus far, much has been learned concerning the behavior of modified PCNA as a key signal integrator in response to DNA damage. In humans and yeast, modified PCNA activates DNA damage bypass via an error-prone or error-free pathway to prevent the breakage of DNA replication forks, which may potentially induce double-strand breaks and subsequent chromosomal rearrangements. However, the exact mechanisms by which these pathways work and by what means the modified PCNA is involved in these processes remain elusive. Thus, the improved understanding of PCNA modification and its implications for DNA damage response may provide us with more insight into the mechanisms by which human cells regulate aberrant recombination events, and cancer initiation and development. The present review focuses on the post-translational modifications of PCNA and its important functions in mediating mammalian cellular response to different types of DNA damage.

  16. Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index

    PubMed Central

    Bedin, Chiara; Romualdi, Chiara; Mainenti, Marta; Mollo, Antonio; Cavicchioli, Laura; Ferro, Silvia; Trez, Davide; De Maria, Raffaella; Nitti, Donato; Saccani, Andrea; Campanella, Michelangelo; Agostini, Marco; Zappulli, Valentina

    2017-01-01

    Circulating cell-free DNA (cfDNA) has been considered an interesting diagnostic/prognostic plasma biomarker in tumor-bearing subjects. In cancer patients, cfDNA can hypothetically derive from tumor necrosis/apoptosis, lysed circulating cells, and some yet unrevealed mechanisms of active release. This study aimed to preliminarily analyze cfDNA in dogs with canine mammary tumors (CMTs). Forty-four neoplastic, 17 non-neoplastic disease-bearing, and 15 healthy dogs were recruited. Necrosis and apoptosis were also assessed as potential source of cfDNA on 78 CMTs diagnosed from the 44 dogs. The cfDNA fragments and integrity index significantly differentiated neoplastic versus non-neoplastic dogs (P<0.05), and allowed the distinction between benign and malignant lesions (P<0.05). Even if without statistical significance, the amount of cfDNA was also affected by tumor necrosis and correlated with tumor size and apoptotic markers expression. A significant (P<0.01) increase of Bcl-2 in malignant tumors was observed, and in metastatic CMTs the evasion of apoptosis was also suggested. This study, therefore, provides evidence that cfDNA could be a diagnostic marker in dogs carrying mammary nodules suggesting that its potential application in early diagnostic procedures should be further investigated. PMID:28081183

  17. Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9.

    PubMed

    Li, Hao; Sheng, Chunyu; Wang, Shan; Yang, Lang; Liang, Yuan; Huang, Yong; Liu, Hongbo; Li, Peng; Yang, Chaojie; Yang, Xiaoxia; Jia, Leili; Xie, Jing; Wang, Ligui; Hao, Rongzhang; Du, Xinying; Xu, Dongping; Zhou, Jianjun; Li, Mingzhen; Sun, Yansong; Tong, Yigang; Li, Qiao; Qiu, Shaofu; Song, Hongbin

    2017-01-01

    The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or "sterile" HBV cure.

  18. Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9

    PubMed Central

    Li, Hao; Sheng, Chunyu; Wang, Shan; Yang, Lang; Liang, Yuan; Huang, Yong; Liu, Hongbo; Li, Peng; Yang, Chaojie; Yang, Xiaoxia; Jia, Leili; Xie, Jing; Wang, Ligui; Hao, Rongzhang; Du, Xinying; Xu, Dongping; Zhou, Jianjun; Li, Mingzhen; Sun, Yansong; Tong, Yigang; Li, Qiao; Qiu, Shaofu; Song, Hongbin

    2017-01-01

    The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or “sterile” HBV cure. PMID:28382278

  19. Effects of freezing-thawing on DNA integrity of boar spermatozoa assessed by the neutral comet assay.

    PubMed

    Fraser, L; Strzezek, J

    2005-12-01

    A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.

  20. 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

  1. 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

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

    PubMed

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

    2015-06-11

    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.

  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. 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.

  5. 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.

  6. Integrating the Affective Domain into the Instructional Design Process

    DTIC Science & Technology

    1992-03-01

    domains. Yet, instructional design models and practices have focused primarily on the acquisition of knowledge and psychomotor skills . Concern for the...the skills involved in the reactive and interactive domains are as amenable to the general principles of instruction as are cognitive and psychomotor ... skills . He also sees a parallel between the automation of affective domain skills (reflexive, conditioned activity versus behavior resulting from a

  7. 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

  8. DNA affects the composition of lipoplex protein corona: a proteomics approach.

    PubMed

    Capriotti, Anna L; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-08-01

    The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.

  9. The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

    PubMed

    Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

    2013-11-01

    We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

  10. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

    SciTech Connect

    Sams, C.F.; Matthews, K.S.

    1988-04-05

    Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

  11. 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.

  12. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    PubMed

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  13. 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.

  14. 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

  15. 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.

  16. Effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males with asthenozoospermia.

    PubMed

    Hussein, T M; Elariny, A F; Elabd, M M; Elgarem, Y F; Elsawy, M M

    2008-10-01

    The aim of this work was to study the possible beneficial effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males and its possible implementation in assisted reproduction. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia. They underwent detailed history taking, complete clinical assessment and hormonal assessment. Patients were asked to bring two semen samples (taken within 1-3 h). Two consecutive samples were assessed with regard to semen volume, sperm count, motility grading, and morphology and sperm DNA integrity using the comet assay. There was a significant improvement in the sperm motility pattern and DNA integrity in the second sample in comparison with the first sample. Therefore, it is concluded that due to its positive impact on sperm motility and DNA integrity, repeated sequential ejaculation is recommended in subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Mutagens manufactured in fungal culture may affect DNA/RNA of producing fungi.

    PubMed

    Paterson, R R M; Lima, N

    2009-04-01

    Self-produced mutagens in culture by fungi may affect DNA analysis of the same fungi. This has not been considered previously. Many fungi produce numerous mutagenic secondary metabolites (SM) in culture. There is a paradox of growing fungi in media to produce representative DNA which also support mutagenic SM. This is a crucial issue in developing diagnostic and phylogenetic methods, especially for closely-related fungi. For example, idh gene analysis of the patulin metabolic pathway in fungi can be interpreted as producing some false negative and positive results in terms of possession, or nonpossession, of the gene from mutated strains. The most obvious mycotoxins and fungi to consider in this regard are aflatoxins and Aspergillus, as aflatoxins are the most mutagenic natural compounds. Many other fungi and SM are relevant. Conditions to grow fungi have not been selected to inhibit SM production although relevant data exist. In fact, fungi repair damaged nucleic acid (NA) and are capable of removing toxins by employing transporter proteins. These and NA repair mechanisms could be inhibited by secondary metabolites. Mutagenic effects may involve inhibition of DNA stabilizing enzymes. There may be an equivalent situation for bacteria. Researchers need to devise methods to reduce SM for valid protocols. More work on how mutagens affect the NA of producing fungus in vitro is required. The current review assesses the potential seriousness of the situation with selected papers.

  2. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.

    PubMed

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael; Rothstein, Rodney; Zhao, Xiaolan

    2007-09-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.

  3. 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.

  4. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus.

    PubMed Central

    Silver, J; Keerikatte, V

    1989-01-01

    We describe a modification of the polymerase chain reaction technique which allows amplification of cellular DNA adjacent to an integrated provirus given sequence information for the provirus only. The modified technique should be generally useful for studies of insertional mutagenesis and other situations in which one wishes to isolate DNA adjacent to a region of known sequence. Images PMID:2704070

  5. Integrative modelling of tumour DNA methylation quantifies the contribution of metabolism

    PubMed Central

    Mehrmohamadi, Mahya; Mentch, Lucas K.; Clark, Andrew G.; Locasale, Jason W.

    2016-01-01

    Altered DNA methylation is common in cancer and often considered an early event in tumorigenesis. However, the sources of heterogeneity of DNA methylation among tumours remain poorly defined. Here we capitalize on the availability of multi-platform data on thousands of human tumours to build integrative models of DNA methylation. We quantify the contribution of clinical and molecular factors in explaining intertumoral variability in DNA methylation. We show that the levels of a set of metabolic genes involved in the methionine cycle is predictive of several features of DNA methylation in tumours, including the methylation of cancer genes. Finally, we demonstrate that patients whose DNA methylation can be predicted from the methionine cycle exhibited improved survival over cases where this regulation is disrupted. This study represents a comprehensive analysis of the determinants of methylation and demonstrates the surprisingly large interaction between metabolism and DNA methylation variation. Together, our results quantify links between tumour metabolism and epigenetics and outline clinical implications. PMID:27966532

  6. Chromosomal Bands Affected by Acute Oil Exposure and DNA Repair Errors

    PubMed Central

    Zock, Jan-Paul; Giraldo, Jesús; Pozo-Rodríguez, Francisco; Espinosa, Ana; Rodríguez-Trigo, Gema; Verea, Hector; Castaño-Vinyals, Gemma; Gómez, Federico P.; Antó, Josep M.; Coll, Maria Dolors; Barberà, Joan Albert; Fuster, Carme

    2013-01-01

    Background In a previous study, we showed that individuals who had participated in oil clean-up tasks after the wreckage of the Prestige presented an increase of structural chromosomal alterations two years after the acute exposure had occurred. Other studies have also reported the presence of DNA damage during acute oil exposure, but little is known about the long term persistence of chromosomal alterations, which can be considered as a marker of cancer risk. Objectives We analyzed whether the breakpoints involved in chromosomal damage can help to assess the risk of cancer as well as to investigate their possible association with DNA repair efficiency. Methods Cytogenetic analyses were carried out on the same individuals of our previous study and DNA repair errors were assessed in cultures with aphidicolin. Results Three chromosomal bands, 2q21, 3q27 and 5q31, were most affected by acute oil exposure. The dysfunction in DNA repair mechanisms, expressed as chromosomal damage, was significantly higher in exposed-oil participants than in those not exposed (p= 0.016). Conclusion The present study shows that breaks in 2q21, 3q27 and 5q31 chromosomal bands, which are commonly involved in hematological cancer, could be considered useful genotoxic oil biomarkers. Moreover, breakages in these bands could induce chromosomal instability, which can explain the increased risk of cancer (leukemia and lymphomas) reported in chronically benzene-exposed individuals. In addition, it has been determined that the individuals who participated in clean-up of the oil spill presented an alteration of their DNA repair mechanisms two years after exposure. PMID:24303039

  7. Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.

    PubMed

    Maettner, R; Sterzik, K; Isachenko, V; Strehler, E; Rahimi, G; Alabart, J L; Sánchez, R; Mallmann, P; Isachenko, E

    2014-06-01

    The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)-selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI-morphological classes (Class 1 versus Class 3, respectively) (P > 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI-selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei.

  8. 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

  9. Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration.

    PubMed

    Yin, Zhiqi; Lapkouski, Mikalai; Yang, Wei; Craigie, Robert

    2012-12-01

    Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV-1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV-1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV-1 DNA integration.

  10. 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)

  11. 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.

  12. 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

  13. Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis.

    PubMed

    Bahga, Supreet S; Han, Crystal M; Santiago, Juan G

    2013-01-07

    We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe. We also demonstrate the feasibility of our assay for multiplexed detection of multiple-length ssDNA targets by simultaneously detecting 39 and 90 nt ssDNA targets.

  14. Time-dependent RNA degradation affecting cDNA array quality in spontaneous canine tumours sampled using standard surgical procedures.

    PubMed

    Von Euler, Henrik; Khoshnoud, Reza; He, Qimin; Khoshnoud, Aida; Fornander, Tommy; Rutqvist, Lars-Erik; Skog, Sven

    2005-12-01

    Heterogeneous gene expression in tumours and the degradation of RNA when sampling under non-RNAse-free conditions may limit the potential benefit of cDNA array studies. This study examines changes in the integrity of RNA by means of RNA gel electrophoresis at various post-operative intervals on canine mammary tumours (n=10) and malignant lymphoma (n=1). The tumours were cut into pieces (3-5 mm diameter, approximately 50 mg) and kept in tubes without RNAse-free buffer at room temperature. No special precautions were taken to avoid the influences of Rnase; rather, normal surgical procedures were used. We found that total RNA of the mammary tumours started to degrade within 30 min of the operation, and the rate of degradation increased up to 4 h, which was the last time point included in this study. RNA in the lymphoma tumours degraded more rapidly, and was completely degraded at 30 min post-operation. The degradation of mRNA in the mammary tumours, as studied by human cDNA arrays, was heterogeneous, i.e. some mRNA degraded completely, some only partially. This indicates that the mRNA degradation rate varied depending on the type of mRNA. However, since we found that gene expression differs depending on the part of the mammary tumour examined, one cannot exclude that the variation in the mRNA degradation rate may simply reflect heterogeneous gene expression within the tumour. We conclude that RNA integrity is unaffected immediately after sampling under non-RNAse-free conditions; however, the tumour sample should be preserved under RNAse-free conditions within 15 min to avoid RNA degradation. This is a much shorter time interval than previously reported in other similar studies; however, these studies generally treated normal tissue, under which 3-5 h non-RNAse-free conditions have been found not to affect RNA quality.

  15. 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

  16. 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

  17. 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

  18. TET1 modulates H4K16 acetylation by controlling auto-acetylation of hMOF to affect gene regulation and DNA repair function

    PubMed Central

    Zhong, Jianing; Li, Xianfeng; Cai, Wanshi; Wang, Yan; Dong, Shanshan; Yang, Jie; Zhang, Jian'an; Wu, Nana; Li, Yuanyuan; Mao, Fengbiao; Zeng, Cheng; Wu, Jinyu; Xu, Xingzhi; Sun, Zhong Sheng

    2017-01-01

    The Ten Eleven Translocation 1 (TET1) protein is a DNA demethylase that regulates gene expression through altering statue of DNA methylation. However, recent studies have demonstrated that TET1 could modulate transcriptional expression independent of its DNA demethylation activity; yet, the detailed mechanisms underlying TET1's role in such transcriptional regulation remain not well understood. Here, we uncovered that Tet1 formed a chromatin complex with histone acetyltransferase Mof and scaffold protein Sin3a in mouse embryonic stem cells by integrative genomic analysis using publicly available ChIP-seq data sets and a series of in vitro biochemical studies in human cell lines. Mechanistically, the TET1 facilitated chromatin affinity and enzymatic activity of hMOF against acetylation of histone H4 at lysine 16 via preventing auto-acetylation of hMOF, to regulate expression of the downstream genes, including DNA repair genes. We found that Tet1 knockout MEF cells exhibited an accumulation of DNA damage and genomic instability and Tet1 deficient mice were more sensitive to x-ray exposure. Taken together, our findings reveal that TET1 forms a complex with hMOF to modulate its function and the level of H4K16Ac ultimately affect gene expression and DNA repair. PMID:27733505

  19. 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.

  20. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

    PubMed Central

    Kienhöfer, Joachim; Häussler, Dagmar Johanna Franziska; Ruckelshausen, Florian; Muessig, Elisabeth; Weber, Klaus; Pimentel, David; Ullrich, Volker; Bürkle, Alexander; Bachschmid, Markus Michael

    2009-01-01

    Mitochondrial DNA (mtDNA) is organized in protein-DNA macrocomplexes called nucleoids. Average nucleoids contain 2–8 mtDNA molecules, which are organized by the histone-like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase γ (Polγ), various other proteins with ill-defined functions have been identified. We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from rat and bovine heart as well as human Jurkat cells. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid fractions. DNA, mitochondrial glutathione peroxidase (GPx1), and Polγ were coimmunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and GPx1 are integral constituents of nucleoids. Interestingly, in cultured bovine endothelial cells the association of SOD2 with mtDNA was absent. Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-induced oxidative damage.—Kienhöfer, J., Häussler, D. J. F., Ruckelshausen, F., Muessig, E., Weber, K., Pimentel, D., Ullrich, V., Bürkle, A., Bachschmid, M. M. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents. PMID:19228881

  1. Integrating Multi-omics Data to Dissect Mechanisms of DNA repair Dysregulation in Breast Cancer

    PubMed Central

    Liu, Chao; Rohart, Florian; Simpson, Peter T.; Khanna, Kum Kum; Ragan, Mark A.; Lê Cao, Kim-Anh

    2016-01-01

    DNA repair genes and pathways that are transcriptionally dysregulated in cancer provide the first line of evidence for the altered DNA repair status in tumours, and hence have been explored intensively as a source for biomarker discovery. The molecular mechanisms underlying DNA repair dysregulation, however, have not been systematically investigated in any cancer type. In this study, we performed a statistical analysis to dissect the roles of DNA copy number alteration (CNA), DNA methylation (DM) at gene promoter regions and the expression changes of transcription factors (TFs) in the differential expression of individual DNA repair genes in normal versus tumour breast samples. These gene-level results were summarised at pathway level to assess whether different DNA repair pathways are affected in distinct manners. Our results suggest that CNA and expression changes of TFs are major causes of DNA repair dysregulation in breast cancer, and that a subset of the identified TFs may exert global impacts on the dysregulation of multiple repair pathways. Our work hence provides novel insights into DNA repair dysregulation in breast cancer. These insights improve our understanding of the molecular basis of the DNA repair biomarkers identified thus far, and have potential to inform future biomarker discovery. PMID:27666291

  2. Detection of Prostate Cancer Progression by Serum DNA Integrity

    DTIC Science & Technology

    2009-04-01

    specimens from patients with PCa. A recent report demonstrated 0%– 18% rates of LOH for various chromosomal loci in plasma DNA; 44% of specimens tested...2004;351: 1802 –3. 4. Bock JL, Klee GG. How sensitive is a prostate- specific antigen measurement? How sensitive does it need to be? Arch Pathol Lab Med...in blood and bone marrow plasma from patients with prostate cancer. Int J Cancer 2007;120:1465–71. 28. Liu L, Yoon JH, Dammann R, Pfeifer GP. Frequent

  3. DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

    PubMed

    Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

    2017-01-25

    The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system.

  4. [Integrated risk evaluation of multiple disasters affecting longyan yield in Fujian Province, East China].

    PubMed

    Chen, Jia-Jin; Wang, Jia-Yi; Li, Li-Chun; Lin, Jing; Yang, Kai; Ma, Zhi-Guo; Xu, Zong-Huan

    2012-03-01

    In this study, an index system for the integrated risk evaluation of multiple disasters on the Longyan production in Fujian Province was constructed, based on the analysis of the major environmental factors affecting the Longyan growth and yield, and from the viewpoints of potential hazard of disaster-causing factors, vulnerability of hazard-affected body, and disaster prevention and mitigation capability of Longyan growth regions in the Province. In addition, an integrated evaluation model of multiple disasters was established to evaluate the risks of the major agro-meteorological disasters affecting the Longyan yield, based on the yearly meteorological data, Longyan planting area and yield, and other socio-economic data in Longyan growth region in Fujian, and by using the integral weight of risk indices determined by AHP and entropy weight coefficient methods. In the Province, the Longyan growth regions with light integrated risk of multiple disasters were distributed in the coastal counties (except Dongshan County) with low elevation south of Changle, the regions with severe and more severe integrated risk were mainly in Zhangping of Longyan, Dongshan, Pinghe, Nanjin, and Hua' an of Zhangzhou, Yongchun and Anxi of Quanzhou, north mountainous areas of Putian and Xianyou, Minqing, Minhou, Luoyuan, and mountainous areas of Fuzhou, and Fuan, Xiapu, and mountainous areas of Ninde, among which, the regions with severe integrated risk were in Dongshan, Zhangping, and other mountainous areas with high altitudes, and the regions with moderate integrated risk were distributed in the other areas of the Province.

  5. A novel non-integrative single-cycle chimeric HIV lentivector DNA vaccine.

    PubMed

    Moussa, Maha; Arrode-Brusés, Géraldine; Manoylov, Iliyan; Malogolovkin, Alexander; Mompelat, Dimitri; Ishimwe, Honorine; Smaoune, Amel; Ouzrout, Bilel; Gagnon, Jean; Chebloune, Yahia

    2015-05-05

    Novel HIV vaccine vectors and strategies are needed to control HIV/AIDS epidemic in humans and eradicate the infection. DNA vaccines alone failed to induce immune responses robust enough to control HIV-1. Development of lentivirus-based DNA vaccines deficient for integration and with a limited replication capacity is an innovative and promising approach. This type of vaccine mimics the early stages of virus infection/replication like the live-attenuated viruses but lacks the inconvenient integration and persistence associated with disease. We developed a novel lentivector DNA vaccine "CAL-SHIV-IN(-)" that undergoes a single round of replication in the absence of integration resulting in augmented expression of vaccine antigens in vivo. Vaccine gene expression is under control of the LTRs of a naturally attenuated lentivirus, Caprine arthritis encephalitis virus (CAEV) the natural goat lentivirus. The safety of this vaccine prototype was increased by the removal of the integrase coding sequences from the pol gene. We examined the functional properties of this lentivector DNA in cell culture and the immunogenicity in mouse models. Viral proteins were expressed in transfected cells, assembled into viral particles that were able to transduce once target permissive cells. Unlike the parental replication-competent SHIV-KU2 that was detected in DNA samples from any of the serial passage infected cells, CAL-SHIV-IN(-) DNA was detected only in target cells of the first round of infection, hence demonstrating the single cycle replication of the vaccine. A single dose DNA immunization of humanized NOD/SCID/β2 mice showed a substantial increase of IFN-γ-ELISPOT in splenocytes compared to the former replication and integration defective Δ4SHIV-KU2 DNA vaccine.

  6. 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.

  7. Survival, DNA Integrity, and Ultrastructural Damage in Antarctic Cryptoendolithic Eukaryotic Microorganisms Exposed to Ionizing Radiation.

    PubMed

    Pacelli, Claudia; Selbmann, Laura; Zucconi, Laura; Raguse, Marina; Moeller, Ralf; Shuryak, Igor; Onofri, Silvano

    2017-02-01

    Life dispersal between planets, planetary protection, and the search for biosignatures are main topics in astrobiology. Under the umbrella of the STARLIFE project, three Antarctic endolithic microorganisms, the melanized fungus Cryomyces antarcticus CCFEE 515, a hyaline strain of Umbilicaria sp. (CCFEE 6113, lichenized fungus), and a Stichococcus sp. strain (C45A, green alga), were exposed to high doses of space-relevant gamma radiation ((60)Co), up to 117.07 kGy. After irradiation survival, DNA integrity and ultrastructural damage were tested. The first was assessed by clonogenic test; viability and dose responses were reasonably described by the linear-quadratic formalism. DNA integrity was evaluated by PCR, and ultrastructural damage was observed by transmission electron microscopy. The most resistant among the tested organisms was C. antarcticus both in terms of colony formation and DNA preservation. Besides, results clearly demonstrate that DNA was well detectable in all the tested organisms even when microorganisms were dead. This high resistance provides support for the use of DNA as a possible biosignature during the next exploration campaigns. Implication in planetary protection and contamination during long-term space travel are put forward. Key Words: Biosignatures-Ionizing radiation-DNA integrity-Eukaryotic microorganisms-Fingerprinting-Mars exploration. Astrobiology 17, 126-135.

  8. Integrating DNA barcode data and taxonomic practice: determination, discovery, and description.

    PubMed

    Goldstein, Paul Z; DeSalle, Rob

    2011-02-01

    DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.

  9. Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield.

    PubMed

    Sanan-Mishra, Neeti; Pham, Xuan Hoi; Sopory, Sudhir K; Tuteja, Narendra

    2005-01-11

    Salt tolerance is an important trait that is required to overcome salinity-induced reduction in plant productivity. We have reported previously the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with the eukaryotic translation initiation factor eIF-4A. Here, we report that PDH45 mRNA is induced in pea seedlings in response to high salt, and its overexpression driven by a constitutive cauliflower mosaic virus-(35)S promoter in tobacco plants confers salinity tolerance, thus suggesting a previously undescribed pathway for manipulating stress tolerance in crop plants. The T(0) transgenic plants showed high levels of PDH45 protein in normal and stress conditions, as compared with WT plants. The T(0) transgenics also showed tolerance to high salinity as tested by a leaf disk senescence assay. The T(1) transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress without any reduction in plant yield in terms of seed weight. Measurement of Na(+) ions in different parts of the plant showed higher accumulation in the old leaves and negligible accumulation in seeds of T(1) transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance is discussed. Overexpression of PDH45 provides a possible example of the exploitation of DNA/RNA unwinding pathways for engineering salinity tolerance without affecting yield in crop plants.

  10. Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response

    PubMed Central

    Kumar, Akhilesh; Birnbaum, Michael D; Patel, Devang M; Morgan, William M; Singh, Jayanti; Barrientos, Antoni; Zhang, Fangliang

    2016-01-01

    Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis. PMID:27685622

  11. Parameters affecting organization and transfection efficiency of amphiphilic copolymers/DNA carriers.

    PubMed

    Roques, Caroline; Bouchemal, Kawthar; Ponchel, Gilles; Fromes, Yves; Fattal, Elias

    2009-08-19

    Amphiphilic block copolymers are attracting increasing interest in the field of gene therapy, especially for transfection of striated muscles. However, little is known about the parameters affecting their transfection efficiency in vivo. These copolymers can self-assemble as micelles in certain conditions. Since micellization strongly depends on the temperature and ionic content of the preparation medium, the present paper aimed at investigating the influence of these parameters in the context of gene delivery. We first assessed the micellization of pluronic L64 and tetronic 304 at various temperatures in water, saline or Tyrode's salts solution. Pluronic L64 can form micelles at temperatures above 37 degrees C in water or at 37 degrees C in the Tyrode's salts solution, in the range of concentration investigated. For tetronic 304, CMC was found to be far below the concentrations used to transfer DNA. Pluronic L64 interacted with DNA only in the presence of micelles. Moreover, in vivo evaluation demonstrated that significantly improved transfection efficiency was obtained at 37 degrees C in Tyrode's salts solution for pluronic L64 based formulations, compared to 4 degrees C and 20 degrees C. Such differences were not recorded with tetronic 304. Finally, optimized formulations of both tetronic 304 and pluronic L64 were able to mediate efficient transfection in dystrophic muscles.

  12. 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

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

    PubMed

    2012-09-06

    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.

  14. Mitochondrial DNA integrity changes with age but does not correlate with learning performance in honey bees.

    PubMed

    Hystad, E M; Amdam, G V; Eide, L

    2014-01-01

    The honey bee is a well-established model organism to study aging, learning and memory. Here, we used young and old forager honey bees to investigate whether age-related learning capacity correlates with mitochondrial function. The bees were selected for age and learning performance and mitochondrial function was evaluated by measuring mtDNA integrity, mtDNA copy number and mitochondrial gene expression. Quite unexpectedly, mtDNA from young bees showed more damage than mtDNA from older bees, but neither mtDNA integrity, nor mtDNA copy number nor mitochondrial gene expression correlated with learning performance. Although not statistically significant (p=0.07) the level of L-rRNA increased with age in good learners whereas it decreased in poor learners. Our results show that learning performance in honey bee does not correlate with absolute mitochondrial parameters like mtDNA damage, copy number or expression of mitochondrial genes, but may be associated with the ability to regulate mitochondrial activity.

  15. Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

    PubMed

    Chung, Mu-En; Yeh, I-Hsin; Sung, Li-Yu; Wu, Meng-Ying; Chao, Yun-Peng; Ng, I-Son; Hu, Yu-Chen

    2017-01-01

    Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.

  16. Scrotal heat stress effects on sperm viability, sperm DNA integrity, and the offspring sex ratio in mice.

    PubMed

    Pérez-Crespo, M; Pintado, B; Gutiérrez-Adán, A

    2008-01-01

    Evidence exists to suggest detrimental effects of heat stress on male fertility. This study was designed to assess the effects of scrotal heat stress on mature and developing sperm in a mouse model. After receiving shock heat treatment (42 degrees C for 30 min), mature spermatozoa were recovered from the epididymis hours (6) or Days (7, 14, 21, 28, 60) later, to determine the variables: number of spermatozoa, sperm viability, motility and progressive motility, sperm DNA integrity as established by the TUNEL method, embryo implantation rate, and sex ratio of the fetuses conceived using the heat-exposed spermatozoa. Our results indicate that transient mild heat treatment does not affect in the same way the different types of male germ cells. Spermatocytes present within the testis at the time of heat stress resulted into a lower concentration of spermatozoa with reduced viability and low motility. Even though, DNA integrity of spermatozoa resulting from spermatocytes was also compromised by heat stress, the higher degree of DNA damage was found among spermatozoa resulting from spermatids present within the testis at the time of heat stress. At last, heat shock effect on spermatozoa present in the epididymis at the time of thermal stress resulted into a sex ratio distortion. These findings point to a higher sensitivity of spermatocytes to heat exposure and also suggest a different response of X and Y chromosome-bearing spermatozoa to heat stress that warrants further investigation.

  17. 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

  18. The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome.

    PubMed Central

    Tinland, B; Schoumacher, F; Gloeckler, V; Bravo-Angel, A M; Hohn, B

    1995-01-01

    The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T-DNA by using a virD2 mutant. This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration. Images PMID:7628458

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

    PubMed Central

    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.

    2016-01-01

    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 ~3 fold, 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 clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

  20. 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…

  1. 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…

  2. 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…

  3. Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

    PubMed Central

    Grandgenett, D P; Inman, R B; Vora, A C; Fitzgerald, M L

    1993-01-01

    Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction. Images PMID:8474165

  4. DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity.

    PubMed

    Sikdar, Nilabja; Banerjee, Soma; Lee, Kyoo-young; Wincovitch, Stephen; Pak, Evgenia; Nakanishi, Koji; Jasin, Maria; Dutra, Amalia; Myung, Kyungjae

    2009-10-01

    Genomic integrity depends on DNA replication, recombination and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of gamma-H2AX, 53BP1 and phosphorylated-ATM that mark chromosomal breaks. Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were upregulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

  5. [Evaluation of the quality of the human spermatozoon: comparison between spermatic DNA integrity and semen variables].

    PubMed

    Cruz, Ibis; Colmenares, Melisa; Berrueta-Carrillo, Leidith; Gomez-Perez, Roald; Montes, Henry; Berrueta, Lisbeth; Salmen, Siham; Osuna, Jesús Alfonso

    2010-03-01

    Semen analysis does not have an absolute predictive value on fertility, however it is a reflection of male fertility potential, which is related to its spermatozoa quality and other semen variables. Great variability in human semen parameters has been demonstrated within a single individual, an observation that could explain why a male with low semen quality can successfully fertilize an egg. Although conventional semen analysis, such as sperm concentration, motility and morphology, provide important information about the clinical status of male fertility, new procedures to predict the sperm functional capability have been developed in the last decade, such as analysis of nuclear DNA integrity, which have improved considerably the clinical diagnosis of male infertility, and increased the knowledge about spermatozoa function. DNA fragmentation consist in interruptions, both in single and double DNA strains, that frequently occur in sperm samples from infertile patients. We have conducted a clinical study in semen samples from patients who have attended the Andrology laboratory of the University of Los Andes, between March 2007 and March 2009. The aim of this study was to compare sperm DNA integrity, analyzed by flow cytometry, with traditional semen parameters. Our results show remarkable correlations between conventional human semen variables and sperm chromatin integrity, contributing to asses an integral evaluation of sperm quality allowing the analysis of its fertilizing potential in clinical studies.

  6. Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

    PubMed

    Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

    2017-02-23

    Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

  7. Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

    PubMed

    Pearce, Jacqueline W; Galle, Laurence E; Kleiboeker, Steve B; Turk, James R; Schommer, Susan K; Dubielizig, Richard R; Mitchell, William J; Moore, Cecil P; Giuliano, Elizabeth A

    2007-11-01

    Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

  8. Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

    PubMed

    Adam, Salomé; Dabin, Juliette; Chevallier, Odile; Leroy, Olivier; Baldeyron, Céline; Corpet, Armelle; Lomonte, Patrick; Renaud, Olivier; Almouzni, Geneviève; Polo, Sophie E

    2016-10-06

    Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

  9. Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

    PubMed

    Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

    2014-06-01

    The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification.

  10. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    PubMed

    Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Sakuma, Tetsushi; Kobayashi, Tomoko; Yamamoto, Takashi; Koyanagi, Yoshio

    2015-01-01

    DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  11. 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

  12. A case of bilateral human herpes virus 6 panuveitis with genomic viral DNA integration

    PubMed Central

    2014-01-01

    Background We report a rare case of bilateral panuveitis from human herpes virus 6 (HHV-6) with genomic viral DNA integration in an immunocompromised man. Findings A 59-year-old man with history of multiple myeloma presented with altered mental status, bilateral eye redness, and blurry vision. Examination revealed bilateral diffuse keratic precipitates, 4+ anterior chamber cell, hypopyon, vitritis, and intraretinal hemorrhages. Intraocular fluid testing by polymerase chain reaction (PCR) was positive for HHV-6. The patient was successfully treated with intravitreal foscarnet and intravenous ganciclovir and foscarnet. Despite clinical improvement, his serum HHV-6 levels remained high, and it was concluded that he had HHV-6 chromosomal integration. Conclusions HHV-6 should be considered in the differential for infectious uveitis in immunocompromised hosts who may otherwise have a negative work-up. HHV-6 DNA integration may lead to difficulties in disease diagnosis and determining disease resolution. PMID:24995045

  13. 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.

  14. Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells

    PubMed Central

    Zhang, Ke; Li, Jin-Tao; Li, Shu-Ying; Zhu, Li-Hua; Zhou, Ling; Zeng, Yi

    2011-01-01

    AIM: To detect human papillomavirus (HPV) DNA in esophageal carcinoma (EC) 109 cells and investigate the relationship between HPV and EC. METHODS: Genomic DNA and total RNA from EC109 cells were isolated. HPV DNA was detected by polymerase chain reaction (PCR) with the general primer sets of My09/11 and GP5 +/6 + for the HPV L1 gene and type-specific primer sets for HPV18 E6 and HPV18 E6-E7. Reverse transcription (RT) of mRNA isolated from EC109 cells was performed to produce a cDNA. And then a PCR-based protocol for the amplification of papillomavirus oncogene transcripts was used to analyze HPV18 DNA and integrated transcripts of HPV18 in the chromosomes of EC109 cells. The final nested PCR products were cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration. RESULTS: HPV18 DNA was detected in EC109 cells by PCR using the general primer sets of My09/11 and GP5 +/6 + for HPV L1 and the type-specific primer sets for HPV18 E6 and E6-E7 to generate products of 450 bp, 150 bp, 335 bp and 944 bp, respectively. Approximately 600 bp of integrated HPV18-specific transcript was identified. The final nested PCR product of integrated HPV18 DNA was cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration. Sequence alignment showed that the HPV18 sequence from EC109 cells was identical to that of the encoded early protein E7-E1 of the standard HPV18 strain X05015, and another partial gene sequence was identical to a partial sequence of human chromosome 8. CONCLUSION: Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis. PMID:22072858

  15. 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

  16. 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.

  17. 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.

  18. Reduced rDNA copy number does not affect "competitive" chromosome pairing in XYY males of Drosophila melanogaster.

    PubMed

    Maggert, Keith A

    2014-03-20

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a "competitive" situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

  19. Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

    PubMed Central

    Maggert, Keith A.

    2014-01-01

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

  20. 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

  1. 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.

  2. Survival, DNA Integrity, and Ultrastructural Damage in Antarctic Cryptoendolithic Eukaryotic Microorganisms Exposed to Ionizing Radiation

    NASA Astrophysics Data System (ADS)

    Pacelli, Claudia; Selbmann, Laura; Zucconi, Laura; Raguse, Marina; Moeller, Ralf; Shuryak, Igor; Onofri, Silvano

    2017-02-01

    Life dispersal between planets, planetary protection, and the search for biosignatures are main topics in astrobiology. Under the umbrella of the STARLIFE project, three Antarctic endolithic microorganisms, the melanized fungus Cryomyces antarcticus CCFEE 515, a hyaline strain of Umbilicaria sp. (CCFEE 6113, lichenized fungus), and a Stichococcus sp. strain (C45A, green alga), were exposed to high doses of space-relevant gamma radiation (60Co), up to 117.07 kGy. After irradiation survival, DNA integrity and ultrastructural damage were tested. The first was assessed by clonogenic test; viability and dose responses were reasonably described by the linear-quadratic formalism. DNA integrity was evaluated by PCR, and ultrastructural damage was observed by transmission electron microscopy. The most resistant among the tested organisms was C. antarcticus both in terms of colony formation and DNA preservation. Besides, results clearly demonstrate that DNA was well detectable in all the tested organisms even when microorganisms were dead. This high resistance provides support for the use of DNA as a possible biosignature during the next exploration campaigns. Implication in planetary protection and contamination during long-term space travel are put forward.

  3. 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

  4. 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.

  5. Extremely low-frequency electromagnetic fields do not affect DNA damage and gene expression profiles of yeast and human lymphocytes.

    PubMed

    Luceri, Cristina; De Filippo, Carlotta; Giovannelli, Lisa; Blangiardo, Marta; Cavalieri, Duccio; Aglietti, Filippo; Pampaloni, Monica; Andreuccetti, Daniele; Pieri, Lapo; Bambi, Franco; Biggeri, Annibale; Dolara, Piero

    2005-09-01

    We studied the effects of extremely low-frequency (50 Hz) electromagnetic fields (EMFs) on peripheral human blood lymphocytes and DBY747 Saccharomyces cerevisiae. Graded exposure to 50 Hz magnetic flux density was obtained with a Helmholtz coil system set at 1, 10 or 100 microT for 18 h. The effects of EMFs on DNA damage were studied with the single-cell gel electrophoresis assay (comet assay) in lymphocytes. Gene expression profiles of EMF-exposed human and yeast cells were evaluated with DNA microarrays containing 13,971 and 6,212 oligonucleotides, respectively. After exposure to the EMF, we did not observe an increase in the amount of strand breaks or oxidated DNA bases relative to controls or a variation in gene expression profiles. The results suggest that extremely low-frequency EMFs do not induce DNA damage or affect gene expression in these two different eukaryotic cell systems.

  6. 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

  7. Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis.

    PubMed

    Toriello, Nicholas M; Liu, Chung N; Blazej, Robert G; Thaitrong, Numrin; Mathies, Richard A

    2007-11-15

    A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

  8. Staying in aerobic shape: how the structural integrity of mitochondria and mitochondrial DNA is maintained.

    PubMed

    Scott, Sidney V; Cassidy-Stone, Ann; Meeusen, Shelly L; Nunnari, Jodi

    2003-08-01

    The structure and integrity of the mitochondrial compartment are features essential for it to function efficiently. The maintenance of mitochondrial structure in cells ranging from yeast to humans has been shown to require both ongoing fission and fusion. Recent characterization of many of the molecular components that direct mitochondrial fission and fusion events have led to a more complete understanding of how these processes take place. Further, mitochondrial fragmentation observed when cells undergo apoptosis requires mitochondrial fission, underlying the importance of mitochondrial dynamics in cellular homeostasis. Mitochondrial structure also impacts mitochondrial DNA inheritance. Recent studies suggest that faithful transmission of mitochondrial DNA to daughter cells might require a mitochondrial membrane tethering apparatus.

  9. DNA sequence context greatly affects the accuracy of bypass across an ultraviolet light 6-4 photoproduct in mammalian cells.

    PubMed

    Shriber, Pola; Leitner-Dagan, Yael; Geacintov, Nicholas; Paz-Elizur, Tamar; Livneh, Zvi

    2015-10-01

    Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells.

  10. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

    PubMed Central

    Heap, John T.; Ehsaan, Muhammad; Cooksley, Clare M.; Ng, Yen-Kuan; Cartman, Stephen T.; Winzer, Klaus; Minton, Nigel P.

    2012-01-01

    Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. PMID:22259038

  11. 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.

  12. 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

  13. 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.

  14. The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells▿

    PubMed Central

    Horner, Stacy M.; DiMaio, Daniel

    2007-01-01

    Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells. PMID:17392356

  15. 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

  16. 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

  17. 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.

  18. Integration of woodchuck hepatitis virus (WHV) DNA at two chromosomal sites (Vk and gag-like) in a hepatocellular carcinoma.

    PubMed

    Yamazoe, M; Nakai, S; Ogasawara, N; Yoshikawa, H

    1991-04-01

    Integration of woodchuck hepatitis virus (WHV) DNA into the liver DNA of a woodchuck infected by the virus was investigated. Clonal viral integration was not detected three months before the appearance of four hepatocellular carcinomas (HCC). Integration of the viral DNA was detected in all four HCCs, of which one was chosen to determine the structure of the viral integration completely in a single tumor. The integration occurred in two sites. One part contains the viral DNA from the middle of the gene encoding surface antigen to two-thirds of the way through the gene encoding X protein (X) with no structural changes. The coding frame of the truncated X gene continues into the chromosomal sequence to make a possible fusion protein. The integration seems to have occurred by recombination within two direct repeats of the viral genome in one junction and by homologous recombination between viral DNA and chromosomal DNA in the other junction. The viral DNA is integrated into a spacer of the immunoglobulin L-chain Vk (IgVk) region without any chromosomal rearrangement accompanying the integration. The viral DNA at the second site has a complex structural rearrangement: part of the preS gene is duplicated and attached to the terminus of the gene encoding core antigen in a head-to-tail fashion, followed by three small fragments derived from other parts of the viral DNA. The integrated preS gene has its own 5' regulatory element and a coding frame consisting of the truncated preS gene, the other parts of viral DNA and the chromosomal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence.

    PubMed

    Holden, Marcia J; Haynes, Ross J; Rabb, Savelas A; Satija, Neena; Yang, Kristina; Blasic, Joseph R

    2009-08-26

    The total amount of DNA in a preparation extracted from tissues can be measured in several ways, each method offering advantages and disadvantages. For the sake of accuracy in quantitation, it is of interest to compare these methodologies and determine if good correlation can be achieved between them. Different answers can also be clues to the physical state of the DNA. In this study, we investigated the lack of correlation between ultraviolet (UV) absorbance and fluorescent (PicoGreen) measurements of the concentration of DNAs isolated from plant tissues. We found that quantitation based on the absorbance-based method correlated with quantitation based on phosphorus content, while the PicoGreen-based method did not. We also found evidence of the production of single-stranded DNA under conditions where the DNA was not fragmented into small pieces. The PicoGreen fluorescent signal was dependent on DNA fragment size but only if the DNA was in pure water, while DNA in buffer was much less sensitive. Finally, we document the high sensitivity of the PicoGreen assays to the detergent known as CTAB (cetyldimethylethylammonium bromide). The CTAB-based method is highly popular for low-cost DNA extraction with many published variations for plant and other tissues. The removal of residual CTAB is important for accurate quantitation of DNA using PicoGreen.

  20. A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

    PubMed Central

    Chiang, C C; Kennell, J C; Wanner, L A; Lambowitz, A M

    1994-01-01

    The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery. Images PMID:7523850

  1. 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

  2. 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

  3. Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides.

    PubMed Central

    Hong, T; Murphy, E; Groarke, J; Drlica, K

    1993-01-01

    The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication. Images PMID:8419642

  4. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

    PubMed

    Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

    2007-01-01

    Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

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

    PubMed

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

    2015-10-06

    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.

  6. 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

  7. Microfluidic chip integrating high throughput continuous-flow PCR and DNA hybridization for bacteria analysis.

    PubMed

    Jiang, Xiran; Shao, Ning; Jing, Wenwen; Tao, Shengce; Liu, Sixiu; Sui, Guodong

    2014-05-01

    Rapid identification of clinical pathogens is the initial and essential step for antimicrobial therapy. Herein, we successfully developed a microfluidic device which combines high-throughput continuous-flow PCR and DNA hybridization for the detection of various bacterial pathogens. Universal primers were designed based on the conserved regions of bacterial 16S ribosomal DNA (16S rDNA), and specific probes were designed from a variable region of 16S rDNA within the amplicon sequences. In the chip operation, after the continuous flow PCR was achieved in the first microfluidic chip, the product was directly introduced into a hybridization chip integrated with microarray containing the immobilized DNA probes. The target-probe hybridization was completed within 1h at 55 °C, and fluorescence signals were obtained as the readout. The presented device is simple, versatile and with less sample consumption compared with traditional instruments. It can perform high-throughput bacteria detections continuously in a single assay, which makes it a promising platform for clinical bacteria identifications.

  8. LSSP-PCR of Trypanosoma cruzi: how the single primer sequence affects the kDNA signature

    PubMed Central

    2013-01-01

    Background Low-stringency single specific primer PCR (LSSP-PCR) is a highly sensitive and discriminating technique that has been extensively used to genetically characterize Trypanosoma cruzi populations in the presence of large amounts of host DNA. To ensure high sensitivity, in most T. cruzi studies, the variable regions of the naturally amplified kinetoplast DNA (kDNA) minicircles were targeted, and this method translated the intraspecific polymorphisms of these molecules into specific and reproducible kDNA signatures. Although the LSSP-PCR technique is reproducible under strict assay conditions, the complex banding pattern generated can be significantly altered by even a single-base change in the target DNA. Our survey of the literature identified eight different primers with similar, if not identical, names that have been used for kDNA amplification and LSSP-PCR of T. cruzi. Although different primer sequences were used in these studies, many of the authors cited the same reference report to justify their primer choice. We wondered whether these changes in the primer sequence could affect also the parasite LSSP-PCR profiles. Findings To answer this question we compared the kDNA signatures obtained from three different and extensively studied T. cruzi populations with the eight primers found in the literature. Our results clearly demonstrate that even minimal modifications in the oligonucleotide sequences, especially in the 3′ or 5′ end, can significantly change the kDNA signature of a T. cruzi strain. Conclusions These results highlight the necessity of careful preservation of primer nomenclature and sequence when reproducing an LSSP-PCR work to avoid confusion and allow comparison of results among different laboratories. PMID:23639061

  9. Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint

    PubMed Central

    Rudzka, Justyna; Campbell, Judith L.; Jonczyk, Piotr; Fijałkowska, Iwona J.

    2017-01-01

    To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ε) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ε in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ε. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ε mutants. PMID:28107343

  10. 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.

  11. A systematic review of multisensory cognitive-affective integration in schizophrenia.

    PubMed

    Tseng, Huai-Hsuan; Bossong, Matthijs G; Modinos, Gemma; Chen, Kuan-Ming; McGuire, Philip; Allen, Paul

    2015-08-01

    The etymology of schizophrenia implies poor functional integration of sensory, cognitive and affective processes. Multisensory integration (MSI) is a spontaneous perceptual-cognitive process by which relevant information from multiple sensory modalities is extracted to generate a holistic experience. Deficits in MSI may hinder prompt and appropriate behavioural responses in a complex and transient environment. Despite extensive investigation of sensory, cognitive and affective processing in patients with schizophrenia, little is known about how MSI is affected in the illness. We systemically searched the PubMed electronic database and reviewed twenty-nine behavioural and neuroimaging studies examining MSI in patients with schizophrenia. The available evidence indicates impaired MSI for non-emotional stimuli in schizophrenia, especially for linguistic information. There is also evidence for altered MSI for emotional stimuli, although findings are inconsistent and may be modality-specific. Brain functional alterations in the superior temporal cortex and inferior frontal cortex appear to underlie the deficits in both non-emotional and emotional MSI. The limitations of the experimental paradigms used and directions for future research are also discussed.

  12. Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

    PubMed

    Bennett, Geoffrey R; Peters, Ryan; Wang, Xiao-hong; Hanne, Jeungphill; Sobol, Robert W; Bundschuh, Ralf; Fishel, Richard; Yoder, Kristine E

    2014-01-01

    Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

  13. 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

  14. 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

  15. Involvement of right STS in audio-visual integration for affective speech demonstrated using MEG.

    PubMed

    Hagan, Cindy C; Woods, Will; Johnson, Sam; Green, Gary G R; Young, Andrew W

    2013-01-01

    Speech and emotion perception are dynamic processes in which it may be optimal to integrate synchronous signals emitted from different sources. Studies of audio-visual (AV) perception of neutrally expressed speech demonstrate supra-additive (i.e., where AV>[unimodal auditory+unimodal visual]) responses in left STS to crossmodal speech stimuli. However, emotions are often conveyed simultaneously with speech; through the voice in the form of speech prosody and through the face in the form of facial expression. Previous studies of AV nonverbal emotion integration showed a role for right (rather than left) STS. The current study therefore examined whether the integration of facial and prosodic signals of emotional speech is associated with supra-additive responses in left (cf. results for speech integration) or right (due to emotional content) STS. As emotional displays are sometimes difficult to interpret, we also examined whether supra-additive responses were affected by emotional incongruence (i.e., ambiguity). Using magnetoencephalography, we continuously recorded eighteen participants as they viewed and heard AV congruent emotional and AV incongruent emotional speech stimuli. Significant supra-additive responses were observed in right STS within the first 250 ms for emotionally incongruent and emotionally congruent AV speech stimuli, which further underscores the role of right STS in processing crossmodal emotive signals.

  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. Feature binding and affect: emotional modulation of visuo-motor integration.

    PubMed

    Colzato, Lorenza S; van Wouwe, Nelleke C; Hommel, Bernhard

    2007-01-28

    The primate cortex represents the external world in a distributed fashion, which calls for a mechanism that integrates and binds the features of a perceived or processed event. Animal and patients studies provide evidence that feature binding in the visual cortex is driven by the muscarinic-cholinergic system, whereas visuo-motor integration may be under dopaminergic control. Consistent with this scenario, we present indication that the binding of visual and action features is modulated by emotions through the probable stimulation of the dopaminergic system. Interestingly, the impact of emotions on binding was restricted to tasks in which shape was task-relevant, suggesting that extracting affective information is not automatic but requires attention to shape.

  18. Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12.

    PubMed Central

    Weiss, B; Cunningham, R P

    1985-01-01

    The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation. An nth insertion mutant and a deletion mutant were studied. nth is located between add and tyrS on the linkage map of E. coli K-12 and was 97% linked to tyrS in a transduction with phage P1. PMID:3886628

  19. Integrating DNA-based data into bioassessments improves our understanding of species distributions and species habitat relationships

    EPA Science Inventory

    The integration of DNA-based identification methods into bioassessments could result in more accurate representations of species distributions and species-habitat relationships. DNA-based approaches may be particularly informative for tracking the distributions of rare and/or inv...

  20. Modification of extraction method for community DNA isolation from salt affected compact wasteland soil samples.

    PubMed

    Zaveri, Purvi; Patel, Rushika; Patel, Meghavi; Sarodia, Devki; Munshi, Nasreen S

    2017-01-01

    To overcome the issue of interferences by salt and compactness in release of bacterial cell required for lysis, method described by Yeates et al. (1998), was optimized for isolation of genomic material (Deoxyribo Nucleic Acid, DNA) from soil microbial community by addition of Al(NH4)SO4. Very low total viable count was observed in the samples tested and hence use of higher amount of soil is required primarily for DNA isolation from wasteland soils. The method proves itself efficient where commercially available bead beating and enzymatic lysis methods could not give isolation of any amount of community genomic DNA due to compact nature and salt concentrations present in soil. •The protocol was found efficient for soil samples with high clay content for microbial community DNA extraction.•Variation in lysis incubation and amount of soil may help with soil samples containing low microbial population.•Addition of Al(NH4)SO4 is crucial step in humic acid removal from extracted DNA samples for soil samples containing high salinity and clay particles.

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

    PubMed

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

    2015-05-11

    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: 9.8-17.8%) and 37.7% (95% CI: 31.9-43.8%) of vaccine recipients, respectively. Three vaccinations (vs. 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.

  2. Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding

    PubMed Central

    Serrao, Erik; Krishnan, Lavanya; Shun, Ming-Chieh; Li, Xiang; Cherepanov, Peter; Engelman, Alan; Maertens, Goedele N.

    2014-01-01

    Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions −3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration. PMID:24520116

  3. Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae.

    PubMed

    Waghmare, Sanjeev K; Caputo, Valentina; Radovic, Slobodanka; Bruschi, Carlo V

    2003-05-01

    Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in [cir+] yeast strains. Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently.

  4. 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.

  5. Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats.

    PubMed

    Galimberti, A; Romano, D F; Genchi, M; Paoloni, D; Vercillo, F; Bizzarri, L; Sassera, D; Bandi, C; Genchi, C; Ragni, B; Casiraghi, M

    2012-05-01

    In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free-ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post-mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already-described taeniid species. Indeed, we detected three well-defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.

  6. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies.

    PubMed

    Singh, Amit Raj; Granek, Rony

    2016-10-14

    We study DNA denaturation by integrating elasticity - as described by the Gaussian network model - with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  7. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies

    NASA Astrophysics Data System (ADS)

    Singh, Amit Raj; Granek, Rony

    2016-10-01

    We study DNA denaturation by integrating elasticity — as described by the Gaussian network model — with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  8. Intronic T-DNA insertion in Arabidopsis NBR1 conditionally affects wild-type transcript level

    PubMed Central

    Rodríguez, Milagros Collados; Wawrzyńska, Anna; Sirko, Agnieszka

    2014-01-01

    Abstract The SALK_135513 line of Arabidopsis thaliana is annotated by GenBank to have the T-DNA insertion in the fourth exon of NBR1 (At4g24690). Careful molecular analyses of the homozygous plants of SALK_135513 line indicated the place of T-DNA insertion in the fourth intron. Unexpectedly, 2 kinds of NBR1 transcripts, the wild-type and the mutated, resulting from alternative splicing events, were detected in those plants. Our findings explain the problems encountered by us with phenotypic evaluation of this line and emphasize the necessity for independent verification of the exact insertion site followed by careful expression studies when working with Arabidopsis T-DNA insertional mutants. PMID:25482782

  9. Intronic T-DNA insertion in Arabidopsis NBR1 conditionally affects wild-type transcript level.

    PubMed

    Rodríguez, Milagros Collados; Wawrzyńska, Anna; Sirko, Agnieszka

    2014-01-01

    The SALK_135513 line of Arabidopsis thaliana is annotated by GenBank to have the T-DNA insertion in the fourth exon of NBR1 (At4g24690). Careful molecular analyses of the homozygous plants of SALK_135513 line indicated the place of T-DNA insertion in the fourth intron. Unexpectedly, 2 kinds of NBR1 transcripts, the wild-type and the mutated, resulting from alternative splicing events, were detected in those plants. Our findings explain the problems encountered by us with phenotypic evaluation of this line and emphasize the necessity for independent verification of the exact insertion site followed by careful expression studies when working with Arabidopsis T-DNA insertional mutants.

  10. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    PubMed

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  11. 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

  12. Selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species

    PubMed Central

    Asghar, Waseem; Velasco, Vanessa; Kingsley, James L.; Shoukat, Muhammad S.; Shafiee, Hadi; Anchan, Raymond M.; Mutter, George L.; Tüzel, Erkan; Demirci, Utkan

    2014-01-01

    Fertilization and reproduction are central to the survival and propagation of a species. Couples who cannot reproduce naturally have to undergo in vitro clinical procedures. An integral part of these clinical procedures includes isolation of healthy sperm from raw semen. Existing sperm sorting methods are not efficient and isolate sperm having high DNA fragmentation and reactive oxygen species, and suffer from multiple manual steps and variations between embryologists. Inspired by in vivo natural sperm sorting mechanisms where vaginal mucus becomes less viscous to form microchannels to guide sperm towards egg, we present a chip that efficiently sorts healthy, motile and morphologically normal sperm without centrifugation. Higher percentage of sorted sperm show significantly lesser reactive oxygen species and DNA fragmentation than the conventional swim-up method. The presented chip is an easy-to-use high throughput sperm sorter that provides standardized sperm sorting assay with less reliance on embryologist’s skills, facilitating reliable operational steps. PMID:24753434

  13. Design of a Synthetic Integral Feedback Circuit: Dynamic Analysis and DNA Implementation.

    PubMed

    Briat, Corentin; Zechner, Christoph; Khammash, Mustafa

    2016-10-21

    The design and implementation of regulation motifs ensuring robust perfect adaptation are challenging problems in synthetic biology. Indeed, the design of high-yield robust metabolic pathways producing, for instance, drug precursors and biofuels, could be easily imagined to rely on such a control strategy in order to optimize production levels and reduce production costs, despite the presence of environmental disturbance and model uncertainty. We propose here a motif that ensures tracking and robust perfect adaptation for the controlled reaction network through integral feedback. Its metabolic load on the host is fully tunable and can be made arbitrarily close to the constitutive limit, the universal minimal metabolic load of all possible controllers. A DNA implementation of the controller network is finally provided. Computer simulations using realistic parameters demonstrate the good agreement between the DNA implementation and the ideal controller dynamics.

  14. Addressing RNA integrity to determine the impact of mitochondrial DNA mutations on brain mitochondrial function with age.

    PubMed

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Strand, Janne M; Stewart, James B; Bjørås, Magnar; Eide, Lars

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations.

  15. Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age

    PubMed Central

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Strand, Janne M.; Stewart, James B.; Bjørås, Magnar; Eide, Lars

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations. PMID:24819950

  16. Prediction of the efficacy of immunotherapy by measuring the integrity of cell-free DNA in plasma in colorectal cancer.

    PubMed

    Kitahara, Masahiro; Hazama, Shoichi; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Kanekiyo, Shinsuke; Inoue, Yuka; Nakajima, Masao; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Iida, Michihisa; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Ueno, Tomio; Yamamoto, Shigeru; Yoshino, Shigefumi; Nagano, Hiroaki

    2016-12-01

    We previously reported a phase II study of a cancer vaccine using five novel peptides recognized by HLA-A*2402-restricted CTL in combination with oxaliplatin-containing chemotherapy (FXV study) as first-line therapy for patients with metastatic colorectal cancer and demonstrated the safety and promising potential of our five-peptide cocktail. The objective of this analysis was to identify predictive biomarkers for identifying patients who are likely to receive a clinical benefit from immunochemotherapy. Circulating cell-free DNA (cfDNA) in plasma has been reported to be a candidate molecular biomarker for the efficacy of anticancer therapy. Unlike uniformly truncated small-sized DNA released from apoptotic normal cells, DNA released from necrotic cancer cells varies in size. The integrity of plasma cfDNA (i.e. the ratio of longer fragments [400 bp] to shorter fragments [100 bp] of cfDNA), may be clinically useful for detecting colorectal cancer progression. We assessed plasma samples collected from 93 patients prior to receiving immunochemotherapy. The cfDNA levels and integrity were analyzed by semi-quantitative real-time PCR. Progression-free survival was significantly better in patients with a low plasma cfDNA integrity value than in those with a high value (P = 0.0027). Surprisingly, in the HLA-A*2402-matched group, patients with a low plasma cfDNA integrity value had significantly better progression-free survival than those with a high value (P = 0.0015). This difference was not observed in the HLA-A*2402-unmatched group. In conclusion, the integrity of plasma cfDNA may provide important clinical information and may be a useful predictive biomarker of the outcome of immunotherapy in metastatic colorectal cancer.

  17. White matter integrity and its association with affective and interpersonal symptoms in borderline personality disorder

    PubMed Central

    Whalley, Heather C.; Nickson, Thomas; Pope, Merrick; Nicol, Katie; Romaniuk, Liana; Bastin, Mark E.; Semple, Scott I.; McIntosh, Andrew M.; Hall, Jeremy

    2015-01-01

    Background Borderline personality disorder (BPD) is a severe psychiatric disorder involving a range of symptoms including marked affective instability and disturbances in interpersonal interactions. Neuroimaging studies are beginning to provide evidence of altered processing in fronto-limbic network deficits in the disorder, however, few studies directly examine structural connections within this circuitry together with their relation to proposed causative processes and clinical features. Methods In the current study, we investigated whether individuals with BPD (n = 20) have deficits in white matter integrity compared to a matched group of healthy controls (n = 18) using diffusion tensor MRI (DTI). We hypothesized that the BPD group would have decreased fractional anisotropy (FA), a measure of white matter integrity, compared to the controls in white matter tracts connecting frontal and limbic regions, primarily the cingulum, fornix and uncinate fasciculus. We also investigated the extent to which any such deficits related to childhood adversity, as measured by the childhood trauma questionnaire, and symptom severity as measured by the Zanarini rating scale for BPD. Results We report decreased white matter integrity in BPD versus controls in the cingulum and fornix. There were no significant relationships between FA and measures of childhood trauma. There were, however, significant associations between FA in the cingulum and clinical symptoms of anger, and in the fornix with affective instability, and measures of avoidance of abandonment from the Zanarini rating scale. Conclusions We report deficits within fronto-limbic connections in individuals with BPD. Abnormalities within the fornix and cingulum were related to severity of symptoms and highlight the importance of these tracts in the pathogenesis of the disorder. PMID:25685714

  18. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

    PubMed

    Barretto, Letícia Siqueira de Sá; Lessio, Camila; Sawaki e Nakamura, Ahy Natally; Lo Turco, Edson Guimarães; da Silva, Camila Gonzaga; Zambon, João Paulo; Gozzo, Fábio César; Pilau, Eduardo Jorge; de Almeida, Fernando Gonçalves

    2014-10-01

    Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

  19. ELF magnetic fields do not affect cell survival and DNA damage induced by ultraviolet B.

    PubMed

    Mizuno, Kohei; Narita, Eijiro; Yamada, Masaru; Shinohara, Naoki; Miyakoshi, Junji

    2014-02-01

    We investigated whether extremely low frequency (ELF) magnetic field exposure has modification effects on cell survival after ultraviolet B (UV-B) irradiation and on repair process of DNA damage induced by UV-B irradiation in WI38VA13 subcloned 2RA and XP2OS(SV) cells. The ELF magnetic field exposure was conducted using a Helmholtz coil-based system that was designed to generate a sinusoidal magnetic field at 5 mT and 60 Hz. Cell survival was assessed by WST assay after UV-B irradiation at 20-80 J/m(2) , ELF magnetic field exposure for 24 h, followed by incubation for 48 h. DNA damage was assessed by quantification of cyclobutane pyrimidine dimer formation and 6-4 photoproduct formation using ELISA after UV-B irradiation at 20-80 J/m(2) followed by ELF magnetic field exposure for 24 h. No significant changes were observed in cell survival between ELF magnetic field and sham exposures. Similarly, DNA damage induced by UV-B irradiation did not change significantly following ELF magnetic field exposure. Our results suggest that ELF magnetic field exposure at 5 mT does not have modification effect on cell survival after UV-B irradiation and on repair process of DNA damage induced by UV-B irradiation.

  20. 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...

  1. 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 ...

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

    PubMed

    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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. Detecting DNA in Eukaryotic Cells Using an Integrated Microfluidics Electronic Sensor

    NASA Astrophysics Data System (ADS)

    Sohn, L. L.; Saleh, O. A.; Facer, G. R.; Carbeck, J. D.; Beavis, A.; Notterman, D. A.

    2000-03-01

    We have developed an integrated electronic sensor fabricated on a microfluidic chip which can measure the dielectric properties of material flowing through a micron-sized channel. We show that this sensor is advantageous over the usual optical detection techniques for microfluidics as it measures the inherent solid-state property of the analytes to be examined. In this talk, we show that we are able to not only identify small volumes of fluids but, more importantly, detect and diffentiate eukaryotic cells on the basis of DNA content.

  8. 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

  9. 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

  10. Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF.

    PubMed

    Tandara, M; Bajić, A; Tandara, L; Bilić-Zulle, L; Šunj, M; Kozina, V; Goluža, T; Jukić, M

    2014-09-01

    Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = -0.69; p < 0.001). Receiver operating characteristic (ROC) analyses show that DFI and big halo are significant (p < 0.001) as prognostic parameters of embryo quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC ± SE for DFI was 0.67 ± 0.06 and 0.75 ± 0.06 for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF.

  11. How lipid hydration and temperature affect the structure of DC-Chol DOPE/DNA lipoplexes

    NASA Astrophysics Data System (ADS)

    Pozzi, Daniela; Amenitsch, Heinz; Caminiti, Ruggero; Caracciolo, Giulio

    2006-05-01

    Effect of lipid hydration on the structure of lamellar lipoplexes made of the cationic lipid 3-[ N-( N, N-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), the neutral 'helper' lipid dioleoylphosphatidylethanolamine (DOPE) and calf-thymus DNA was investigated by synchrotron small angle X-ray diffraction (SAXD). Here, we show that lipid hydration is the key factor regulating the equilibrium structure of lipoplexes. Thermotropic behavior was also investigated between 5 and 65 °C. Both the membrane thickness and the water layer thickness were found to decrease linearly as a function of temperature while the one dimensional DNA rod lattice between lipid bilayers was found to enlarge. Structural results were interpreted in terms of recently proposed theoretical models.

  12. Resveratrol affects DNA damage induced by ionizing radiation in human lymphocytes in vitro.

    PubMed

    Basso, Emiliano; Regazzo, Giulia; Fiore, Mario; Palma, Valentina; Traversi, Gianandrea; Testa, Antonella; Degrassi, Francesca; Cozzi, Renata

    2016-08-01

    Resveratrol (3,4',5-trihydroxystilbene; RSV) acts on cancer cells in several ways, inducing cell cycle delay and apoptotic death, and enhancing ionizing radiation (IR)-mediated responses. However, fewer studies have examined RSV effects on normal cells. We have treated human lymphocytes in vitro with RSV, either alone or combined with IR, to evaluate its potential use as a radioprotector. We measured the effects of RSV on induction of DNA damage, repair kinetics, and modulation of histone deacetylase activity.

  13. 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

  14. DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

    PubMed

    Marilley, M; Radebaugh, C A; Geiss, G K; Laybourn, P J; Paule, M R

    2002-08-01

    Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

  15. 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.

  16. Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration.

    PubMed

    McAllister, Robert G; Liu, Jiahui; Woods, Matthew W; Tom, Sean K; Rupar, C Anthony; Barr, Stephen D

    2014-08-26

    The blood-brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.

  17. I feel who I see: visual body identity affects visual-tactile integration in peripersonal space.

    PubMed

    Salomon, R; van Elk, M; Aspell, J E; Blanke, O

    2012-09-01

    Recent studies have shown the importance of integrating multisensory information in the body representation for constituting self-consciousness. However, one idea that has received only scant attention is that our body representation is also constituted by knowledge of bodily visual characteristics (i.e. 'what I look like'). Here in two experiments we used a full body crossmodal congruency task in which visual distractors were presented on a photograph of the participant, another person, who was either familiar or unfamiliar, or an object. Results revealed that during the 'self-condition' CCEs were enhanced compared to the 'other condition'. The CCE was similar for unfamiliar and familiar others. CCEs for the object condition were significantly smaller. The results show that presentation of an irrelevant image of a body affects multimodal processing and that the effect is enhanced when that image is of the self. The results hold intriguing implications for body representation in social situations.

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

    PubMed

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

    2016-08-05

    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.

  19. 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

  20. 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.

  1. Multisensory integration of drumming actions: musical expertise affects perceived audiovisual asynchrony.

    PubMed

    Petrini, Karin; Dahl, Sofia; Rocchesso, Davide; Waadeland, Carl Haakon; Avanzini, Federico; Puce, Aina; Pollick, Frank E

    2009-09-01

    We investigated the effect of musical expertise on sensitivity to asynchrony for drumming point-light displays, which varied in their physical characteristics (Experiment 1) or in their degree of audiovisual congruency (Experiment 2). In Experiment 1, 21 repetitions of three tempos x three accents x nine audiovisual delays were presented to four jazz drummers and four novices. In Experiment 2, ten repetitions of two audiovisual incongruency conditions x nine audiovisual delays were presented to 13 drummers and 13 novices. Participants gave forced-choice judgments of audiovisual synchrony. The results of Experiment 1 show an enhancement in experts' ability to detect asynchrony, especially for slower drumming tempos. In Experiment 2 an increase in sensitivity to asynchrony was found for incongruent stimuli; this increase, however, is attributable only to the novice group. Altogether the results indicated that through musical practice we learn to ignore variations in stimulus characteristics that otherwise would affect our multisensory integration processes.

  2. 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.

  3. Integration of clinical point-of-care requirements in a DNA microarray genotyping test.

    PubMed

    Van Dorst, Bieke; Cremers, Amelieke; Jans, Karolien; Van Domburg, Trees; Steegen, Kim; Huang, Chengjun; Dorrer, Christian; Lagae, Liesbet; Ferwerda, Gerben; Stuyver, Lieven J

    2014-11-15

    Various proof-of-concept studies have shown the potential of biosensors with a high multiplex detection capability for the readout of DNA microarrays in a lab-on-a-chip. This is particularly interesting for the development of point-of-care genotyping tests, to screen for multiple pathogens and/or antibiotic resistance patterns. In this paper, an assay workflow is presented, suited for the development of novel lab-on-a-chips with an integrated DNA microarray. Besides the description of the different assay steps (DNA purification, amplification and detection), a control strategy is presented according to recommendations of the US Food and Drug Administration (FDA). To use a lab-on-a-chip for diagnostic applications, the optimization and evaluation of the assay performance with clinical samples is very important. Therefore, appropriate quantification methods are described, which allow optimization and evaluation of the separate assay steps, as well as total assay performance. In order to demonstrate and evaluate the total workflow, blood samples spiked with Streptococcus pneumoniae were tested. All blood samples with ≥ 10(3)CFU S. pneumoniae per ml of human blood were successfully detected by this genotyping assay.

  4. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells*

    PubMed Central

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-01-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host–pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick–Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These

  5. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

    PubMed

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-12-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results

  6. Integrative DNA, RNA, and protein evidence connects TREML4 to coronary artery calcification.

    PubMed

    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

    2014-07-03

    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.

  7. Soil drying procedure affects the DNA quantification of Lactarius vinosus but does not change the fungal community composition.

    PubMed

    Castaño, Carles; Parladé, Javier; Pera, Joan; Martínez de Aragón, Juan; Alday, Josu G; Bonet, José Antonio

    2016-11-01

    Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.

  8. 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

  9. Impairment of sensory-motor integration in patients affected by RLS.

    PubMed

    Rizzo, Vincenzo; Aricò, I; Liotta, G; Ricciardi, L; Mastroeni, C; Morgante, F; Allegra, R; Condurso, R; Girlanda, P; Silvestri, R; Quartarone, A

    2010-12-01

    Much evidence suggests that restless legs syndrome (RLS) is a disorder characterized by an unsuppressed response to sensory urges due to abnormalities in inhibitory pathways that specifically link sensory input and motor output. Therefore, in the present study, we tested sensory-motor integration in patients with RLS, measured by short latency afferent inhibition (SAI) and long latency afferent inhibition (LAI). SAI and LAI were determined using transcranial magnetic stimulation before and after 1 month of dopaminergic treatment in RLS patients. Ten naïve patients with idiopathic RLS and ten healthy age-matched controls were recruited. Patients with secondary causes for RLS (e.g. renal failure, anaemia, low iron and ferritin) were excluded, as well as those with other sleep disorders. Untreated RLS patients demonstrated deficient SAI in the human motor cortex, which proved revertible toward normal values after dopaminergic treatment. We demonstrated an alteration of sensory-motor integration, which is normalized by dopaminergic treatment, in patients affected by RLS. It is likely that the reduction of SAI might contribute significantly to the release of the involuntary movements and might account for the sensory urge typical of this condition.

  10. Visually Induced Inhibition of Return Affects the Integration of Auditory and Visual Information.

    PubMed

    Van der Stoep, N; Van der Stigchel, S; Nijboer, T C W; Spence, C

    2016-08-02

    Multisensory integration (MSI) and exogenous spatial attention can both speedup responses to perceptual events. Recently, it has been shown that audiovisual integration at exogenously attended locations is reduced relative to unattended locations. This effect was observed at short cue-target intervals (200-250 ms). At longer intervals, however, the initial benefits of exogenous shifts of spatial attention at the cued location are often replaced by response time (RT) costs (also known as Inhibition of Return, IOR). Given these opposing cueing effects at shorter versus longer intervals, we decided to investigate whether MSI would also be affected by IOR. Uninformative exogenous visual spatial cues were presented between 350 and 450 ms prior to the onset of auditory, visual, and audiovisual targets. As expected, IOR was observed for visual targets (invalid cue RT < valid cue RT). For auditory and audiovisual targets, neither IOR nor any spatial cueing effects were observed. The amount of relative multisensory response enhancement and race model inequality violation was larger for uncued as compared with cued locations indicating that IOR reduces MSI. The results are discussed in the context of changes in unisensory signal strength at cued as compared with uncued locations.

  11. 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

  12. 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.

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

    PubMed Central

    Xu, Ruiping; Ke, Yang

    2016-01-01

    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

  14. 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

    2016-02-02

    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.

  15. 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

  16. Integrative Review of the Supportive Care Needs of Arab People Affected by Cancer

    PubMed Central

    Alananzeh, Ibrahim; Levesque, Janelle; Kwok, Cannas; Everett, Bronwyn

    2016-01-01

    This review aimed to identify the unmet supportive care needs to conduct an integrative review of the literature, to identify the unmet supportive care needs of Arab people affected by cancer (patients and caregivers), and the impact of these needs on quality of life and psychosocial well-being. In July 2015 databases, search engines and electronic list servers were searched, with no limit on the year of publication. Reference lists of included articles and published reviews were also hand searched. Six studies met the inclusion criteria. Most studies examined the supportive care/unmet needs of Arab cancer patients and their family caregivers. Language, communication, information, and the need to get relief from dependency were the most frequently reported unmet needs among Arab cancer patients. For immigrant Arab patients, physical unmet needs were higher than other migrant groups and native Anglo-Australians. Arab caregivers’ unmet needs included concerns about providing suitable care for their family member, sharing their experience with other caregivers, obtaining information, and, in the case of pediatric cancers, dealing with siblings’ emotional reactions. The existing literature exploring the unmet supportive care needs of Arab people affected by cancer is limited suggesting that comprehensive studies are needed to enhance our understanding of these needs and to inform service planning. PMID:27981153

  17. 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.

  18. 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

  19. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

    PubMed

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  20. An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control.

    PubMed

    Inuzuka, M; Wada, Y

    1988-02-08

    Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

  1. Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site.

    PubMed

    Hasegawa, Maki; Yamaguchi, Shuichi; Aizawa, Shiro; Ikeda, Hidetoshi; Tatsumi, Kouichi; Noda, Yuko; Hirokawa, Katsuiku; Kitagawa, Masanobu

    2005-08-01

    Retroviral DNA integration is mediated by the viral protein integrase. However, elements of the host DNA repair machinery such as the phosphatidylinositol 3-kinase (PI-3K)-related protein kinase family system would play a role in the integration of viral DNA into the host DNA. Here, we show that a host PI-3K-related protein kinase, DNA-dependent protein kinase (DNA-PK), plays a role in the specific integration of retroviral DNA and induction of retroviral diseases in vivo. DNA-PK-deficient scid mice inoculated with Friend leukemia virus (FLV) exhibited a random integration into their genomic DNA and expressed the viral envelope protein gp70. However, the specific integration of FLV at Spi-1 or Fli-1 sites did not occur in association with the significant resistance of scid mice to FLV-induced leukemogenesis. In contrast, the knockout of another member of the PI-3K-related protein kinase family, encoded by the ataxia telangiectasia mutated (ATM) gene, resulted in mice as sensitive to FLV-induced leukemogenesis as the wild type mice. FLV was specifically integrated into the DNA at Spi-1 and Fli-1 sites with significant expression of these transcription factors. These findings indicated that DNA-PK would be essential for controlling the in vivo integration of FLV at specific sites as well as the susceptibility to FLV-induced leukemogenesis.

  2. Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

    PubMed Central

    Yi, Xia; de Vries, Hilda I.; Siudeja, Katarzyna; Rana, Anil; Lemstra, Willy; Brunsting, Jeanette F.; Kok, Rob M.; Smulders, Yvo M.; Schaefer, Matthias; Dijk, Freark; Shang, Yongfeng; Eggen, Bart J.L.; Kampinga, Harm H.

    2009-01-01

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress. PMID:19056684

  3. 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.

  4. 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

  5. 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

    2012-06-18

    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.

  6. 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)

  7. Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

    PubMed Central

    Morin, N; Delsert, C; Klessig, D F

    1989-01-01

    The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed. Images PMID:2585602

  8. How does the DNA sequence affect the Hill curve of transcriptional response?

    PubMed

    Sheinman, M; Kafri, Y

    2012-10-01

    The Hill coefficient is often used as a direct measure of the cooperativity of binding processes. It is an essential tool for probing properties of reactions in many biochemical systems. Here, we analyze existing experimental data and demonstrate that the Hill coefficient characterizing the binding of transcription factors to their cognate sites can in fact be larger than one-the standard indication of cooperativity-even in the absence of any standard cooperative binding mechanism. We demonstrate that this effect occurs due to the disordered binding energy of transcription factors to the DNA molecule and the steric repulsion between the different copies of the transcription factor. We show that the enhanced Hill coefficient implies a significant reduction in the number of copies of the transcription factors which is needed to occupy a cognate site and, in many cases, can explain existing estimates for number of copies of the transcription factors in cells.

  9. 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

    2015-03-05

    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.

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

    PubMed

    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.

  11. 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

  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.

  13. Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA.

    PubMed

    Dyda, Fred; Hickman, Alison B

    2015-01-01

    It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA transposition reactions and the crystal structure of the Cas1/Cas2 complex.

  14. 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.

  15. 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

  16. An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

    PubMed Central

    Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

    2012-01-01

    During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

  17. 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.

  18. 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

  19. Integrated analysis of gene expression and DNA methylation changes induced by hepatocyte growth factor in human hepatocytes.

    PubMed

    Xie, Cheng-Rong; Sun, Hongguang; Wang, Fu-Qiang; Li, Zhao; Yin, Yi-Rui; Fang, Qin-Liang; Sun, Yu; Zhao, Wen-Xiu; Zhang, Sheng; Zhao, Wen-Xing; Wang, Xiao-Min; Yin, Zhen-Yu

    2015-09-01

    Hepatocellular carcinoma (HCC) is the one of most common malignant tumors. The tumor microenvironment has a role in not only supporting growth and survival of tumor cells, but also triggering tumor recurrence and metastasis. Hepatocyte growth factor (HGF), one of the important growth factors in the tumor microenvironment, has an important role in angiogenesis, tumorigenesis and regeneration. However, the exact mechanism by which HGF regulates HCC initiation and development via epigenetic reprogramming has remained elusive. The present study focused on the epigenetic modification and target tumor-suppressive genes of HGF treatment in HCC. Expression profiling and DNA methylation array were performed to investigate the function of HGF and examine global genomic DNA methylation changes, respectively. Integrated analysis of gene expression and DNA methylation revealed potential tumor suppressor genes (TSGs) in HCC. The present study showed the multiple functions of HGF in tumorous and non‑tumorous pathways and global genomic DNA methylation changes. HGF treatment upregulated the expression of DNA methyltransferase 1 (DNMT1). Overexpression of DNMT1 in HCC patients correlated with the malignant potential and poor prognosis of HCC. Furthermore, integration analysis of gene expression and DNA methylation changes revealed novel potential tumor suppressor genes TSGs including MYOCD, PANX2 and LHX9. The present study has provided mechanistic insight into epigenetic repression of TSGs through HGF‑induced DNA hypermethylation.

  20. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

    PubMed Central

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-01

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity. PMID:28134787

  1. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

    PubMed

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-26

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  2. An Integrative Genomic Island Affects the Adaptations of the Piezophilic Hyperthermophilic Archaeon Pyrococcus yayanosii to High Temperature and High Hydrostatic Pressure

    PubMed Central

    Li, Zhen; Li, Xuegong; Xiao, Xiang; Xu, Jun

    2016-01-01

    Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure and sharp temperature and chemical gradients. Horizontal gene transfer is thought to play an important role in the microbial adaptation to such an extreme environment. In this study, a 21.4-kb DNA fragment was identified as a genomic island, designated PYG1, in the genomic sequence of the piezophilic hyperthermophile Pyrococcus yayanosii. According to the sequence alignment and functional annotation, the genes in PYG1 could tentatively be divided into five modules, with functions related to mobility, DNA repair, metabolic processes and the toxin-antitoxin system. Integrase can mediate the site-specific integration and excision of PYG1 in the chromosome of P. yayanosii A1. Gene replacement of PYG1 with a SimR cassette was successful. The growth of the mutant strain ΔPYG1 was compared with its parent strain P. yayanosii A2 under various stress conditions, including different pH, salinity, temperature, and hydrostatic pressure. The ΔPYG1 mutant strain showed reduced growth when grown at 100°C, while the biomass of ΔPYG1 increased significantly when cultured at 80 MPa. Differential expression of the genes in module III of PYG1 was observed under different temperature and pressure conditions. This study demonstrates the first example of an archaeal integrative genomic island that could affect the adaptation of the hyperthermophilic piezophile P. yayanosii to high temperature and high hydrostatic pressure. PMID:27965650

  3. An integrative approach to species discovery in odonates: from character-based DNA barcoding to ecology.

    PubMed

    Damm, Sandra; Schierwater, Bernd; Hadrys, Heike

    2010-09-01

    Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision-making. Such an approach can enhance both species identification and species discovery. The character-based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA-barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.

  4. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice

    PubMed Central

    Chabory, Eléonore; Damon, Christelle; Lenoir, Alain; Kauselmann, Gary; Kern, Hedrun; Zevnik, Branko; Garrel, Catherine; Saez, Fabrice; Cadet, Rémi; Henry-Berger, Joelle; Schoor, Michael; Gottwald, Ulrich; Habenicht, Ursula; Drevet, Joël R.; Vernet, Patrick

    2009-01-01

    The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5–/– epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5–/– male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability. PMID:19546506

  5. 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.

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

    PubMed Central

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

    2016-01-01

    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

  7. Drosophila bloom helicase maintains genome integrity by inhibiting recombination between divergent DNA sequences.

    PubMed

    Kappeler, Michael; Kranz, Elisabeth; Woolcock, Katrina; Georgiev, Oleg; Schaffner, Walter

    2008-12-01

    DNA double strand breaks (DSB) can be repaired either via a sequence independent joining of DNA ends or via homologous recombination. We established a detection system in Drosophila melanogaster to investigate the impact of sequence constraints on the usage of the homology based DSB repair via single strand annealing (SSA), which leads to recombination between direct repeats with concomitant loss of one repeat copy. First of all, we find the SSA frequency to be inversely proportional to the spacer length between the repeats, for spacers up to 2.4 kb in length. We further show that SSA between divergent repeats (homeologous SSA) is suppressed in cell cultures and in vivo in a sensitive manner, recognizing sequence divergences smaller than 0.5%. Finally, we demonstrate that the suppression of homeologous SSA depends on the Bloom helicase (Blm), encoded by the Drosophila gene mus309. Suppression of homeologous recombination is a novel function of Blm in ensuring genomic integrity, not described to date in mammalian systems. Unexpectedly, distinct from its function in Saccharomyces cerevisiae, the mismatch repair factor Msh2 encoded by spel1 does not suppress homeologous SSA in Drosophila.

  8. 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

    2016-08-02

    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.

  9. 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

  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. 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.

  12. An integrated telemedicine platform for the assessment of affective physiological states.

    PubMed

    Katsis, Christos D; Ganiatsas, George; Fotiadis, Dimitrios I

    2006-08-01

    AUBADE is an integrated platform built for the affective assessment of individuals. The system performs evaluation of the emotional state by classifying vectors of features extracted from: facial Electromyogram, Respiration, Electrodermal Activity and Electrocardiogram. The AUBADE system consists of: (a) a multisensorial wearable, (b) a data acquisition and wireless communication module, (c) a feature extraction module, (d) a 3D facial animation module which is used for the projection of the obtained data through a generic 3D face model; whereas the end-user will be able to view the facial expression of the subject in real time, (e) an intelligent emotion recognition module, and (f) the AUBADE databases where the acquired signals along with the subject's animation videos are saved. The system is designed to be applied to human subjects operating under extreme stress conditions, in particular car racing drivers, and also to patients suffering from neurological and psychological disorders. AUBADE's classification accuracy into five predefined emotional classes (high stress, low stress, disappointment, euphoria and neutral face) is 86.0%. The pilot system applications and components are being tested and evaluated on Maserati's car racing drivers.

  13. α-Synuclein Oligomers Induced by Docosahexaenoic Acid Affect Membrane Integrity

    PubMed Central

    Fecchio, Chiara; De Franceschi, Giorgia; Relini, Annalisa; Greggio, Elisa; Dalla Serra, Mauro; Bubacco, Luigi; Polverino de Laureto, Patrizia

    2013-01-01

    A key feature of Parkinson disease is the aggregation of α-synuclein and its intracellular deposition in fibrillar form. Increasing evidence suggests that the pathogenicity of α-synuclein is correlated with the activity of oligomers formed in the early stages of its aggregation process. Oligomers toxicity seems to be associated with both their ability to bind and affect the integrity of lipid membranes. Previously, we demonstrated that α-synuclein forms oligomeric species in the presence of docosahexaenoic acid and that these species are toxic to cells. Here we studied how interaction of these oligomers with membranes results in cell toxicity, using cellular membrane-mimetic and cell model systems. We found that α-synuclein oligomers are able to interact with large and small unilamellar negatively charged vesicles acquiring an increased amount of α-helical structure, which induces small molecules release. We explored the possibility that oligomers effects on membranes could be due to pore formation, to a detergent-like effect or to fibril growth on the membrane. Our biophysical and cellular findings are consistent with a model where α-synuclein oligomers are embedded into the lipid bilayer causing transient alteration of membrane permeability. PMID:24312431

  14. Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor

    PubMed Central

    Michel, Fabrice; Crucifix, Corinne; Granger, Florence; Eiler, Sylvia; Mouscadet, Jean-François; Korolev, Sergei; Agapkina, Julia; Ziganshin, Rustam; Gottikh, Marina; Nazabal, Alexis; Emiliani, Stéphane; Benarous, Richard; Moras, Dino; Schultz, Patrick; Ruff, Marc

    2009-01-01

    Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies. PMID:19229293

  15. Microsatellite DNA suggests that group size affects sex-biased dispersal patterns in red colobus monkeys.

    PubMed

    Miyamoto, Michael M; Allen, Julie M; Gogarten, Jan F; Chapman, Colin A

    2013-05-01

    Dispersal is a major life history trait of social organisms influencing the behavioral and genetic structure of their groups. Unfortunately, primate dispersal is difficult to quantify, because of the rarity of these events and our inability to ascertain if individuals dispersed or died when they disappear. Socioecological models have been partially developed to understand the ecological causes of different dispersal systems and their social consequences. However, these models have yielded confusing results when applied to folivores. The folivorous red colobus monkey (Procolobus rufomitratus) in Kibale National Park, Uganda is thought to exhibit female-biased dispersal, although both sexes have been observed to disperse and there remains considerable debate over the selective pressures favoring the transfers of males and females and the causes of variation in the proportion of each sex to leave the natal group. We circumvent this problem by using microsatellite DNA data to investigate the prediction that female dispersal will be more frequent in larger groups as compared to smaller ones. The rationale for this prediction is that red colobus exhibit increased within-group competition in bigger groups, which should favor higher female dispersal rates and ultimately lower female relatedness. Genetic data from two unequally sized neighboring groups of red colobus demonstrate increased female relatedness within the smaller group, suggesting females are less likely to disperse when there is less within-group competition. We suggest that the dispersal system is mediated to some degree by scramble competition and group size. Since red colobus group sizes have increased throughout Kibale by over 50% in the last decade, these changes may have major implications for the genetic structure and ultimately the population viability of this endangered primate.

  16. Microsatellite DNA Suggests that Group Size Affects Sex-biased Dispersal Patterns in Red Colobus Monkeys

    PubMed Central

    Miyamoto, Michael M.; Allen, Julie M.; Gogarten, Jan F.; Chapman, Colin A.

    2013-01-01

    Dispersal is a major life history trait of social organisms influencing the behavioral and genetic structure of their groups. Unfortunately, primate dispersal is difficult to quantify, because of the rarity of these events and our inability to ascertain if individuals dispersed or died when they disappear. Socioecological models have been partially developed to understand the ecological causes of different dispersal systems and their social consequences. However, these models have yielded confusing results when applied to folivores. The folivorous red colobus monkey (Procolobus rufomitratus) in Kibale National Park, Uganda is thought to exhibit female-biased dispersal, although both sexes have been observed to disperse and there remains considerable debate over the selective pressures favoring the transfers of males and females and the causes of variation in the proportion of each sex to leave the natal group. We circumvent this problem by using microsatellite DNA data to investigate the prediction that female dispersal will be more frequent in larger groups as compared to smaller ones. The rationale for this prediction is that red colobus exhibit increased within-group competition in bigger groups, which should favor higher female dispersal rates and ultimately lower female relatedness. Genetic data from two unequally sized neighboring groups of red colobus demonstrate increased female relatedness within the smaller group, suggesting females are less likely to disperse when there is less within-group competition. We suggest that the dispersal system is mediated to some degree by scramble competition and group size. Since red colobus group sizes have increased throughout Kibale by over 50% in the last decade, these changes may have major implications for the genetic structure and ultimately the population viability of this endangered primate. PMID:23307485

  17. Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

    PubMed

    Haghpanah, Tahereh; Salehi, Mohammad; Ghaffari Novin, Marefat; Masteri Farahani, Reza; Fadaei-Fathabadi, Fatemeh; Dehghani-Mohammadabadi, Maryam; Azimi, Hadi

    2016-01-01

    Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ≤30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p < 0.05). The rate of fertilization, cleavage, and embryo quality score were not significantly different between the two groups (all p > 0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p > 0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte.

  18. Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

    PubMed Central

    Pokrzywinski, Kaytee L.; Biel, Thomas G.; Kryndushkin, Dmitry; Rao, V. Ashutosh

    2016-01-01

    Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis. PMID:28030582

  19. Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation.

    PubMed

    Spencer, Shawal; Gugliotta, Agustina; Koenitzer, Jennifer; Hauser, Hansjörg; Wirth, Dagmar

    2015-02-10

    Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation.

  20. Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans.

    PubMed

    Wolff, J N; Nafisinia, M; Sutovsky, P; Ballard, J W O

    2013-01-01

    Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS-PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.

  1. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    PubMed Central

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications. PMID:28112157

  2. Microfluidic device for integrated restriction digestion reaction and resulting DNA fragment analysis.

    PubMed

    Xie, Hua; Li, Bowei; Zhong, Runtao; Qin, Jianhua; Zhu, Yisheng; Lin, Bingcheng

    2008-12-01

    A microfluidic system combining temperature-controlled reactor, analyte delivery, chip electrophoresis (CE) separation, and fluorescence detection was developed, in which the heaters, resistance temperature detectors, enzymatic reactors, CE channels, and pneumatic valves/pumps were integrated onto a single glass-PDMS chip. The microdevice was used to perform the digestion reaction, followed by on-line electrophoresis separation and detection of the resulting fragments with endonuclease BamHI and FokI as models. Pneumatic valves/pumps served not only for isolating the reaction region from the separation medium to prevent contamination, but also for delivering and quantitatively diluting the fluid from the reaction chamber to the CE section. Thus enzymatic reaction and electrophoresis separation could be insulated and connected as needed. A dynamic coating procedure with the use of PVP and mannitol was firstly adopted for glass-PDMS hybrid chip-based DNA separations, leading to an improved separation efficiency with reproducible migration time and theoretical plates. The expected 263- and 287-bp digestion products of BamHI and FokI were definitely verified by the size-based electrophoretic separation and detection. The whole integrated reaction-CE system can be manipulated in a simple manner with good reproducibility, which is expected to be applied in other on-line analysis of various biochemical reactions.

  3. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  4. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules.

    PubMed

    Wang, Chao; Nam, Sung-Wook; Cotte, John M; Jahnes, Christopher V; Colgan, Evan G; Bruce, Robert L; Brink, Markus; Lofaro, Michael F; Patel, Jyotica V; Gignac, Lynne M; Joseph, Eric A; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-23

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  5. 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…

  6. Variation in DNA repair gene XRCC3 affects susceptibility to astrocytomas and glioblastomas.

    PubMed

    Custódio, A C; Almeida, L O; Pinto, G R; Santos, M J; Almeida, J R W; Clara, C A; Rey, J A; Casartelli, C

    2012-02-10

    The gene XRCC3 (X-ray cross complementing group 3) has the task of repairing damage that occurs when there is recombination between homologous chromosomes. Repair of recombination between homologous chromosomes plays an important role in maintaining genome integrity, although it is known that double-strand breaks are the main inducers of chromosomal aberrations. Changes in the XRCC3 protein lead to an increase in errors in chromosome segregation due to defects in centrosomes, resulting in aneuploidy and other chromosomal aberrations, such as small increases in telomeres. We examined XRCC3 Thr241Met polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. The individuals of the control group (N = 100) were selected from the general population of the São Paulo State. Odds ratio and 95%CI were calculated using a logistic regression model. Patients who had the allele Met of the XRCC3 Thr241Met polymorphism had a significantly increased risk of tumor development (odds ratio = 3.13; 95% confidence interval = 1.50-6.50). There were no significant differences in overall survival of patients. We suggest that XRCC3 Thr241Met polymorphism is involved in susceptibility for developing astrocytomas and glioblastomas.

  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.

  8. 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.

  9. When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage.

    PubMed

    Serrano, Diego; D'Amours, Damien

    2014-09-01

    The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA damage response dominates over the biological drive to proliferate, and may even result in programmed cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is observed in all eukaryotic species where the process has been studied, including normal and cancer cells in humans. Polo-like kinases (PLKs) are critical regulators of the adaptation response to DNA damage and they play key roles at the interface of cell cycle and checkpoint-related decisions in cells. Here, we review recent progress in defining the specific roles of PLKs in the adaptation process and how this conserved family of eukaryotic kinases can integrate the fundamental need to preserve genomic integrity with effective cellular proliferation.

  10. Disruption of a DNA topoisomerase I gene affects morphogenesis in Arabidopsis.

    PubMed

    Takahashi, Taku; Matsuhara, Shio; Abe, Mitsutomo; Komeda, Yoshibumi

    2002-09-01

    The genesis of phyllotaxis, which often is associated with the Fibonacci series of numbers, is an old unsolved puzzle in plant morphogenesis. Here, we show that disruption of an Arabidopsis topoisomerase (topo) I gene named TOP1alpha affects phyllotaxis and plant architecture. The divergence angles and internode lengths between two successive flowers were more random in the top1alpha mutant than in the wild type. The top1alpha plants sporadically produced multiple flowers from one node, and the number of floral organ primordia often was different. The mutation also caused the twisting of inflorescences and individual flowers and the serration of leaf margins. These morphological abnormalities indicate that TOP1alpha may play a critical role in the maintenance of a regular pattern of organ initiation. The top1alpha mutant transformed with the RNA interference construct for TOP1beta, another topo I gene arrayed tandemly with TOP1alpha, was found to be lethal at young seedling stages, suggesting that topo I activity is essential in plants.

  11. 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, ...

  12. 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)

  13. 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

    2014-03-06

    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.

  14. 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

  15. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

    PubMed Central

    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 Hg2+ 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

  16. 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

  17. 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…

  18. Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes.

    PubMed

    Heinzmann, Julia; Mattern, Felix; Aldag, Patrick; Bernal-Ulloa, Sandra Milena; Schneider, Tamara; Haaf, Thomas; Niemann, Heiner

    2015-10-01

    To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4- to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans.

  19. 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

  20. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    PubMed

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  1. Structural integrity of resin-modified glass ionomers as affected by the delay or omission of light activation.

    PubMed

    de Gee, A J; Leloup, G; Werner, A; Vreven, J; Davidson, C L

    1998-08-01

    Since light activation of resin-modified glass ionomers as a means of polymerizing the HEMA is usually done shortly after mixing occurs, the acid-base reaction will proceed mainly within a formed HEMA-polymer matrix. Delaying or omitting light activation may alter the structure and consequently its integrity. The aim of this study was to investigate the effect on the structural integrity of Fuji II LC, Photac-Fil, and Vitremer by delaying or omitting light initiation as compared with the integrity when light activation is performed 2 min after mixing occurs. We evaluated integrity by three-body wear experiments, conducted 8 hrs after sample preparation, to establish the integrity in the early phase of hardening, as well as after 1 wk and after 4 mos, to follow the materials throughout the process of maturation. When light activation was delayed for 1 hr, the structural integrity of Fuji II LC and Photac-Fil improved significantly in the early stages of hardening. In the case of Vitremer, an hour's delay of light activation significantly decreased integrity, which declined further when light activation was omitted. Fuji II LC was not affected by the omission of light activation, while Photac-Fil was markedly weakened. After 4 mos of aging, most of the samples of each product which had been cured by the different methods attained equal integrity, with the exception of the non-light-activated Vitremer samples, which remained weaker. We concluded that the structural integrity of resin-modified glass-ionomer cements benefits from a chemical integration of the polyalkenoate and poly-HEMA networks, as in Vitremer. Improvement in the structural integrity in the early phase for cements with a mechanical entanglement of the matrices, as in Fuji II LC and Photac-Fil, requires an acid-base reaction, a considerable portion of which may take place before activation of the HEMA polymerization.

  2. 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

  3. 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…

  4. 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…

  5. Spiking Phineas Gage: A Neurocomputational Theory of Cognitive-Affective Integration in Decision Making

    ERIC Educational Resources Information Center

    Wagar, Brandon M.; Thagard, Paul

    2004-01-01

    The authors present a neurological theory of how cognitive information and emotional information are integrated in the nucleus accumbens during effective decision making. They describe how the nucleus accumbens acts as a gateway to integrate cognitive information from the ventromedial prefrontal cortex and the hippocampus with emotional…

  6. Factors Affecting Counselor Educators' Integration of Educational Technology: A Path Analysis

    ERIC Educational Resources Information Center

    Kennedy, John F.

    2011-01-01

    This study used path analysis to explore the effects of individual and institutional-level factors on counselor educators' integration of technology in counselor education. The study fills a gap in the literature by providing a research-based path model describing counselor educators' integration of technology in counselor education. Counselor…

  7. 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…

  8. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

    PubMed

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-11-15

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

  9. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

  10. 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.

  11. Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection.

    PubMed

    Men, Nguyen Thi; Kikuchi, Kazuhiro; Nakai, Michiko; Fukuda, Atsunori; Tanihara, Fuminori; Noguchi, Junko; Kaneko, Hiroyuki; Linh, Nguyen Viet; Nguyen, Bui Xuan; Nagai, Takashi; Tajima, Atsushi

    2013-12-01

    Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the in vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI.

  12. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

    PubMed Central

    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  13. 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.

  14. Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

    PubMed

    Hatano, Takashi; Sano, Daisuke; Takahashi, Hideaki; Hyakusoku, Hiroshi; Isono, Yasuhiro; Shimada, Shoko; Sawakuma, Kae; Takada, Kentaro; Oikawa, Ritsuko; Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Itoh, Fumio; Myers, Jeffrey N; Oridate, Nobuhiko

    2017-04-01

    Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

  15. An Unnecessary Divorce: Integrating the Study of Affect and Emotion in New Media

    ERIC Educational Resources Information Center

    Nelson, Julie D.

    2016-01-01

    Rhetoric and composition scholars' almost exclusive reliance on Brian Massumi's definition of affect has spurred a theoretical and practical divorce between "affect" and "emotion" in our field. This article returns to Lynn Worsham's "Going Postal" and argues that to fully scrutinize and respond to what she calls…

  16. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo

    PubMed Central

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L.; Stavans, Joel

    2014-01-01

    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

  17. Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

    PubMed

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

    2011-02-07

    Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

  18. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics.

    PubMed

    Doolittle-Hall, Janet M; Cunningham Glasspoole, Danielle L; Seaman, William T; Webster-Cyriaque, Jennifer

    2015-11-10

    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.

  19. 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

  20. Inhibitors of Apoptosis Affect DNA Degradation and Repair in Sulfur Mustard (HD)-Exposed Human Epidermal Keratinocytes (HEK)

    DTIC Science & Technology

    2003-07-01

    accompanied by DNA ligase I activation via DNA-dependent protein kinase (DNA-PK) mediated phosphorylation, and is retarded in the presence of a poly (ADP...ATCC No. HB 11726). Bovine DNA ligase I monoclonal antibody was a kind gift from Dr. Tomas Lindahl of the Imperial Cancer Research Fund, UK...metabolic 33P labeling of DNA ligase in HEK and other cells: The experimental and control cells were washed with 37oC saline and then exposed to 1 mM HD

  1. Diet and Cell Size Both Affect Queen-Worker Differentiation through DNA Methylation in Honey Bees (Apis mellifera, Apidae)

    PubMed Central

    Shi, Yuan Yuan; Huang, Zachary Y.; Zeng, Zhi Jiang; Wang, Zi Long; Wu, Xiao Bo; Yan, Wei Yu

    2011-01-01

    Background Young larvae of the honey bee (Apis mellifera) are totipotent; they can become either queens (reproductives) or workers (largely sterile helpers). DNA methylation has been shown to play an important role in this differentiation. In this study, we examine the contributions of diet and cell size to caste differentiation. Methodology/Principal Findings We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62; as well as morphological characteristics of adult bees developed either from larvae fed with worker jelly or royal jelly; and larvae raised in either queen or worker cells. We show that both diet type and cell size contributed to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62. Conclusions/Significance We confirm previous findings that Dnmt3 plays a critical role in honey bee caste differentiation. Further, we show for the first time that cell size also plays a role in influencing larval development when diet is kept the same. PMID:21541319

  2. Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology.

    PubMed

    Azakami, Hiroyuki; Akimichi, Hiromi; Usui, Masakatsu; Yumoto, Hiromichi; Ebisu, Shigeyuki; Kato, Akio

    2005-05-23

    Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.

  3. DNA Methylation Affects the Efficiency of Transcription Activator-Like Effector Nucleases-Mediated Genome Editing in Rice

    PubMed Central

    Kaya, Hidetaka; Numa, Hisataka; Nishizawa-Yokoi, Ayako; Toki, Seiichi; Habu, Yoshiki

    2017-01-01

    Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N∗) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N∗ module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N∗ modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N∗-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N∗-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs. PMID:28348570

  4. 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

  5. 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.

  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

    2013-04-07

    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. Negative affect predicts social functioning across schizophrenia and bipolar disorder: Findings from an integrated data analysis.

    PubMed

    Grove, Tyler B; Tso, Ivy F; Chun, Jinsoo; Mueller, Savanna A; Taylor, Stephan F; Ellingrod, Vicki L; McInnis, Melvin G; Deldin, Patricia J

    2016-09-30

    Most people with a serious mental illness experience significant functional impairment despite ongoing pharmacological treatment. Thus, in order to improve outcomes, a better understanding of functional predictors is needed. This study examined negative affect, a construct comprised of negative emotional experience, as a predictor of social functioning across serious mental illnesses. One hundred twenty-seven participants with schizophrenia, 113 with schizoaffective disorder, 22 with psychosis not otherwise specified, 58 with bipolar disorder, and 84 healthy controls (N=404) completed self-report negative affect measures. Elevated levels of negative affect were observed in clinical participants compared with healthy controls. For both clinical and healthy control participants, negative affect measures were significantly correlated with social functioning, and consistently explained significant amounts of variance in functioning. For clinical participants, this relationship persisted even after accounting for cognition and positive/negative symptoms. The findings suggest that negative affect is a strong predictor of outcome across these populations and treatment of serious mental illnesses should target elevated negative affect in addition to cognition and positive/negative symptoms.

  8. 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.

  9. 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

  10. Polymorphisms in DNA polymerase γ affect the mtDNA stability and the NRTI-induced mitochondrial toxicity in Saccharomyces cerevisiae

    PubMed Central

    Baruffini, Enrico; Ferrari, Jessica; Dallabona, Cristina; Donnini, Claudia; Lodi, Tiziana

    2015-01-01

    Several pathological mutations have been identified in human POLG gene, encoding for the catalytic subunit of Pol γ, the solely mitochondrial replicase in animals and fungi. However, little is known regarding non-pathological polymorphisms found in this gene. Here we studied, in the yeast model Saccharomyces cerevisiae, eight human polymorphisms. We found that most of them are not neutral but enhanced both mtDNA extended mutability and the accumulation of mtDNA point mutations, either alone or in combination with a pathological mutation. In addition, we found that the presence of some SNPs increased the stavudine and/or zalcitabine-induced mtDNA mutability and instability. PMID:25462018

  11. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

    SciTech Connect

    Peng, Jamy C.

    2007-01-01

    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

  12. 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

  13. 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…

  14. Report: Recommendation to Strengthen Management Integrity Processes Affecting Recovery Act Activities

    EPA Pesticide Factsheets

    Report #09-X-0145, April 27, 2009. The EPA OIG has completed a review of the Agency’s Fiscal Year (FY) 2009 Management Integrity guidance for reporting on internal control reviews and preparing the annual assurance letters sent to the Administrator.

  15. Factors affecting implant mobility at placement and integration of mobile implants at uncovering.

    PubMed

    Orenstein, I H; Tarnow, D P; Morris, H F; Ochi, S

    1998-12-01

    This study examined 1) factors that contributed to implant stability at placement and 2) the likelihood for an implant that was mobile at placement to osseointegrate. Eighty-one (3.1%) of 2,641 implants placed by the Dental Implant Clinical Research Group between 1991 and 1995 were found to be mobile at placement. Seventy-six (93.8%) of the 81 mobile implants were integrated at uncovering compared to 97.5% for the 2,560 immobile implants. Variables that influenced mobility at placement included patient age, implant design and material, anterior-posterior jaw location, bone density, and use of a bone tap. Hydroxyapatite (HA)-coated implants were slightly more likely to be mobile at placement (P = 0.324) than non-hydroxypatite (HA)-coated implants. Of the 54 HA-coated implants that were mobile at placement, all (100%) integrated, while only 17 (81.5%) of the 22 mobile non-HA-coated implants integrated (P = 0.003). Mean electronic mobility testing device values (PTVs) at uncovering for all implants mobile or immobile at placement that integrated were -2.9 and -3.6 respectively. PTVs for HA-coated implants that were mobile (-3.5 PTV) or immobile (-4.0 PTV) at placement differed by 0.5 PTV, whereas non-HA-coated implants exhibited a greater difference of 1.2 PTVs at uncovering. HA-coated implants, regardless of mobility at placement, integrated more frequently and exhibited greater stability than non HA-coated implants.

  16. Fabrication and characterization of high-K dielectric integrated silicon nanowire sensor for DNA sensing application (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Jayakumar, Ganesh; Legallais, Maxime; Hellström, Per-Erik; Mouis, Mireille; Stambouli, Valérie; Ternon, Céline; Östling, Mikael

    2016-09-01

    1D silicon nanowires (SiNW) are attractive for charge based DNA sensing applications due to their small size and large surface to volume ratio. An ideal portable biosensor is expected to have repeatable and reliable sensitivity, selectivity, low production cost and small feature size. Instead of using tools such as e-beam that are capital and time intensive, we propose a low cost CMOS self-aligned-double-patterning I-line lithography process to fabricate 60 nm wide SiNW. DNA probes are grafted on a thin dielectric layer that is deposited on top of the SiNW surface. Here we used HfO2 instead of the usual SiO2. Indeed, compared to SiO2, HfO2 has been reported to have higher amount of OH groups on its surface leading to enhanced signal quality. We also report preliminary biosensor characterizations. After HfO2 functionalization and single-stranded DNA probe grafting onto the SiNWs, the sensors were first put in contact with fluorophore labelled complementary DNA targets in order to test the efficiency of DNA hybridization optically. Then, a sequence of hybridization, de-hybridization and re-hybridization steps was followed by Id-Vg measurements in order to measure the electrical response of the sensors to target DNA as well as recycling capability. After each step, SiNW devices exhibited a threshold voltage shift larger than device-to-device dispersion, showing that both complementary DNA hybridization and de-hybridization can be electrically detected. These results are very encouraging as they open new frontiers for heterogeneous integration of liquid interacting array of nano sensors with CMOS circuits to fabricate a complete lab on chip.

  17. Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities.

    PubMed

    Bennetts, L E; De Iuliis, G N; Nixon, B; Kime, M; Zelski, K; McVicar, C M; Lewis, S E; Aitken, R J

    2008-05-10

    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male

  18. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®.

    PubMed

    Lee, Sin Hang

    2014-03-01

    Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.

  19. 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

  20. 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...

  1. The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

    PubMed

    O'Driscoll, Mark

    2017-01-01

    Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  2. Elg1 forms an alternative RFC complex important for DNA replication and genome integrity.

    PubMed

    Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

    2003-08-15

    Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.

  3. A model for the control of DNA integrity by the sperm nuclear matrix

    PubMed Central

    Gawecka, Joanna E; Ribas-Maynou, Jordi; Benet, Jordi; Ward, W Steven

    2015-01-01

    The highly condensed chromatin of mammalian spermatozoa is usually considered to be biologically inert before fertilization. However, we have demonstrated that even in this compacted state, sperm chromatin is subject to degradation at open configurations associated with the nuclear matrix through a process we have termed sperm chromatin fragmentation (SCF). This suggests that a mechanism exists to monitor the health of spermatozoa during transit through the male reproductive tract and to destroy the genome of defective sperm cells. The site of DNA damage in SCF, the matrix attachment sites, are the same that we hypothesize initiate DNA synthesis in the zygote. When sperm that have damaged DNA are injected into the oocyte, the newly created zygote responds by delaying DNA synthesis in the male pronucleus and, if the damage is severe enough, arresting the embryo's development. Here we present a model for paternal DNA regulation by the nuclear matrix that begins during sperm maturation and continues through early embryonic development. PMID:25926613

  4. 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…

  5. Longitudinal Evaluation of the Structural Integrity of Teeth Affected by Molar Incisor Hypomineralisation.

    PubMed

    Bullio Fragelli, Camila Maria; Jeremias, Fabiano; Feltrin de Souza, Juliana; Paschoal, Marco Aurélio; de Cássia Loiola Cordeiro, Rita; Santos-Pinto, Lourdes

    2015-01-01

    The aim of this prospective cohort study was to evaluate the risk of posteruptive breakdown and the development of caries lesions in teeth with molar incisor hypomineralisation (MIH). A total of 367 permanent incisors and first molars, affected and not affected by MIH lesions, of 45 children with MIH from Araraquara, São Paulo, Brazil, were evaluated at intervals from 6 to 12 months by assessing the severity of MIH, the presence of tooth caries lesions and the treatment needed. During the study period, all patients received preventive care. The data were analysed using Fisher's exact test and actuarial method survival analysis. Significant associations were also found in teeth between the presence of MIH and a DMFT index >0 in all periods and also between the need for treatment and the presence of MIH. The teeth affected by MIH opacities were healthy in 99% of incisors and 93% of molars at the end of the 12-month period. Due to the high likelihood of maintaining the tooth structure in opacities, the complete or premature removal of the affected area is not justified.

  6. 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.

  7. 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

  8. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

    PubMed

    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.

  9. 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

  10. Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models.

    PubMed

    Bonczkowski, Pawel; De Scheerder, Marie-Angélique; Malatinkova, Eva; Borch, Alexandra; Melkova, Zora; Koenig, Renate; De Spiegelaere, Ward; Vandekerckhove, Linos

    2016-12-02

    To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency.

  11. Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

    PubMed Central

    Giunta, Simona

    2017-01-01

    Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation. PMID:28167779

  12. Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T.

    PubMed

    Giunta, Simona; Funabiki, Hironori

    2017-02-21

    Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

  13. Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

    PubMed Central

    Bonczkowski, Pawel; De Scheerder, Marie-Angélique; Malatinkova, Eva; Borch, Alexandra; Melkova, Zora; Koenig, Renate; De Spiegelaere, Ward; Vandekerckhove, Linos

    2016-01-01

    To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency. PMID:27910923

  14. H4K16 acetylation affects recombination and ncRNA transcription at rDNA in Saccharomyces cerevisiae.

    PubMed

    Cesarini, Elisa; D'Alfonso, Anna; Camilloni, Giorgio

    2012-07-01

    Transcription-associated recombination is an important process involved in several aspects of cell physiology. In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae, RNA polymerase II transcription-dependent recombination has been demonstrated among the repeated units. In this study, we investigate the mechanisms controlling this process at the chromatin level. On the basis of a small biased screening, we found that mutants of histone deacetylases and chromatin architectural proteins alter both the amount of Pol II-dependent noncoding transcripts and recombination products at rDNA in a coordinated manner. Of interest, chromatin immunoprecipitation analyses in these mutants revealed a corresponding variation of the histone H4 acetylation along the rDNA repeat, particularly at Lys-16. Here we provide evidence that a single, rapid, and reversible posttranslational modification-the acetylation of the H4K16 residue-is involved in the coordination of transcription and recombination at rDNA.

  15. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication

    PubMed Central

    Albert, Benjamin; Hafner, Lukas; Lezaja, Aleksandra; Costanzo, Michael; Boone, Charlie; Shore, David

    2016-01-01

    The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common. PMID:27820830

  16. 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

  17. 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

  18. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    PubMed

    Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B; Kato, Takamitsu A; Shih, Hung-Ying; Xie, Xian-Jin; Wilson, Paul F; Brogan, John R; Kurimasa, Akihiro; Chen, David J; Bedford, Joel S; Chen, Benjamin P C

    2014-01-01

    We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  19. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence

    PubMed Central

    Lorenc, Michał T.; Dudley, Kevin J.; Hellens, Roger P.

    2017-01-01

    Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5’ terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker. PMID:28231340

  20. The widely used Nicotiana benthamiana 16c line has an unusual T-DNA integration pattern including a transposon sequence.

    PubMed

    Philips, Joshua G; Naim, Fatima; Lorenc, Michał T; Dudley, Kevin J; Hellens, Roger P; Waterhouse, Peter M

    2017-01-01

    Nicotiana benthamiana is employed around the world for many types of research and one transgenic line has been used more extensively than any other. This line, 16c, expresses the Aequorea victoria green fluorescent protein (GFP), highly and constitutively, and has been a major resource for visualising the mobility and actions of small RNAs. Insights into the mechanisms studied at a molecular level in N. benthamiana 16c are likely to be deeper and more accurate with a greater knowledge of the GFP gene integration site. Therefore, using next generation sequencing, genome mapping and local alignment, we identified the location and characteristics of the integrated T-DNA. As suggested from previous molecular hybridisation and inheritance data, the transgenic line contains a single GFP-expressing locus. However, the GFP coding sequence differs from that originally reported. Furthermore, a 3.2 kb portion of a transposon, appears to have co-integrated with the T-DNA. The location of the integration mapped to a region of the genome represented by Nbv0.5scaffold4905 in the www.benthgenome.com assembly, and with less integrity to Niben101Scf03641 in the www.solgenomics.net assembly. The transposon is not endogenous to laboratory strains of N. benthamiana or Agrobacterium tumefaciens strain GV3101 (MP90), which was reportedly used in the generation of line 16c. However, it is present in the popular LBA4404 strain. The integrated transposon sequence includes its 5' terminal repeat and a transposase gene, and is immediately adjacent to the GFP gene. This unexpected genetic arrangement may contribute to the characteristics that have made the 16c line such a popular research tool and alerts researchers, taking transgenic plants to commercial release, to be aware of this genomic hitchhiker.

  1. Connecting the dots: how local structure affects global integration in infants.

    PubMed

    Palomares, Melanie; Pettet, Mark; Vildavski, Vladimir; Hou, Chuan; Norcia, Anthony

    2010-07-01

    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- to 5.5-month-old infants are sensitive to the global structure of Glass patterns by measuring visual-evoked potentials. 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.

  2. 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

  3. Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis▿

    PubMed Central

    Ward, Katherine N.; Leong, Hoe Nam; Thiruchelvam, Anton D.; Atkinson, Claire E.; Clark, Duncan A.

    2007-01-01

    The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log10 copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log10 copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (≥3.5 log10 copies/ml) or whole blood (≥6.0 log10 copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log10 copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log10 copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood. PMID:17229866

  4. Nucleoid organization and the maintenance of DNA integrity in E. coli, B. subtilis and D. radiodurans.

    PubMed

    Frenkiel-Krispin, Daphna; Minsky, Abraham

    2006-11-01

    For enzymatic activities to be effectively carried out, basic prerequisites must be met. Many enzymatic tasks require continuous consumption and dissipation of energy, sometimes in massive amounts. Some activities, such as DNA replication, transcription, and repair through homologous recombination rely upon templates that provide the information required for these transactions. Yet, circumstances where intracellular energy pools are severely depleted, or where intact templates are not available, frequently occur. Moreover, the fact that in order to reach their targets, enzymes must cope with an extremely crowded and viscous cellular milieu that drastically slows down their diffusion is often neglected. These impediments are particularly evident under stress conditions such as prolonged starvation or continuous exposure to DNA-damaging agents. Here we survey recent studies, which imply that when enzymatically-mediated DNA repair pathways are hindered, alternative strategies are deployed, whose common denominator is the reorganization of bacterial nucleoids into morphologies that promote DNA repair and protection.

  5. 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.

  6. 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...

  7. Maintaining Genetic Integrity Under Extreme Conditions: Novel DNA Damage Repair Biology in the Archaea

    DTIC Science & Technology

    2013-11-23

    is a nuclease while Rad50 consists of two globular DNA-binding domains. Together, these two proteins form a complex that binds to and tethers DNA...with nre, deleting the rad50 and mre11 genes in H. volcanii results in increased resistance to phleomycin. Under these circumstances, there is also...equivalent, otherwise ∆nre cells would be resistant rather than sensitive to phleomycin. Instead we hypothesise that Nre acts at a late stage in the

  8. 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

  9. Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

    PubMed Central

    Lorenzi, Julio C. C.; Cohen, Yehuda Z.; Cohn, Lillian B.; Kreider, Edward F.; Oliveira, Thiago; Lavine, Christy L.; Horwitz, Joshua A.; Settler, Allison; Jankovic, Mila; Seaman, Michael S.; Chakraborty, Arup K.; Hahn, Beatrice H.; Caskey, Marina; Nussenzweig, Michel C.

    2016-01-01

    HIV-1–infected individuals harbor a latent reservoir of infected CD4+ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones. PMID:27872306

  10. 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

  11. Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA.

    PubMed

    Lorenzi, Julio C C; Cohen, Yehuda Z; Cohn, Lillian B; Kreider, Edward F; Barton, John P; Learn, Gerald H; Oliveira, Thiago; Lavine, Christy L; Horwitz, Joshua A; Settler, Allison; Jankovic, Mila; Seaman, Michael S; Chakraborty, Arup K; Hahn, Beatrice H; Caskey, Marina; Nussenzweig, Michel C

    2016-12-06

    HIV-1-infected individuals harbor a latent reservoir of infected CD4(+) T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.

  12. 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

  13. 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

  14. Integration of DNA Copy Number Alterations and Transcriptional Expression Analysis in Human Gastric Cancer

    PubMed Central

    Coral, Ho; Yuen, Siu Tsan; Chu, Kent Man; Law, Simon; Zhang, Lianhai; Ji, Jiafu; Leung, Suet Yi; Chen, Xin

    2012-01-01

    Background Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level. Principal Findings We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis. Conclusions This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic

  15. Vaccinia-related kinase 1 (VRK1) confers resistance to DNA-damaging agents in human breast cancer by affecting DNA damage response

    PubMed Central

    Salzano, Marcella; Vázquez-Cedeira, Marta; Sanz-García, Marta; Valbuena, Alberto; Blanco, Sandra; Fernández, Isabel F.; Lazo, Pedro A.

    2014-01-01

    Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs. PMID:24731990

  16. 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.

  17. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot?

    PubMed Central

    Moraes, C T; Ciacci, F; Bonilla, E; Jansen, C; Hirano, M; Rao, N; Lovelace, R E; Rowland, L P; Schon, E A; DiMauro, S

    1993-01-01

    We identified two patients with pathogenic single nucleotide changes in two different mitochondrial tRNA genes: the first mutation in the tRNA(Asn) gene, and the ninth known mutation in the tRNA(Leu(UUR)) gene. The mutation in tRNA(Asn) was associated with isolated ophthalmoplegia, whereas the mutation in tRNA(Leu(UUR)) caused a neurological syndrome resembling MERRF (myoclonus epilepsy and ragged-red fibers) plus optic neuropathy, retinopathy, and diabetes. Both mutations were heteroplasmic, with higher percentages of mutant mtDNA in affected tissues, and undetectable levels in maternal relatives. Analysis of single muscle fibers indicated that morphological and biochemical alterations appeared only when the proportions of mutant mtDNA exceeded 90% of the total cellular mtDNA pool. The high incidence of mutations in the tRNA(Leu(UUR)) gene suggests that this region is an "etiologic hot spot" in mitochondrial disease. Images PMID:8254046

  18. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis.

    PubMed

    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

    2014-07-01

    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.

  19. 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

  20. Lycopene and resveratrol improve post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA integrity.

    PubMed

    Bucak, M N; Ataman, M B; Başpınar, N; Uysal, O; Taşpınar, M; Bilgili, A; Öztürk, C; Güngör, Ş; İnanç, M E; Akal, E

    2015-06-01

    We focussed on evaluating the protective effect of lycopene and resveratrol on post-thaw bull sperm and oxidative stress parameters. Nine ejaculates for each bull were used in the study. Each ejaculate, splitted into three equal aliquots and diluted at 37 °C with base extenders containing lycopene (1 × 10(-3)  g ml(-1) ) and resveratrol (1 mm), and no antioxidant (control), was cooled to 5 °C and then frozen. Frozen straws were thawed in a water bath for evaluation. The supplementation of the semen extender with lycopene and resveratrol increased the percentages of post-thawed computer-assisted sperm analysis (CASA) motility (55.8 ± 3.8 and 61.9 ± 4.0%) and progressive motility (38 ± 2.4 and 37 ± 8.8), compared with the controls (50.7 ± 2.65 and 33.3 ± 3.74%, respectively, P < 0.05). Resveratrol provided a higher ALH (4.3 ± 0.1), in comparison with the control (3.9 ± 0.3, P < 0.05). The supplementation of the semen extender with lycopene and resveratrol produced a higher mitochondrial activity (24.6 ± 2.9 and 30.1 ± 6.5% respectively), compared with that of the control (11.8 ± 9.5%, P < 0.05). It was determined that both antioxidants resulted in a lower percentage of sperm with damaged DNA than that of the control (P < 0.05). Sperm motion characteristics except for ALH, acrosome integrity, sperm viability and oxidative stress parameters were not affected by the adding of lycopene and resveratrol.

  1. Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity.

    PubMed Central

    Buetti, E

    1994-01-01

    In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. Images PMID:8289800

  2. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections.

    PubMed

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-03-23

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals.

  3. 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.

  4. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    PubMed Central

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  5. 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%.

  6. DNA Replication Is an Integral Part of the Mouse Oocyte’s Reprogramming Machinery

    PubMed Central

    Wang, Bingyuan; Pfeiffer, Martin J.; Schwarzer, Caroline; Araúzo-Bravo, Marcos J.; Boiani, Michele

    2014-01-01

    Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24–36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle. PMID:24836291

  7. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    PubMed

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  8. 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.

  9. 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

  10. Anticoagulant factor V: factors affecting the integration of novel scientific discoveries into the broader framework.

    PubMed

    LaBonte, Michelle L

    2014-09-01

    Since its initial discovery in the 1940s, factor V has long been viewed as an important procoagulant protein in the coagulation cascade. However, in the later part of the 20th century, two different scientists proposed novel anticoagulant roles for factor V. Philip Majerus proposed the first anticoagulant function for factor V in 1983, yet ultimately it was not widely accepted by the broader scientific community. In contrast, Björn Dahlbäck proposed a different anticoagulant role for factor V in 1994. While this role was initially contested, it was ultimately accepted and integrated into the scientific framework. In this paper, I present a detailed historical account of these two anticoagulant discoveries and propose three key reasons why Dahlbäck's anticoagulant role for factor V was accepted whereas Majerus' proposed role was largely overlooked. Perhaps most importantly, Dahlbäck's proposed anticoagulant role was of great clinical interest because the discovery involved the study of an important subset of patients with thrombophilia. Soon after Dahlbäck's 1994 work, this patient population was shown to possess the factor V Leiden mutation. Also key in the ultimate acceptance of the second proposed anticoagulant role was the persistence of the scientist who made the discovery and the interest in and ability of others to replicate and reinforce this work. This analysis of two different yet similar discoveries sheds light on factors that play an important role in how new discoveries are incorporated into the existing scientific framework.

  11. Integration of Ethylene and Light Signaling Affects Hypocotyl Growth in Arabidopsis

    PubMed Central

    Yu, Yanwen; Huang, Rongfeng

    2017-01-01

    As an ideal model for studying ethylene effects on cell elongation, Arabidopsis hypocotyl growth is widely used due to the unique characteristic that ethylene stimulates hypocotyl elongation in the light but inhibits it in the dark. Although the contrasting effect of ethylene on hypocotyl growth has long been known, the molecular basis of this effect has only gradually been identified in recent years. In the light, ethylene promotes the expression of PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and the degradation of ELONGATED HYPOCOTYL 5 (HY5) protein, thus stimulating hypocotyl growth. In the dark, ETHYLENE RESPONSE FACTOR 1 (ERF1) and WAVE-DAMPENED 5 (WDL5) induced by ethylene are responsible for its inhibitory effect on hypocotyl elongation. Moreover, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and PHYTOCHROME B (phyB) mediate the light-suppressed ethylene response in different ways. Here, we review several pivotal advances associated with ethylene-regulated hypocotyl elongation, focusing on the integration of ethylene and light signaling during seedling emergence from the soil. PMID:28174592

  12. Does the Agaricus blazei Murill mushroom have properties that affect the immune system? An integrative review.

    PubMed

    Lima, Cristiane Urcina Joanna Oliveira; Cordova, Cláudio Olavo de Almeida; Nóbrega, Otávio de Tolêdo; Funghetto, Silvana Schwerz; Karnikowski, Margô Gomes de Oliveira

    2011-01-01

    There has been a significant increase in the use of mushrooms for therapeutic and medicinal purposes, in particular, use of the species Agaricus blazei Murrill, a basidiomycota of Brazilian origin. The objective of this study was to identify scientific evidence regarding the influence of A. blazei Murrill on the immune system. We undertook an integrative review of indexed publications published between 2000 and 2009, using the following question as a guideline: "What evidence can be found in the literature regarding the influence of A. blazei Murrill on the immune system?" Fourteen studies verified that there is in vitro and in vivo research demonstrating this mushroom's influence on the immune system. All research was characterized as evidence level 7 (preclinical study [animals/in vitro]). The research shows that A. blazei Murrill functions through bioactive compounds via mechanisms that are not yet entirely clear, although it has been shown that they promote action on the innate and adaptive immunological response, activation of the complement system, and synthesis of pro- and anti-inflammatory cytokines and even aid in diapedesis. Despite broad scientific evidence demonstrating relevant immunomodulatory properties of A. blazei Murrill, randomized clinical trials with human subjects are still needed in order for the mushroom to be put into clinical practice.

  13. 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

  14. Optimization of RNA isolation from Brittle Leaf Disease affected date palm leaves and construction of a subtractive cDNA library.

    PubMed

    Saïdi, Mohammed Najib; Gargouri-Bouzid, Radhia; Rayanni, Mariem; Drira, Noureddine

    2009-01-01

    A simple and efficient method was described here for the isolation of high-quality RNA from date palm leaves affected with Brittle Leaf Disease (BLD) and containing high amount of phenolic compounds. The procedure was based on the use of a non-ionic detergent Nonidet-P40 (NP-40), Polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in the extraction buffer in order to isolate cytoplasmic RNA and to prevent the oxidation of phenolic compounds. This method allowed the isolation of intact RNA, suitable for cDNA synthesis and library construction. Differential screening of the subtractive cDNA library from affected leaf RNA led to the identification of some BLD-induced genes.

  15. Integrative analysis of DNA methylation and gene expression data identifies EPAS1 as a key regulator of COPD.

    PubMed

    Yoo, Seungyeul; Takikawa, Sachiko; Geraghty, Patrick; Argmann, Carmen; Campbell, Joshua; Lin, Luan; Huang, Tao; Tu, Zhidong; Foronjy, Robert F; Feronjy, Robert; Spira, Avrum; Schadt, Eric E; Powell, Charles A; Zhu, Jun

    2015-01-01

    Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a 'causal' role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.

  16. Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

    PubMed Central

    Reijns, Martin A.M.; Rabe, Björn; Rigby, Rachel E.; Mill, Pleasantine; Astell, Katy R.; Lettice, Laura A.; Boyle, Shelagh; Leitch, Andrea; Keighren, Margaret; Kilanowski, Fiona; Devenney, Paul S.; Sexton, David; Grimes, Graeme; Holt, Ian J.; Hill, Robert E.; Taylor, Martin S.; Lawson, Kirstie A.; Dorin, Julia R.; Jackson, Andrew P.

    2012-01-01

    Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells. PMID:22579044

  17. An integrated system of ABO typing and multiplex STR testing for forensic DNA analysis.

    PubMed

    Jiang, Xianhua; He, Juan; Jia, Fei; Shen, Hongying; Zhao, Jinling; Chen, Chuguang; Bai, Liping; Liu, Feng; Hou, Guangwei; Guo, Faye

    2012-12-01

    A new amplification system for ABO and STR genotyping in a single reaction has been successfully developed. Two types of information can be obtained from a biological sample at one time. One is the classical information of ABO blood group typing for screening suspects and the other is STR information for individual identification. The system allows for the simultaneous detection of 15 autosomal STR loci (containing all CODIS STR loci as well as Penta D and Penta E), six ABO genotypes (O/O, B/B, A/A, A/O, A/B, and B/O) and the gender-determining locus Amelogenin. Primers are designed so that the amplicons are distributed ranging from 75bp to 500bp within a four-dye fluorescent design, leaving a fourth dye for the internal size standard. With 30 cycles, the results showed that the optimal amount of DNA template for this multiplex ranges from 250pg to 2ng and the lowest detection threshold is 125pg (as low as 63pg for ABO loci). For the DNA template outside the optimal detection range, we could adjust the number of cycles to obtain the robust profiles. Mixture studies showed that over 83% of minor alleles were detected at 1:9 ratios. The full profiles were still observed when 4ng of degraded DNA was digested by DNase I and 1ng undegraded DNA was added to 40μM haematin. Polymerase chain reaction (PCR)-based conditions including the concentrations of primers, magnesium and the Taq polymerase as well as volume, cycle numbers and annealing temperature were examined and optimised. In addition, the system was validated by 364 bloodstain samples and 32 common casework samples. According to the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, our system demonstrates good detection performance and is an ideal tool for forensic DNA typing with potential application.

  18. Toward emotion aware computing: an integrated approach using multichannel neurophysiological recordings and affective visual stimuli.

    PubMed

    Frantzidis, Christos A; Bratsas, Charalampos; Papadelis, Christos L; Konstantinidis, Evdokimos; Pappas, Costas; Bamidis, Panagiotis D

    2010-05-01

    This paper proposes a methodology for the robust classification of neurophysiological data into four emotional states collected during passive viewing of emotional evocative pictures selected from the International Affective Picture System. The proposed classification model is formed according to the current neuroscience trends, since it adopts the independency of two emotional dimensions, namely arousal and valence, as dictated by the bidirectional emotion theory, whereas it is gender-specific. A two-step classification procedure is proposed for the discrimination of emotional states between EEG signals evoked by pleasant and unpleasant stimuli, which also vary in their arousal/intensity levels. The first classification level involves the arousal discrimination. The valence discrimination is then performed. The Mahalanobis (MD) distance-based classifier and support vector machines (SVMs) were used for the discrimination of emotions. The achieved overall classification rates were 79.5% and 81.3% for the MD and SVM, respectively, significantly higher than in previous studies. The robust classification of objective emotional measures is the first step toward numerous applications within the sphere of human-computer interaction.

  19. 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.

  20. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family.

    PubMed

    Preston, G M; Agre, P

    1991-12-15

    CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

  1. Associations among Cognitive Functions, Plasma DNA, and White Matter Integrity in Patients with Early-Onset Parkinson's Disease

    PubMed Central

    Chen, Yueh-Sheng; Chen, Meng-Hsiang; Lu, Cheng-Hsien; Chen, Pei-Chin; Chen, Hsiu-Ling; Yang, I-Hsiao; Tsai, Nai-Wen; Lin, Wei-Che

    2017-01-01

    Early-onset Parkinson's disease (EOPD) patients are symptomatic at a relatively young age, and the impacts of the disease on both the patients and their caregivers are dramatic. Few studies have reported on the cognitive impairments seen in EOPD, and the results of these studies have been diverse. Furthermore, it is still unclear what microstructural white matter (WM) changes are present in EOPD patients. As such, we conducted this study to investigate the microstructural WM changes experienced by EOPD patients and their association with cognitive function and plasma DNA levels. We enrolled 24 EOPD patients and 33 sex- and age-matched healthy volunteers who underwent complete neuro-psychological testing (NPT) to evaluate their cognitive function and diffusion tensor imaging (DTI) scanning to determine their fiber integrity. The plasma DNA measurements included measurements of nuclear and mitochondrial DNA levels. Fractional anisotropy (FA) maps were compared using voxel-based statistics to determine differences between the two groups. The differences in DTI indices and NPT scores were correlated after adjusting for age, sex, and education. Our results demonstrate that patients with EOPD have elevated nuclear DNA levels and wide spectrums of impairments in NPT, especially in the executive function and visuospatial function domains. Exploratory group-wise comparisons of the DTI indices revealed that the patients with EOPD exhibited lower DTI parameters in several brain locations. These poorer DTI parameters were associated with worse cognitive performances and elevated plasma nuclear DNA levels, especially in the anterior thalamic radiation region. Our findings suggest that the thalamus and its adjacent anterior thalamic radiation may be important in the pathogenesis of EOPD, as they appear to become involved in the disease process at an early stage. PMID:28174514

  2. The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific.

    PubMed

    Raule, Nicola; Sevini, Federica; Li, Shengting; Barbieri, Annalaura; Tallaro, Federica; Lomartire, Laura; Vianello, Dario; Montesanto, Alberto; Moilanen, Jukka S; Bezrukov, Vladyslav; Blanché, Hélène; Hervonen, Antti; Christensen, Kaare; Deiana, Luca; Gonos, Efstathios S; Kirkwood, Tom B L; Kristensen, Peter; Leon, Alberta; Pelicci, Pier Giuseppe; Poulain, Michel; Rea, Irene M; Remacle, Josè; Robine, Jean Marie; Schreiber, Stefan; Sikora, Ewa; Eline Slagboom, Peternella; Spazzafumo, Liana; Antonietta Stazi, Maria; Toussaint, Olivier; Vaupel, James W; Rose, Giuseppina; Majamaa, Kari; Perola, Markus; Johnson, Thomas E; Bolund, Lars; Yang, Huanming; Passarino, Giuseppe; Franceschi, Claudio

    2014-06-01

    To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.

  3. Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template.

    PubMed

    Fisher, Timothy S; Darden, Tom; Prasad, Vinayaka R

    2003-05-01

    Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.

  4. 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

  5. Integrated Analysis of Transcriptomic and Proteomic Datasets Reveals Information on Protein Expressivity and Factors Affecting Translational Efficiency.

    PubMed

    Wang, Jiangxin; Wu, Gang; Chen, Lei; Zhang, Weiwen

    2016-01-01

    Integrated analysis of large-scale transcriptomic and proteomic data can provide important insights into the metabolic mechanisms underlying complex biological systems. In this chapter, we present methods to address two aspects of issues related to integrated transcriptomic and proteomic analysis. First, due to the fact that proteomic datasets are often incomplete, and integrated analysis of partial proteomic data may introduce significant bias. To address these issues, we describe a zero-inflated Poisson (ZIP)-based model to uncover the complicated relationships between protein abundances and mRNA expression levels, and then apply them to predict protein abundance for the proteins not experimentally detected. The ZIP model takes into consideration the undetected proteins by assuming that there is a probability mass at zero representing expressed proteins that were undetected owing to technical limitations. The model validity is demonstrated using biological information of operons, regulons, and pathways. Second, weak correlation between transcriptomic and proteomic datasets is often due to biological factors affecting translational processes. To quantify the effects of these factors, we describe a multiple regression-based statistical framework to quantitatively examine the effects of various translational efficiency-related sequence features on mRNA-protein correlation. Using the datasets from sulfate-reducing bacteria Desulfovibrio vulgaris, the analysis shows that translation-related sequence features can contribute up to 15.2-26.2% of the total variation of the correlation between transcriptomic and proteomic datasets, and also reveals the relative importance of various features in translation process.

  6. 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.

  7. 8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme

    PubMed Central

    Kiryutin, Alexey S.; Kasymov, Rustem D.; Petrova, Darya V.; Endutkin, Anton V.; Popov, Alexander V.; Yurkovskaya, Alexandra V.; Fedechkin, Stanislav O.; Brockerman, Jacob A.; Zharkov, Dmitry O.; Smirnov, Serge L.

    2016-01-01

    8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew—Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar—phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein—DNA interface. PMID:27749894

  8. Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching.

    PubMed

    Benmerzouga, Imaan; Concepción-Acevedo, Jeniffer; Kim, Hee-Sook; Vandoros, Anthula V; Cross, George A M; Klingbeil, Michele M; Li, Bibo

    2013-01-01

    Binding of the Origin Recognition Complex (ORC) to replication origins is essential for initiation of DNA replication, but ORC has non-essential functions outside of DNA replication, including in heterochromatic gene silencing and telomere maintenance. Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis, uses antigenic variation as a major virulence mechanism to evade the host's immune attack by expressing its major surface antigen, the Variant Surface Glycoprotein (VSG), in a monoallelic manner. An Orc1/Cdc6 homologue has been identified in T. brucei, but its role in DNA replication has not been directly confirmed and its potential involvement in VSG repression or switching has not been thoroughly investigated. In this study, we show that TbOrc1 is essential for nuclear DNA replication in mammalian-infectious bloodstream and tsetse procyclic forms (BF and PF). Depletion of TbOrc1 resulted in derepression of telomere-linked silent VSGs in both BF and PF, and increased VSG switching particularly through the in situ transcriptional switching mechanism. TbOrc1 associates with telomere repeats but appears to do so independently of two known T. brucei telomere proteins, TbRAP1 and TbTRF. We conclude that TbOrc1 has conserved functions in DNA replication and is also required to control telomere-linked VSG expression and VSG switching.

  9. 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...

  10. Integrated assessment of oxidative stress and