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Sample records for egfr-initiated signalling dna

  1. Physical signals for protein–DNA recognition

    International Nuclear Information System (INIS)

    Cao, Xiao-Qin; Zeng, Jia; Yan, Hong

    2009-01-01

    This paper discovers consensus physical signals around eukaryotic splice sites, transcription start sites, and replication origin start and end sites on a genome-wide scale based on their DNA flexibility profiles calculated by three different flexibility models. These salient physical signals are localized highly rigid and flexible DNAs, which may play important roles in protein–DNA recognition by the sliding search mechanism. The found physical signals lead us to a detailed hypothetical view of the search process in which a DNA-binding protein first finds a genomic region close to the target site from an arbitrary starting location by three-dimensional (3D) hopping and intersegment transfer mechanisms for long distances, and subsequently uses the one-dimensional (1D) sliding mechanism facilitated by the localized highly rigid DNAs to accurately locate the target flexible binding site within 30 bp (base pair) short distances. Guided by these physical signals, DNA-binding proteins rapidly search the entire genome to recognize a specific target site from the 3D to 1D pathway. Our findings also show that current promoter prediction programs (PPPs) based on DNA physical properties may suffer from lots of false positives because other functional sites such as splice sites and replication origins have similar physical signals as promoters do

  2. Signal replication in a DNA nanostructure

    Science.gov (United States)

    Mendoza, Oscar; Houmadi, Said; Aimé, Jean-Pierre; Elezgaray, Juan

    2017-01-01

    Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

  3. Genomic signal processing for DNA sequence clustering.

    Science.gov (United States)

    Mendizabal-Ruiz, Gerardo; Román-Godínez, Israel; Torres-Ramos, Sulema; Salido-Ruiz, Ricardo A; Vélez-Pérez, Hugo; Morales, J Alejandro

    2018-01-01

    Genomic signal processing (GSP) methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.

  4. A Universal Fast Colorimetric Method for DNA Signal Detection with DNA Strand Displacement and Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Xin Li

    2015-01-01

    Full Text Available DNA or gene signal detection is of great significance in many fields including medical examination, intracellular molecular monitoring, and gene disease signal diagnosis, but detection of DNA or gene signals in a low concentration with instant visual results remains a challenge. In this work, a universal fast and visual colorimetric detection method for DNA signals is proposed. Specifically, a DNA signal amplification “circuit” based on DNA strand displacement is firstly designed to amplify the target DNA signals, and then thiol modified hairpin DNA strands and gold nanoparticles are used to make signal detection results visualized in a colorimetric manner. If the target DNA signal exists, the gold nanoparticles aggregate and settle down with color changing from dark red to grey quickly; otherwise, the gold nanoparticles’ colloids remain stable in dark red. The proposed method provides a novel way to detect quickly DNA or gene signals in low concentrations with instant visual results. When applied in real-life, it may provide a universal colorimetric method for gene disease signal diagnosis.

  5. DNA Damage Signals and Space Radiation Risk

    Science.gov (United States)

    Cucinotta, Francis A.

    2011-01-01

    Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

  6. ATM signaling and genomic stability in response to DNA damage

    International Nuclear Information System (INIS)

    Lavin, Martin F.; Birrell, Geoff; Chen, Philip; Kozlov, Sergei; Scott, Shaun; Gueven, Nuri

    2005-01-01

    DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed

  7. Biomimetic Molecular Signaling using DNA Walkers on Microparticles.

    Science.gov (United States)

    Damase, Tulsi Ram; Spencer, Adam; Samuel, Bamidele; Allen, Peter B

    2017-06-22

    We report the release of catalytic DNA walkers from hydrogel microparticles and the detection of those walkers by substrate-coated microparticles. This might be considered a synthetic biology analog of molecular signal release and reception. One type of particles was coated with components of a DNA one-step strand displacement (OSD) reaction to release the walker. A second type of particle was coated with substrate (or "track") for the molecular walker. We distinguish these particle types using fluorescence barcoding: we synthesized and distinguished multiple particle types with multicolor fluorescence microscopy and automated image analysis software. This represents a step toward amplified, multiplex, and microscopically localized detection based on DNA nanotechnology.

  8. DNA Nanobiosensors: An Outlook on Signal Readout Strategies

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    Arun Richard Chandrasekaran

    2017-01-01

    Full Text Available A suite of functionalities and structural versatility makes DNA an apt material for biosensing applications. DNA-based biosensors are cost-effective and sensitive and have the potential to be used as point-of-care diagnostic tools. Along with robustness and biocompatibility, these sensors also provide multiple readout strategies. Depending on the functionality of DNA-based biosensors, a variety of output strategies have been reported: fluorescence- and FRET-based readout, nanoparticle-based colorimetry, spectroscopy-based techniques, electrochemical signaling, gel electrophoresis, and atomic force microscopy.

  9. Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization.

    Science.gov (United States)

    do Nascimento, Cássio; Muller, Katia; Sato, Sandra; Albuquerque Junior, Rubens Ferreira

    2012-04-01

    Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at -20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p  0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.

  10. DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas

    DEFF Research Database (Denmark)

    Herrtwich, Laura; Nanda, Indrajit; Evangelou, Konstantinos

    2016-01-01

    to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid...

  11. The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

    Science.gov (United States)

    Agarwal, Poonam; Miller, Kyle M

    2016-10-01

    DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

  12. Assembly of Slx4 signaling complexes behind DNA replication forks.

    Science.gov (United States)

    Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

    2015-08-13

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

  13. Electrochemical DNA biosensor based on MNAzyme-mediated signal amplification

    International Nuclear Information System (INIS)

    Diao, Wei; Tang, Min; Ding, Xiaojuan; Zhang, Ye; Yang, Jianru; Cheng, Wenbin; Mo, Fei; Wen, Bo; Xu, Lulu; Yan, Yurong

    2016-01-01

    The authors describe an electrochemical sensing strategy for highly sensitive and specific detection of target (analyte) DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme (MNAzyme). MNAzymes were formed by multicomponent complexes which produce amplified “output” signals in response to specific “input” signal. In the presence of target nucleic acid, multiple partial enzymes (partzymes) oligonucleotides are assembled to form active MNAzymes. These can cleave H0 substrate into two pieces, thereby releasing the activated MNAzyme to undergo an additional cycle of amplification. Here, the two pieces contain a biotin-tagged sequence and a byproduct. The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode. By employing streptavidinylated alkaline phosphatase as an enzyme label, an electrochemical signal is obtained. The electrode, if operated at a working potential of 0.25 V (vs. Ag/AgCl) in solution of pH 7.5, covers the 100 pM to 0.25 μM DNA concentration range, with a 79 pM detection limit. In our perception, the strategy introduced here has a wider potential in that it may be applied to molecular diagnostics and pathogen detection. (author)

  14. Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

    Science.gov (United States)

    Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

    2016-12-15

    In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Non coding RNA: sequence-specific guide for chromatin modification and DNA damage signaling

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    Sofia eFrancia

    2015-11-01

    Full Text Available Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports suggest that ncRNAs are involved in DDR signaling and homology-mediated DNA repair. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair.

  16. Genomic signal processing methods for computation of alignment-free distances from DNA sequences.

    Science.gov (United States)

    Borrayo, Ernesto; Mendizabal-Ruiz, E Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P; Morales, J Alejandro

    2014-01-01

    Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments.

  17. A biological inspired fuzzy adaptive window median filter (FAWMF) for enhancing DNA signal processing.

    Science.gov (United States)

    Ahmad, Muneer; Jung, Low Tan; Bhuiyan, Al-Amin

    2017-10-01

    Digital signal processing techniques commonly employ fixed length window filters to process the signal contents. DNA signals differ in characteristics from common digital signals since they carry nucleotides as contents. The nucleotides own genetic code context and fuzzy behaviors due to their special structure and order in DNA strand. Employing conventional fixed length window filters for DNA signal processing produce spectral leakage and hence results in signal noise. A biological context aware adaptive window filter is required to process the DNA signals. This paper introduces a biological inspired fuzzy adaptive window median filter (FAWMF) which computes the fuzzy membership strength of nucleotides in each slide of window and filters nucleotides based on median filtering with a combination of s-shaped and z-shaped filters. Since coding regions cause 3-base periodicity by an unbalanced nucleotides' distribution producing a relatively high bias for nucleotides' usage, such fundamental characteristic of nucleotides has been exploited in FAWMF to suppress the signal noise. Along with adaptive response of FAWMF, a strong correlation between median nucleotides and the Π shaped filter was observed which produced enhanced discrimination between coding and non-coding regions contrary to fixed length conventional window filters. The proposed FAWMF attains a significant enhancement in coding regions identification i.e. 40% to 125% as compared to other conventional window filters tested over more than 250 benchmarked and randomly taken DNA datasets of different organisms. This study proves that conventional fixed length window filters applied to DNA signals do not achieve significant results since the nucleotides carry genetic code context. The proposed FAWMF algorithm is adaptive and outperforms significantly to process DNA signal contents. The algorithm applied to variety of DNA datasets produced noteworthy discrimination between coding and non-coding regions contrary

  18. Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Kashino, Genro [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom); Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Suzuki, Keiji [Division of Radiation Biology, Department of Radiology and Radiation Biology, Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Matsuda, Naoki [Division of Radiation Biology and Protection, Center for Frontier Life Sciences, Nagasaki University, Nagasaki 852-8102 (Japan); Kodama, Seiji [Radiation Biology Laboratory, Radiation Research Center, Frontier Science Innovation Center, Organization for University-Industry-Government Cooperation, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570 (Japan); Ono, Koji [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Watanabe, Masami [Laboratory of Radiation Biology, Division of Radiation Life Science, Department of Radiation Life Science and Radiation Medical Science, Kyoto University Research Reactor Institute, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Prise, Kevin M [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom) and Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Lisburn Road, Belfast BT9 7AB (United Kingdom)]. E-mail: prise@gci.ac.uk

    2007-06-01

    Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.

  19. Estrogen signalling and the DNA damage response in hormone dependent breast cancers

    Directory of Open Access Journals (Sweden)

    C Elizabeth Caldon

    2014-05-01

    Full Text Available Estrogen is necessary for the normal growth and development of breast tissue, but high levels of estrogen are a major risk factor for breast cancer. One mechanism by which estrogen could contribute to breast cancer is via the induction of DNA damage. This perspective discusses the mechanisms by which estrogen alters the DNA damage response (DDR and DNA repair through the regulation of key effector proteins including ATM, ATR, CHK1, BRCA1 and p53 and the feedback on estrogen receptor signalling from these proteins. We put forward the hypothesis that estrogen receptor signalling converges to suppress effective DNA repair and apoptosis in favour of proliferation. This is important in hormone-dependent breast cancer as it will affect processing of estrogen-induced DNA damage, as well as other genotoxic insults. DDR and DNA repair proteins are frequently mutated or altered in estrogen responsive breast cancer which will further change the processing of DNA damage. Finally the action of estrogen signalling on DNA damage is also relevant to the therapeutic setting as the suppression of a DNA damage response by estrogen has the potential to alter the response of cancers to anti-hormone treatment or chemotherapy that induces DNA damage.

  20. The basic chemistry of exercise-induced DNA oxidation: oxidative damage, redox signalling and their interplay

    Directory of Open Access Journals (Sweden)

    James Nathan Cobley

    2015-06-01

    Full Text Available Acute exercise increases reactive oxygen and nitrogen species generation. This phenomenon is associated with two major outcomes: (1 redox signalling and (2 macromolecule damage. Mechanistic knowledge of how exercise-induced redox signalling and macromolecule damage are interlinked is limited. This review focuses on the interplay between exercise-induced redox signalling and DNA damage, using hydroxyl radical (·OH and hydrogen peroxide (H2O2 as exemplars. It is postulated that the biological fate of H2O2 links the two processes and thus represents a bifurcation point between redox signalling and damage. Indeed, H2O2 can participate in two electron signalling reactions but its diffusion and chemical properties permit DNA oxidation following reaction with transition metals and ·OH generation. It is also considered that the sensing of DNA oxidation by repair proteins constitutes a non-canonical redox signalling mechanism. Further layers of interaction are provided by the redox regulation of DNA repair proteins and their capacity to modulate intracellular H2O2 levels. Overall, exercise-induced redox signalling and DNA damage may be interlinked to a greater extent than was previously thought but this requires further investigation.

  1. p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

    DEFF Research Database (Denmark)

    Borisova, Marina E; Voigt, Andrea; Tollenaere, Maxim A X

    2018-01-01

    quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins......Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ...

  2. DNA Damage Signalling and Repair Inhibitors: The Long-Sought-After Achilles’ Heel of Cancer

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    Denis Velic

    2015-11-01

    Full Text Available For decades, radiotherapy and chemotherapy were the two only approaches exploiting DNA repair processes to fight against cancer. Nowadays, cancer therapeutics can be a major challenge when it comes to seeking personalized targeted medicine that is both effective and selective to the malignancy. Over the last decade, the discovery of new targeted therapies against DNA damage signalling and repair has offered the possibility of therapeutic improvements in oncology. In this review, we summarize the current knowledge of DNA damage signalling and repair inhibitors, their molecular and cellular effects, and future therapeutic use.

  3. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    Energy Technology Data Exchange (ETDEWEB)

    Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

    2012-08-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  4. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    International Nuclear Information System (INIS)

    Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

    2012-01-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  5. Branched-DNA signal amplification combined with paper chromatography hybridization assay and used in hepatitis B virus DNA detection

    International Nuclear Information System (INIS)

    Fu, F.Z.; Liu, L.X.; Wang, W.Q.; Sun, S. H.; Liu, L.B.

    2002-01-01

    Nucleic acids detection method is vital to the clinical pathogen diagnosis. The established method can be classified into target direct amplification and signal amplification format according to the target DNA or RNA being directly amplified or not. Those methods have advantages and disadvantages respectively in the clinical application. In the United States of American, branched-DNA as a strong signal amplifier is broadly used in the quantification of the nucleic acids. To gain satisfied sensitivity, some expensive label molecular and instruments should be adopted. Personnel should be special trained to perform. Hence, those can't be widely carried out in the Third World. To avoid those disadvantages, we used the branched-DNA amplifier in the paper chromatography hybridization assay. Methods: Branched DNA signal amplifier and series of probes complementary to the nucleic acid sequence of hepatitis B virus (HBV) have been synthesized. HBV-DNA or it's capture probe were immobilized on the high flow nitrocellulose strip. Having loaded at one end of the strip in turn, probes or HBV-DNA in the hybridization solution migrate to the opposite end of the strip by capillary forces and hybridizes to the immobilized DNA. The branched-DNA signal amplifier and probe labeled with biotin or 32P were then loaded. Through streptavidin-alkaline phosphatase (SA-AP) conjugate and NBT/BCIP ( the specific chromogenic substrate of AP) or autoradiography, the result can be visualized by color reaction or image production on the X-ray film. Results: The sensitivity of this HBV-DNA detection method used probe labeled with biotin and 32P are 1ng and 10pg. The method using the probe labeled with biotin is simple and rapid (2h) without depending on special instruments, it also avoids the pollution of EtBr which can lead to tumor. And the method using the probe labeled with 32P is simple and sensitive, with the exception of long time autoradiography and the inconvenient isotopic disposal

  6. AFM Imaging of Hybridization Chain Reaction Mediated Signal Transmission between Two DNA Origami Structures.

    Science.gov (United States)

    Helmig, Sarah; Gothelf, Kurt Vesterager

    2017-10-23

    Signal transfer is central to the controlled exchange of information in biology and advanced technologies. Therefore, the development of reliable, long-range signal transfer systems for artificial nanoscale assemblies is of great scientific interest. We have designed such a system for the signal transfer between two connected DNA nanostructures, using the hybridization chain reaction (HCR). Two sets of metastable DNA hairpins, one of which is immobilized at specific points along tracks on DNA origami structures, are polymerized to form a continuous DNA duplex, which is visible using atomic force microscopy (AFM). Upon addition of a designed initiator, the initiation signal is efficiently transferred more than 200 nm from a specific location on one origami structure to an end point on another origami structure. The system shows no significant loss of signal when crossing from one nanostructure to another and, therefore, has the potential to be applied to larger multi-component DNA assemblies. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents.

    Science.gov (United States)

    Hickman, M J; Samson, L D

    1999-09-14

    All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O(6)-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine (O(6)CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSalpha branch of the DNA mismatch repair pathway (but not the MutSbeta branch) is absolutely required for signaling the initiation of apoptosis in response to O(6)MeGs and is partially required for signaling apoptosis in response to O(6)CEGs. Further, O(6)MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutSalpha-dependent. Despite this, MutSalpha-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.

  8. Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

    Science.gov (United States)

    Choi, Jun-Hyuk; Lindsey-Boltz, Laura A; Kemp, Michael; Mason, Aaron C; Wold, Marc S; Sancar, Aziz

    2010-08-03

    ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

  9. Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas

    DEFF Research Database (Denmark)

    Bartek, Jiri; Fornara, Olesja; Merchut-Maya, Joanna Maria

    2017-01-01

    suppressor activation, across our medulloblastoma cohort. Most tumours showed high proliferation (Ki67 marker), variable oxidative DNA damage (8-oxoguanine lesions) and formation of 53BP1 nuclear 'bodies', the latter indicating (along with ATR-Chk1 signalling) endogenous replication stress. The bulk...... cell replication stress and DNA repair. Collectively, the scenario we report here likely fuels genomic instability and evolution of medulloblastoma resistance to standard-of-care genotoxic treatments....... eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (γH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour...

  10. Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents

    OpenAIRE

    Hickman, Mark J.; Samson, Leona D.

    1999-01-01

    All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O6-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O6-methylguanine (O6MeG) and O6-chloroethylguanine (O6CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSα branch of the DNA mismatch repair pathway (but not the MutSβ branch) is absolutely required for signaling the ...

  11. Chloroethylating nitrosoureas in cancer therapy: DNA damage, repair and cell death signaling.

    Science.gov (United States)

    Nikolova, Teodora; Roos, Wynand P; Krämer, Oliver H; Strik, Herwig M; Kaina, Bernd

    2017-08-01

    Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O 6 -chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Noncanonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage.

    Science.gov (United States)

    Marteijn, Jurgen A; Vermeulen, Wim; Tresini, Maria

    2017-01-01

    Environmental genotoxins and metabolic byproducts generate DNA lesions that can cause genomic instability and disrupt tissue homeostasis. To ensure genomic integrity, cells employ mechanisms that convert signals generated by stochastic DNA damage into organized responses, including activation of repair systems, cell cycle checkpoints, and apoptotic mechanisms. DNA damage response (DDR) signaling pathways coordinate these responses and determine cellular fates in part, by transducing signals that modulate RNA metabolism. One of the master DDR coordinators, the Ataxia Telangiectasia Mutated (ATM) kinase, has a fundamental role in mediating DNA damage-induced changes in mRNA synthesis. ATM acts by modulating a variety of RNA metabolic pathways including nascent RNA splicing, a process catalyzed by the spliceosome. Interestingly, ATM and the spliceosome influence each other's activity in a reciprocal manner by a pathway that initiates when transcribing RNA polymerase II (RNAPII) encounters DNA lesions that prohibit forward translocation. In response to stalling of RNAPII assembly of late-stage spliceosomes is disrupted resulting in increased splicing factor mobility. Displacement of spliceosomes from lesion-arrested RNA polymerases facilitates formation of R-loops between the nascent RNA and DNA adjacent to the transcription bubble. R-loops signal for noncanonical ATM activation which in quiescent cells occurs in absence of detectable dsDNA breaks. In turn, activated ATM signals to regulate spliceosome dynamics and AS genome wide.This chapter describes the use of fluorescence microscopy methods that can be used to evaluate noncanonical ATM activation by transcription-blocking DNA damage. First, we present an immunofluorescence-detection method that can be used to evaluate ATM activation by autophosphorylation, in fixed cells. Second, we present a protocol for Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged splicing factors, a highly sensitive and

  13. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    International Nuclear Information System (INIS)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A.

    2015-01-01

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer

  14. The Impact of Hedgehog Signaling Pathway on DNA Repair Mechanisms in Human Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Erhong; Hanna, Ann; Samant, Rajeev S.; Shevde, Lalita A., E-mail: lsamant@uab.edu [Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, WTI320D, 1824 6th Avenue South, Birmingham, AL 35233 (United States)

    2015-07-21

    Defined cellular mechanisms have evolved that recognize and repair DNA to protect the integrity of its structure and sequence when encountering assaults from endogenous and exogenous sources. There are five major DNA repair pathways: mismatch repair, nucleotide excision repair, direct repair, base excision repair and DNA double strand break repair (including non-homologous end joining and homologous recombination repair). Aberrant activation of the Hedgehog (Hh) signaling pathway is a feature of many cancer types. The Hh pathway has been documented to be indispensable for epithelial-mesenchymal transition, invasion and metastasis, cancer stemness, and chemoresistance. The functional transcription activators of the Hh pathway include the GLI proteins. Inhibition of the activity of GLI can interfere with almost all DNA repair types in human cancer, indicating that Hh/GLI functions may play an important role in enabling tumor cells to survive lethal types of DNA damage induced by chemotherapy and radiotherapy. Thus, Hh signaling presents an important therapeutic target to overcome DNA repair-enabled multi-drug resistance and consequently increase chemotherapeutic response in the treatment of cancer.

  15. Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

    International Nuclear Information System (INIS)

    Nakada, Shinichiro

    2016-01-01

    The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

  16. Proteomic Identification of DNA-PK Involvement within the RET Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Lyle J Burdine

    Full Text Available Constitutive activation of the Rearranged during Transfection (RET proto-oncogene leads to the development of MEN2A medullary thyroid cancer (MTC. The relatively clear genotype/phenotype relationship seen with RET mutations and the development of MEN2A is unusual in the fact that a single gene activity can drive the progression towards metastatic disease. Despite knowing the oncogene responsible for MEN2A, MTC, like most tumors of neural crest origin, remains largely resistant to chemotherapy. Constitutive activation of RET in a SK-N-MC cell line model reduces cell sensitivity to chemotherapy. In an attempt to identify components of the machinery responsible for the observed RET induced chemoresistance, we performed a proteomic screen of histones and associated proteins in cells with a constitutively active RET signaling pathway. The proteomic approach identified DNA-PKcs, a DNA damage response protein, as a target of the RET signaling pathway. Active DNA-PKcs, which is phosphorylated at site serine 2056 and localized to chromatin, was elevated within our model. Treatment with the RET inhibitor RPI-1 significantly reduced s2056 phosphorylation in RET cells as well as in a human medullary thyroid cancer cell line. Additionally, inhibition of DNA-PKcs activity diminished the chemoresistance observed in both cell lines. Importantly, we show that activated DNA-PKcs is elevated in medullary thyroid tumor samples and that expression correlates with expression of RET in thyroid tumors. These results highlight one mechanism by which RET signaling likely primes cells for rapid response to DNA damage and suggests DNA-PKcs as an additional target in MTC.

  17. Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

    Science.gov (United States)

    Kato, Kazuki; Omura, Hiroki; Ishitani, Ryuichiro; Nureki, Osamu

    2017-06-20

    The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

  18. Highly sensitive "signal-on" electrochemiluminescent biosensor for the detection of DNA based on dual quenching and strand displacement reaction.

    Science.gov (United States)

    Lou, Jing; Wang, Zhaoyin; Wang, Xiao; Bao, Jianchun; Tu, Wenwen; Dai, Zhihui

    2015-10-07

    A "signal-on" electrochemiluminescent DNA biosensing platform was proposed based on the dual quenching and strand displacement reaction. This novel "signal-on" detection strategy revealed its sensitivity in achieving a detection limit of 2.4 aM and its selectivity in distinguishing single nucleotide polymorphism of target DNA.

  19. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    International Nuclear Information System (INIS)

    Fukumoto, Yasunori; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint

  20. The preliminary study on the inductory signal triggering the error-prone DNA repair function in mammalian cells

    International Nuclear Information System (INIS)

    Su Zaozhong; Luo Zuyu

    1989-01-01

    The nature of the signal triggering error-prone DNA repair function in mammalian cells was studied from two notions: (1) Does the inducing signal result from the direct hitting the cellular targets by DNA-damaging agents? (2) Is inhibition of DNA replication a prerequisite condition for the triggering effect? Thus, the ultraviolet (UV)-irradiated exogenous DNAs were introduced into human and rat cells by transfection. The results showed that this transfection was able to induce the error-prone repair as efficient as direct UV-irradiation to cells. Moreover, the two inductory treaetments expressed similar kinetics and dose-responses. No matter whether the introduced DNAs initiated replication, they exhibited the incuctory activity. Therefore, it can be considered that DNA lesions itself, not the direct interaction of DNA-damaging agents with specific cellular targets, serve as a triggering signal for the inductory process. Inhibition of DNA replication is not a prerequisite for the inductory signal

  1. Herbivore-Induced DNA Demethylation Changes Floral Signalling and Attractiveness to Pollinators in Brassica rapa.

    Directory of Open Access Journals (Sweden)

    Roman T Kellenberger

    Full Text Available Plants have to fine-tune their signals to optimise the trade-off between herbivore deterrence and pollinator attraction. An important mechanism in mediating plant-insect interactions is the regulation of gene expression via DNA methylation. However, the effect of herbivore-induced DNA methylation changes on pollinator-relevant plant signalling has not been systematically investigated. Here, we assessed the impact of foliar herbivory on DNA methylation and floral traits in the model crop plant Brassica rapa. Methylation-sensitive amplified fragment length polymorphism (MSAP analysis showed that leaf damage by the caterpillar Pieris brassicae was associated with genome-wide methylation changes in both leaves and flowers of B. rapa as well as a downturn in flower number, morphology and scent. A comparison to plants with jasmonic acid-induced defence showed similar demethylation patterns in leaves, but both the floral methylome and phenotype differed significantly from P. brassicae infested plants. Standardised genome-wide demethylation with 5-azacytidine in five different B. rapa full-sib groups further resulted in a genotype-specific downturn of floral morphology and scent, which significantly reduced the attractiveness of the plants to the pollinator bee Bombus terrestris. These results suggest that DNA methylation plays an important role in adjusting plant signalling in response to changing insect communities.

  2. Persistent Amplification of DNA Damage Signal Involved in Replicative Senescence of Normal Human Diploid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Masatoshi Suzuki

    2012-01-01

    Full Text Available Foci of phosphorylated histone H2AX and ATM are the surrogate markers of DNA double strand breaks. We previously reported that the residual foci increased their size after irradiation, which amplifies DNA damage signals. Here, we addressed whether amplification of DNA damage signal is involved in replicative senescence of normal human diploid fibroblasts. Large phosphorylated H2AX foci (>1.5 μm diameter were specifically detected in presenescent cells. The frequency of cells with large foci was well correlated with that of cells positive for senescence-associated β-galactosidase staining. Hypoxic cell culture condition extended replicative life span of normal human fibroblast, and we found that the formation of large foci delayed in those cells. Our immuno-FISH analysis revealed that large foci partially localized at telomeres in senescent cells. Importantly, large foci of phosphorylated H2AX were always colocalized with phosphorylated ATM foci. Furthermore, Ser15-phosphorylated p53 showed colocalization with the large foci. Since the treatment of senescent cells with phosphoinositide 3-kinase inhibitor, wortmannin, suppressed p53 phosphorylation, it is suggested that amplification of DNA damage signaling sustains persistent activation of ATM-p53 pathway, which is essential for replicative senescence.

  3. Enzyme-free and label-free ultrasensitive electrochemical detection of DNA and adenosine triphosphate by dendritic DNA concatamer-based signal amplification.

    Science.gov (United States)

    Liu, Shufeng; Lin, Ying; Liu, Tao; Cheng, Chuanbin; Wei, Wenji; Wang, Li; Li, Feng

    2014-06-15

    Hybridization chain reaction (HCR) strategy has been well developed for the fabrication of various biosensing platforms for signal amplification. Herein, a novel enzyme-free and label-free ultrasensitive electrochemical DNA biosensing platform for the detection of target DNA and adenosine triphosphate (ATP) was firstly proposed, in which three auxiliary DNA probes were ingeniously designed to construct the dendritic DNA concatamer via HCR strategy and used as hexaammineruthenium(III) chloride (RuHex) carrier for signal amplification. With the developed dendritic DNA concatamer-based signal amplification strategy, the DNA biosensor could achieve an ultrasensitive electrochemical detection of DNA and ATP with a superior detection limit as low as 5 aM and 20 fM, respectively, and also demonstrate a high selectivity for DNA and ATP detection. The currently proposed dendritic DNA concatamer opens a promising direction to construct ultrasensitive DNA biosensing platform for biomolecular detection in bioanalysis and clinical biomedicine, which offers the distinct advantages of simplicity and cost efficiency owing to no need of any kind of enzyme, chemical modification or labeling. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. The Chromatin Scaffold Protein SAFB1 Renders Chromatin Permissive for DNA Damage Signaling

    DEFF Research Database (Denmark)

    Altmeyer, Matthias; Toledo Lazaro, Luis Ignacio; Gudjonsson, Thorkell

    2013-01-01

    Although the general relevance of chromatin modifications for genotoxic stress signaling, cell-cycle checkpoint activation, and DNA repair is well established, how these modifications reach initial thresholds in order to trigger robust responses remains largely unexplored. Here, we identify...... the chromatin-associated scaffold attachment factor SAFB1 as a component of the DNA damage response and show that SAFB1 cooperates with histone acetylation to allow for efficient γH2AX spreading and genotoxic stress signaling. SAFB1 undergoes a highly dynamic exchange at damaged chromatin in a poly......(ADP-ribose)-polymerase 1- and poly(ADP-ribose)-dependent manner and is required for unperturbed cell-cycle checkpoint activation and guarding cells against replicative stress. Altogether, our data reveal that transient recruitment of an architectural chromatin component is required in order to overcome physiological...

  5. TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji; An, You Sun; Yi, Jae Youn, E-mail: yjy_71@kcch.re.kr

    2016-08-05

    The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.

  6. Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

    Science.gov (United States)

    Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

    2017-06-15

    Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Correlation dynamics and enhanced signals for the identification of serial biomolecules and DNA bases

    International Nuclear Information System (INIS)

    Ahmed, Towfiq; Haraldsen, Jason T; Balatsky, Alexander V; Rehr, John J; Di Ventra, Massimiliano; Schuller, Ivan

    2014-01-01

    Nanopore-based sequencing has demonstrated a significant potential for the development of fast, accurate, and cost-efficient fingerprinting techniques for next generation molecular detection and sequencing. We propose a specific multilayered graphene-based nanopore device architecture for the recognition of single biomolecules. Molecular detection and analysis can be accomplished through the detection of transverse currents as the molecule or DNA base translocates through the nanopore. To increase the overall signal-to-noise ratio and the accuracy, we implement a new ‘multi-point cross-correlation’ technique for identification of DNA bases or other molecules on the single molecular level. We demonstrate that the cross-correlations between each nanopore will greatly enhance the transverse current signal for each molecule. We implement first-principles transport calculations for DNA bases surveyed across a multilayered graphene nanopore system to illustrate the advantages of the proposed geometry. A time-series analysis of the cross-correlation functions illustrates the potential of this method for enhancing the signal-to-noise ratio. This work constitutes a significant step forward in facilitating fingerprinting of single biomolecules using solid state technology. (paper)

  8. Correlation dynamics and enhanced signals for the identification of serial biomolecules and DNA bases

    Science.gov (United States)

    Ahmed, Towfiq; Haraldsen, Jason T.; Rehr, John J.; Di Ventra, Massimiliano; Schuller, Ivan; Balatsky, Alexander V.

    2014-03-01

    Nanopore-based sequencing has demonstrated a significant potential for the development of fast, accurate, and cost-efficient fingerprinting techniques for next generation molecular detection and sequencing. We propose a specific multilayered graphene-based nanopore device architecture for the recognition of single biomolecules. Molecular detection and analysis can be accomplished through the detection of transverse currents as the molecule or DNA base translocates through the nanopore. To increase the overall signal-to-noise ratio and the accuracy, we implement a new ‘multi-point cross-correlation’ technique for identification of DNA bases or other molecules on the single molecular level. We demonstrate that the cross-correlations between each nanopore will greatly enhance the transverse current signal for each molecule. We implement first-principles transport calculations for DNA bases surveyed across a multilayered graphene nanopore system to illustrate the advantages of the proposed geometry. A time-series analysis of the cross-correlation functions illustrates the potential of this method for enhancing the signal-to-noise ratio. This work constitutes a significant step forward in facilitating fingerprinting of single biomolecules using solid state technology.

  9. AFM Imaging of Hybridization Chain Reaction-Mediated Signal Transmission Between two DNA Origami Structures

    DEFF Research Database (Denmark)

    Helmig, Sarah Wendelbo; Gothelf, Kurt Vesterager

    2017-01-01

    transfer between two connected DNA nanostructures, using the hybridization chain reaction (HCR). Two sets of metastable DNA hairpins - of which one is immobilized in specific points along tracks on DNA origami structures - are polymerized to form a continuous DNA duplex, which is visible using atomic force...... microscopy (AFM). Upon addition of a designed initiator, the initiation signal is efficiently transferred >200 nm from a specific location on one origami structure to an end point on another origami structure. The system shows no significant loss of signal when crossing from one nanostructure to another...

  10. Analysis of variants in DNA damage signalling genes in bladder cancer

    Directory of Open Access Journals (Sweden)

    Bishop D Timothy

    2008-07-01

    Full Text Available Abstract Background Chemicals from occupational exposure and components of cigarette smoke can cause DNA damage in bladder urothelium. Failure to repair DNA damage by DNA repair proteins may result in mutations leading to genetic instability and the development of bladder cancer. Immunohistochemistry studies have shown DNA damage signal activation in precancerous bladder lesions which is lost on progression, suggesting that the damage signalling mechanism acts as a brake to further tumorigenesis. Single nucleotide polymorphisms (SNPs in DSB signalling genes may alter protein function. We hypothesized that SNPs in DSB signalling genes may modulate predisposition to bladder cancer and influence the effects of environmental exposures. Methods We recruited 771 cases and 800 controls (573 hospital-based and 227 population-based from a previous case-control study and interviewed them regarding their smoking habits and occupational history. DNA was extracted from a peripheral blood sample and genotyping of 24 SNPs in MRE11, NBS1, RAD50, H2AX and ATM was undertaken using an allelic discrimination method (Taqman. Results Smoking and occupational dye exposure were strongly associated with bladder cancer risk. Using logistic regression adjusting for age, sex, smoking and occupational dye exposure, there was a marginal increase in risk of bladder cancer for an MRE11 3'UTR SNP (rs2155209, adjusted odds ratio 1.54 95% CI (1.13–2.08, p = 0.01 for individuals homozygous for the rare allele compared to those carrying the common homozygous or heterozygous genotype. However, in the hospital-based controls, the genotype distribution for this SNP deviated from Hardy-Weinberg equilibrium. None of the other SNPs showed an association with bladder cancer and we did not find any significant interaction between any of these polymorphisms and exposure to smoking or dye exposure. Conclusion Apart from a possible effect for one MRE11 3'UTR SNP, our study does not support

  11. FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

    Science.gov (United States)

    Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

    2014-01-01

    Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

  12. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...... and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...

  13. Using physicochemical and compositional characteristics of DNA sequence for prediction of genomic signals

    KAUST Repository

    Mulamba, Pierre Abraham

    2014-12-01

    The challenge in finding genes in eukaryotic organisms using computational methods is an ongoing problem in the biology. Based on various genomic signals found in eukaryotic genomes, this problem can be divided into many different sub­‐problems such as identification of transcription start sites, translation initiation sites, splice sites, poly (A) signals, etc. Each sub-­problem deals with a particular type of genomic signals and various computational methods are used to solve each sub-­problem. Aggregating information from all these individual sub-­problems can lead to a complete annotation of a gene and its component signals. The fundamental principle of most of these computational methods is the mapping principle – building an input-­output model for the prediction of a particular genomic signal based on a set of known input signals and their corresponding output signal. The type of input signals used to build the model is an essential element in most of these computational methods. The common factor of most of these methods is that they are mainly based on the statistical analysis of the basic nucleotide sequence string composition. 4 Our study is based on a novel approach to predict genomic signals in which uniquely generated structural profiles that combine compressed physicochemical properties with topological and compositional properties of DNA sequences are used to develop machine learning predictive models. The compression of the physicochemical properties is made using principal component analysis transformation. Our ideas are evaluated through prediction models of canonical splice sites using support vector machine models. We demonstrate across several species that the proposed methodology has resulted in the most accurate splice site predictors that are publicly available or described. We believe that the approach in this study is quite general and has various applications in other biological modeling problems.

  14. Stimulus-Responsive Plasmonic Chiral Signals of Gold Nanorods Organized on DNA Origami.

    Science.gov (United States)

    Jiang, Qiao; Liu, Qing; Shi, Yuefeng; Wang, Zhen-Gang; Zhan, Pengfei; Liu, Jianbing; Liu, Chao; Wang, Hui; Shi, Xinghua; Zhang, Li; Sun, Jiashu; Ding, Baoquan; Liu, Minghua

    2017-11-08

    In response to environmental variations, living cells need to arrange the conformational changes of macromolecules to achieve the specific biofunctions. Inspired by natural molecular machines, artificial macromolecular assemblies with controllable nanostructures and environmentally responsive functions can be designed. By assembling macromolecular nanostructures with noble metal nanoparticles, environmental information could be significantly amplified and modulated. However, manufacturing dynamic plasmonic nanostructures that are efficiently responsive to different stimuli is still a challenging task. Here we demonstrate a stimulus-responsive plasmonic nanosystem based on DNA origami-organized gold nanorods (GNRs). L-shaped GNR dimers were assembled on rhombus-shaped DNA origami templates. The geometry and chiral signals of the GNR nanoarchitectures respond to multiple stimuli, including glutathione reduction, restriction enzyme action, pH change, or photoirradiation. While the glutathione reduction or restriction enzyme caused irreversible changes in the plasmonic circular dichroism (CD) signals, both pH and light irradiation triggered reversible changes in the plasmonic CD. Our system transduces external stimuli into conformational changes and circular dichroism responses in near-infrared (NIR) wavelengths. By this approach, programmable optical reporters for essential biological signals can be fabricated.

  15. Failure of signal transduction pathway of DNA damage in hereditary microcephaly

    International Nuclear Information System (INIS)

    Miyamoto, Tatsuo; Matsuura, Shinya

    2009-01-01

    Mechanisms underlying the brain size determination are considered from an aspect of DNA-damage signaling recently revealed by studies on hereditary microcephaly (M), in relation to the radiation-induced M. International Commission of Radiological Protection (ICRP) assesses the risk of M by in utero exposure as 40%/Sv, the threshold dose is about 0.2 Gy (deterministic effect), A-bomb M is conceived to be due to the exposure at 8-5 weeks of gestation, and M is induced by radiation at 10 days after fertilization in the mouse. Recent studies on causing genes of M have revealed its particular connection with signaling pathways: in ataxia-telangiectasia (AT), genes of ATM; in Seckel syndrome, of ATR (AT and Rad3-related) and pericentrin (PCNT); Nijmegen syndrome (NBS), of NBS1; NBS-like disease, of Rad50 and Mre11; AT-like disease, of Mre11; Lig4 syndrome, of Lig4; immunodeficiency combined with M, of XLF; primary M, of MCPH1, ASPM, CdkRap2, CENP-J and STIL. Single and double strand breaks of DNA respectively activate the signaling pathway of ATR where PCNT and MCPH1 participate, and pathway of ATM where NBS1, Mre11 and Rad50 do. PCNT is a major protein, pericentrin, composing the centrosome, of which defect results in the Seckel disease with spindle dysfunction. At present, M can be thus said to be of the cellular common features of failure of ATM/ATR signaling and of dysfunction of centrosome. As well, ASPM gene expression is recently reported to be suppressed by radiation. Thus future studies on M will spread to wider biological field of cell and development as well as radiation and inheritance. (K.T.)

  16. Estrogen-induced DNA synthesis in vascular endothelial cells is mediated by ROS signaling

    Directory of Open Access Journals (Sweden)

    Felty Quentin

    2006-04-01

    to be dose dependent. Conclusion We have shown that estrogen exposure stimulates the rapid production of intracellular ROS and they are involved in growth signaling of endothelial cells. It appears that the early estrogen signaling does not require estrogen receptor genomic signaling because we can inhibit estrogen-induced DNA synthesis by antioxidants. Findings of this study may further expand research defining the underlying mechanism of how estrogen may promote vascular lesions. It also provides important information for the design of new antioxidant-based drugs or new antioxidant gene therapy to protect the cardiovascular health of individuals sensitive to estrogen.

  17. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

    Science.gov (United States)

    Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

    2010-01-01

    The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

  18. DMPD: Signal transduction pathways mediated by the interaction of CpG DNA withToll-like receptor 9. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14751759 Signal transduction pathways mediated by the interaction of CpG DNA withTo...;16(1):17-22. (.png) (.svg) (.html) (.csml) Show Signal transduction pathways mediated by the interaction of... CpG DNA withToll-like receptor 9. PubmedID 14751759 Title Signal transduction pathways media

  19. Signalign: An Ontology of DNA as Signal for Comparative Gene Structure Prediction Using Information-Coding-and-Processing Techniques.

    Science.gov (United States)

    Yu, Ning; Guo, Xuan; Gu, Feng; Pan, Yi

    2016-03-01

    Conventional character-analysis-based techniques in genome analysis manifest three main shortcomings-inefficiency, inflexibility, and incompatibility. In our previous research, a general framework, called DNA As X was proposed for character-analysis-free techniques to overcome these shortcomings, where X is the intermediates, such as digit, code, signal, vector, tree, graph network, and so on. In this paper, we further implement an ontology of DNA As Signal, by designing a tool named Signalign for comparative gene structure analysis, in which DNA sequences are converted into signal series, processed by modified method of dynamic time warping and measured by signal-to-noise ratio (SNR). The ontology of DNA As Signal integrates the principles and concepts of other disciplines including information coding theory and signal processing into sequence analysis and processing. Comparing with conventional character-analysis-based methods, Signalign can not only have the equivalent or superior performance, but also enrich the tools and the knowledge library of computational biology by extending the domain from character/string to diverse areas. The evaluation results validate the success of the character-analysis-free technique for improved performances in comparative gene structure prediction.

  20. DNA and protein co-administration induces tolerogenic dendritic cells through DC-SIGN mediated negative signals.

    Science.gov (United States)

    Li, Jinyao; Geng, Shuang; Liu, Xiuping; Liu, Hu; Jin, Huali; Liu, Chang-Gong; Wang, Bin

    2013-10-01

    We previously demonstrated that DNA and protein co-administration induced differentiation of immature dendritic cells (iDCs) into CD11c(+)CD40(low)IL-10(+) regulatory DCs (DCregs) via the caveolin-1 (Cav-1) -mediated signal pathway. Here, we demonstrate that production of IL-10 and the low expression of CD40 play a critical role in the subsequent induction of regulatory T cells (Tregs) by the DCregs. We observed that DNA and protein were co-localized with DC-SIGN in caveolae and early lysosomes in the treated DCs, as indicated by co-localization with Cav-1 and EEA-1 compartment markers. DNA and protein also co-localized with LAMP-2. Gene-array analysis of gene expression showed that more than a thousand genes were significantly changed by the DC co-treatment with DNA + protein compared with controls. Notably, the level of DC-SIGN expression was dramatically upregulated in pOVA + OVA co-treated DCs. The expression levels of Rho and Rho GNEF, the down-stream molecules of DC-SIGN mediated signal pathway, were also greatly upregulated. Further, the level of TLR9, the traditional DNA receptor, was significantly downregulated. These results suggest that DC-SIGN as the potential receptor for DNA and protein might trigger the negative pathway to contribute the induction of DCreg combining with Cav-1 mediated negative signal pathway.

  1. Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

    Science.gov (United States)

    Hu, Pan; Yang, Bin

    2016-01-15

    Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb(2+) as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb(2+) in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Molecular beacon based biosensor for the sequence-specific detection of DNA using DNA-capped gold nanoparticles-streptavidin conjugates for signal amplification

    International Nuclear Information System (INIS)

    Fang, Xian; Jiang, Wei; Han, Xiaowei; Zhang, Yuzhong

    2013-01-01

    We describe a highly sensitive and selective molecular beacon-based electrochemical impedance biosensor for the sequence-specific detection of DNA. DNA-capped conjugates between gold nanoparticles (Au-NPs) and streptavidin are used for signal amplification. The molecular beacon was labeled with a thiol at its 5′ end and with biotin at its 3′ end, and then immobilized on the surface of a bare gold electrode through the formation of Au-S bonds. Initially, the molecular beacon is present in the “closed” state, and this shields the biotin from being approached by streptavidin due to steric hindrance. In the presence of the target DNA, the target DNA molecules hybridize with the loop and cause a conformational change that moves the biotin away from the surface of the electrode. The biotin thereby becomes accessible for the reporter (the DNA-streptavidin capped Au-NPs), and this results in a distinct increase in electron transfer resistance. Under optimal conditions, the increase in resistance is linearly related to the logarithm of the concentration of complementary target DNA in the range from 1.0 fM to 0.1 μM, with a detection limit of 0.35 fM (at an S/N of 3). This biosensor exhibits good selectivity, and acceptable stability and reproducibility. (author)

  3. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

    Science.gov (United States)

    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  4. Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

    International Nuclear Information System (INIS)

    Zhang, Ruoxi; Fang, Liurong; Wang, Dang; Cai, Kaimei; Zhang, Huan; Xie, Lilan; Li, Yi; Chen, Huanchun; Xiao, Shaobo

    2015-01-01

    To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

  5. Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ruoxi [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Fang, Liurong, E-mail: fanglr@mail.hzau.edu.cn [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Wang, Dang; Cai, Kaimei; Zhang, Huan [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Xie, Lilan; Li, Yi [College of Life Science and Technology, Wuhan Institute of Bioengineering, Wuhan 430415 (China); Chen, Huanchun; Xiao, Shaobo [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China)

    2015-11-15

    To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

  6. Requirement for aspartate-cleaved bid in apoptosis signaling by DNA-damaging anti-cancer regimens

    NARCIS (Netherlands)

    Werner, Arlette B.; Tait, Stephen W. G.; de Vries, Evert; Eldering, Eric; Borst, Jannie

    2004-01-01

    Lymphoid malignancies can escape from DNA-damaging anti-cancer drugs and gamma-radiation by blocking apoptosis-signaling pathways. How these regimens induce apoptosis is incompletely defined, especially in cells with nonfunctional p53. We report here that the BH3-only Bcl-2 family member Bid is

  7. Mitochondrial DNA signals of late glacial recolonization of Europe from near eastern refugia.

    Science.gov (United States)

    Pala, Maria; Olivieri, Anna; Achilli, Alessandro; Accetturo, Matteo; Metspalu, Ene; Reidla, Maere; Tamm, Erika; Karmin, Monika; Reisberg, Tuuli; Hooshiar Kashani, Baharak; Perego, Ugo A; Carossa, Valeria; Gandini, Francesca; Pereira, Joana B; Soares, Pedro; Angerhofer, Norman; Rychkov, Sergei; Al-Zahery, Nadia; Carelli, Valerio; Sanati, Mohammad Hossein; Houshmand, Massoud; Hatina, Jiři; Macaulay, Vincent; Pereira, Luísa; Woodward, Scott R; Davies, William; Gamble, Clive; Baird, Douglas; Semino, Ornella; Villems, Richard; Torroni, Antonio; Richards, Martin B

    2012-05-04

    Human populations, along with those of many other species, are thought to have contracted into a number of refuge areas at the height of the last Ice Age. European populations are believed to be, to a large extent, the descendants of the inhabitants of these refugia, and some extant mtDNA lineages can be traced to refugia in Franco-Cantabria (haplogroups H1, H3, V, and U5b1), the Italian Peninsula (U5b3), and the East European Plain (U4 and U5a). Parts of the Near East, such as the Levant, were also continuously inhabited throughout the Last Glacial Maximum, but unlike western and eastern Europe, no archaeological or genetic evidence for Late Glacial expansions into Europe from the Near East has hitherto been discovered. Here we report, on the basis of an enlarged whole-genome mitochondrial database, that a substantial, perhaps predominant, signal from mitochondrial haplogroups J and T, previously thought to have spread primarily from the Near East into Europe with the Neolithic population, may in fact reflect dispersals during the Late Glacial period, ∼19-12 thousand years (ka) ago. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  8. Control of electrochemical signals from quantum dots conjugated to organic materials by using DNA structure in an analog logic gate.

    Science.gov (United States)

    Chen, Qi; Yoo, Si-Youl; Chung, Yong-Ho; Lee, Ji-Young; Min, Junhong; Choi, Jeong-Woo

    2016-10-01

    Various bio-logic gates have been studied intensively to overcome the rigidity of single-function silicon-based logic devices arising from combinations of various gates. Here, a simple control tool using electrochemical signals from quantum dots (QDs) was constructed using DNA and organic materials for multiple logic functions. The electrochemical redox current generated from QDs was controlled by the DNA structure. DNA structure, in turn, was dependent on the components (organic materials) and the input signal (pH). Independent electrochemical signals from two different logic units containing QDs were merged into a single analog-type logic gate, which was controlled by two inputs. We applied this electrochemical biodevice to a simple logic system and achieved various logic functions from the controlled pH input sets. This could be further improved by choosing QDs, ionic conditions, or DNA sequences. This research provides a feasible method for fabricating an artificial intelligence system. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    Directory of Open Access Journals (Sweden)

    Naoto Tatewaki

    Full Text Available Ataxia telangiectasia mutated (ATM kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR. The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15 and Chk1 (Ser317 was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

  10. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    Energy Technology Data Exchange (ETDEWEB)

    Jarvis, Ian W.H., E-mail: Ian.Jarvis@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Bergvall, Christoffer, E-mail: Christoffer.Bergvall@anchem.su.se [Department of Analytical Chemistry, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm (Sweden); Bottai, Matteo, E-mail: Matteo.Bottai@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Westerholm, Roger, E-mail: Roger.Westerholm@anchem.su.se [Department of Analytical Chemistry, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm (Sweden); Stenius, Ulla, E-mail: Ulla.Stenius@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Dreij, Kristian, E-mail: Kristian.Dreij@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden)

    2013-02-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  11. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    International Nuclear Information System (INIS)

    Jarvis, Ian W.H.; Bergvall, Christoffer; Bottai, Matteo; Westerholm, Roger; Stenius, Ulla; Dreij, Kristian

    2013-01-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  12. MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala [Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm (Sweden); Glas, Rickard, E-mail: rickard.glas@ki.se [Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm (Sweden)

    2010-08-27

    Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  13. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells.

    Science.gov (United States)

    Landvik, N E; Arlt, V M; Nagy, E; Solhaug, A; Tekpli, X; Schmeiser, H H; Refsnes, M; Phillips, D H; Lagadic-Gossmann, D; Holme, J A

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system. Copyright 2009 Elsevier B.V. All rights reserved.

  14. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Landvik, N.E. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Arlt, V.M.; Nagy, E. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Solhaug, A. [Section for Toxicology, Department of Feed and Food Safety, National Veterinary Institute Pb 750 Sentrum, N-0106 Oslo (Norway); Tekpli, X. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Schmeiser, H.H. [Research Group Genetic Alteration in Carcinogenesis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Refsnes, M. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Phillips, D.H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Lagadic-Gossmann, D. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Holme, J.A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway)

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ({sup 32}P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of I{kappa}B-{alpha} (suggesting activation of NF-{kappa}B) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-{kappa}B play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

  15. Human longevity and variation in GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidant pathway genes: cross sectional and longitudinal studies.

    Science.gov (United States)

    Soerensen, Mette; Dato, Serena; Tan, Qihua; Thinggaard, Mikael; Kleindorp, Rabea; Beekman, Marian; Jacobsen, Rune; Suchiman, H Eka D; de Craen, Anton J M; Westendorp, Rudi G J; Schreiber, Stefan; Stevnsner, Tinna; Bohr, Vilhelm A; Slagboom, P Eline; Nebel, Almut; Vaupel, James W; Christensen, Kaare; McGue, Matt; Christiansen, Lene

    2012-05-01

    Here we explore association with human longevity of common genetic variation in three major candidate pathways: GH/IGF-1/insulin signaling, DNA damage signaling and repair and pro/antioxidants by investigating 1273 tagging SNPs in 148 genes composing these pathways. In a case-control study of 1089 oldest-old (age 92-93) and 736 middle-aged Danes we found 1 pro/antioxidant SNP (rs1002149 (GSR)), 5 GH/IGF-1/INS SNPs (rs1207362 (KL), rs2267723 (GHRHR), rs3842755 (INS), rs572169 (GHSR), rs9456497 (IGF2R)) and 5 DNA repair SNPs (rs11571461 (RAD52), rs13251813 (WRN), rs1805329 (RAD23B), rs2953983 (POLB), rs3211994 (NTLH1)) to be associated with longevity after correction for multiple testing. In a longitudinal study with 11 years of follow-up on survival in the oldest-old Danes we found 2 pro/antioxidant SNPs (rs10047589 (TNXRD1), rs207444 (XDH)), 1 GH/IGF-1/INS SNP (rs26802 (GHRL)) and 3 DNA repair SNPs (rs13320360 (MLH1), rs2509049 (H2AFX) and rs705649 (XRCC5)) to be associated with mortality in late life after correction for multiple testing. When examining the 11 SNPs from the case-control study in the longitudinal data, rs3842755 (INS), rs13251813 (WRN) and rs3211994 (NTHL1) demonstrated the same directions of effect (ppolymorphisms. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Signalling detection of DNA damage induced by low doses of ionizing radiation in human lymphocytes

    International Nuclear Information System (INIS)

    Valente, M.

    2011-01-01

    Individuals spontaneously present different sensitivities to ionizing radiation, measured by the severity of their post-radiotherapy side-effects. Cells from some patients with extreme clinical radiosensitivity have shown altered cellular radiosensitivity measured by different endpoints as apoptosis or DNA damage. Linking clinical and cellular sensitivity is of fundamental importance to establish a clinical test capable of predicting a person's radiosensitivity from a sample. Easily sampled, peripheral blood lymphocytes (PBL) are an appealing cellular model to study individual radiosensitivity as they have been shown to be the most radiosensitive hematopoietic cells. DNA damages and repair can be visualized by observing the kinetics of appearance and disappearance of gamma-H2AX foci on DNA double-strand breaks through immunofluorescence microscopy. The experimental strategy chosen here was to follow lymphocyte gamma-H2AX foci kinetics in response to different levels of irradiation as delayed gamma-H2AX foci disappearance has been observed in cells of individuals with high clinical radiosensitivity. For our initial study we irradiated in vitro samples of radiotherapy patients with different clinical radiosensitivities. The groups of distinct clinical sensitivities showed no corresponding differences in their cellular gamma-H2AX response. In addition, several samples were lost, mainly due to the long transportation period before being treated in our lab. To render this method usable for clinical applications, several changes were made: after improving sample viability, speed was increased by automation of image acquisition (Metasystem) and gamma-H2AX focus scoring (freeware CellProfiler). This technique was able to detect doses as low as 0.005 Gy and gave similar results to manual focus scoring. The possibility of discriminating different lymphocyte subsets (CD4, CD8 and CD19) during analysis was added to identify among the lymphocyte subsets the one producing more

  17. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...

  18. The Possible Crosstalk of MOB2 With NDR1/2 Kinases in Cell Cycle and DNA Damage Signaling.

    Science.gov (United States)

    Gundogdu, Ramazan; Hergovich, Alexander

    2016-09-06

    This article is the authors' opinion of the roles of the signal transducer Mps one binder 2 (MOB2) in the control of cell cycle progression and the DNA Damage Response (DDR). We recently found that endogenous MOB2 is required to prevent the accumulation of endogenous DNA damage in order to prevent the undesired, and possibly detrimental, activation of cell cycle checkpoints. In this regard, it is noteworthy that MOB2 has been linked biochemically to the regulation of the NDR1/2 (aka STK38/STK38L) protein kinases, which themselves have functions at different steps of the cell cycle. Therefore, we are speculating in this article about the possible connections of MOB2 with NDR1/2 kinases in cell cycle and DDR Signaling.

  19. Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

    Science.gov (United States)

    Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

    2012-05-01

    We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem

  20. Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore); Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore)

    2012-05-01

    Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

  1. Gestational Diabetes Alters Offspring DNA Methylation Profiles in Human and Rat: Identification of Key Pathways Involved in Endocrine System Disorders, Insulin Signaling, Diabetes Signaling, and ILK Signaling.

    Science.gov (United States)

    Petropoulos, Sophie; Guillemin, Claire; Ergaz, Zivanit; Dimov, Sergiy; Suderman, Matthew; Weinstein-Fudim, Liza; Ornoy, Asher; Szyf, Moshe

    2015-06-01

    Gestational diabetes is associated with risk for metabolic disease later in life. Using a cross-species approach in rat and humans, we examined the hypothesis that gestational diabetes during pregnancy triggers changes in the methylome of the offspring that might be mediating these risks. We show in a gestation diabetes rat model, the Cohen diabetic rat, that gestational diabetes triggers wide alterations in DNA methylation in the placenta in both candidate diabetes genes and genome-wide promoters, thus providing evidence for a causal relationship between diabetes during pregnancy and DNA methylation alterations. There is a significant overlap between differentially methylated genes in the placenta and the liver of the rat offspring. Several genes differentially methylated in rat placenta exposed to maternal diabetes are also differentially methylated in the human placenta of offspring exposed to gestational diabetes in utero. DNA methylation changes inversely correlate with changes in expression. The changes in DNA methylation affect known functional gene pathways involved in endocrine function, metabolism, and insulin responses. These data provide support to the hypothesis that early-life exposures and their effects on metabolic disease are mediated by DNA methylation changes. This has important diagnostic and therapeutic implications.

  2. Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

    International Nuclear Information System (INIS)

    Kang, Khong Bee; Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong

    2012-01-01

    Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)–Akt-DNA–dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H 2 AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H 2 AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G 2 /M arrest and increased γ-H 2 AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H 2 AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are resistant to irradiation

  3. A centrosome-autonomous signal that involves centriole disengagement permits centrosome duplication in G2 phase after DNA damage.

    LENUS (Irish Health Repository)

    2010-11-15

    DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1\\/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2\\/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2\\/unirradiated G2 fusions. Chicken-human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

  4. Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

    Science.gov (United States)

    Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

    2015-06-16

    A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

  5. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    Science.gov (United States)

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  6. A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

    Science.gov (United States)

    Hannon, Eilis; Chand, Annisa N; Evans, Mark D; Wong, Chloe C Y; Grubb, Matthew S; Mill, Jonathan

    2015-07-01

    Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type Ca V 1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.

  7. Ultrasound-induced DNA damage and signal transductions indicated by gammaH2AX

    Science.gov (United States)

    Furusawa, Yukihiro; Fujiwara, Yoshisada; Zhao, Qing-Li; Hassan, Mariame Ali; Ogawa, Ryohei; Tabuchi, Yoshiaki; Takasaki, Ichiro; Takahashi, Akihisa; Ohnishi, Takeo; Kondo, Takashi

    2011-09-01

    Ultrasound (US) has been shown to induce cancer cell death via different forms including apoptosis. Here, we report the potential of low-intensity pulsed US (LIPUS) to induce genomic DNA damage and subsequent DNA damage response. Using the ionizing radiation-induced DNA double-strand breaks (DSBs) as the positive control, we were able to observe the induction of DSBs (as neutral comet tails) and the subsequent formation of gammaH2AX-positive foci (by immunofluorescence detection) in human leukemia cells following exposure to LIPUS. The LIPUS-induced DNA damage arose most likely from the mechanical, but not sonochemical, effect of cavitation, based on our observation that the suppression of inertial cavitation abrogated the gammH2AX foci formation, whereas scavenging of free radical formation (e.g., hydroxyl radical) had no protective effect on it. Treatment with the specific kinase inhibitor of ATM or DNA-PKcs, which can phosphorylate H2AX Ser139, revealed that US-induced gammaH2AX was inhibited more effectively by the DNA-PK inhibitor than ATM kinase inhibitor. Notably, these inhibitor effects were opposite to those with radiation-induced gammH2AX. In conclusion, we report, for the first time that US can induce DNA damage and the DNA damage response as indicated by gammaH2AX was triggered by the cavitational mechanical effects. Thus, it is expected that the data shown here may provide a better understanding of the cellular responses to US.

  8. Persistence and dynamics of DNA damage signal amplification determined by microcolony formation and live-cell imaging

    International Nuclear Information System (INIS)

    Oka, Yasuyoshi; Yamauchi, Motohiro; Suzuki, Masatoshi; Yamashita, Shunichi; Suzuki, Keiji

    2011-01-01

    Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm 2 ). Cells were grown for up to 3 days, and phosphorylated ataxia-telangiectasia mutated (ATM) at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process. (author)

  9. Differential Processing of Low and High LET Radiation Induced DNA Damage: Investigation of Switch from ATM to ATR Signaling

    Science.gov (United States)

    Saha, Janapriya; Wang, Minli; Hada, Megumi; Cucinotta, Francis A.

    2011-01-01

    The members of the phosphatidylinositol kinase-like kinase family of proteins namely ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are directly responsible for the maintenance of genomic integrity by mounting DDR through signaling and facilitating the recruitment of repair factors at the sites of DNA damage along with coordinating the deployment of cell cycle checkpoints to permit repair by phosphorylating Checkpoint kinase Chk1, Chk2 and p53. High LET radiation from GCR (Galactic Cosmic Rays) consisting mainly of protons and high energy and charged (HZE) particles from SPE (Solar Particle Event) pose a major health risk for astronauts on their space flight missions. The determination of these risks and the design of potential safeguards require sound knowledge of the biological consequences of lesion induction and the capability of the cells to counter them. We here strive to determine the coordination of ATM and ATR kinases at the break sites directly affecting checkpoint signaling and DNA repair and whether differential processing of breaks induced by low and high LET radiation leads to possible augmentation of swap of these damage sensors at the sites of DNA damage. Exposure of cells to IR triggers rapid autophosphorylation of serine-1981 that causes dimer dissociation and initiates monomer formation of ATM. ATM kinase activity depends on the disruption of the dimer, which allows access and phosphorylation of downstream ATM substrates like Chk2. Evidence suggests that ATM is activated by the alterations in higher-order chromatin structure although direct binding of ATM to DSB ends may be a crucial step in its activation. On the other hand, in case of ATR, RPA (replication protein A)-coated ssDNA (single-stranded DNA) generated as a result of stalled DNA replication or during processing of chromosomal lesions is crucial for the localization of ATR to sites of DNA damage in association with ATR-interacting protein (ATRIP). Although the

  10. Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage

    DEFF Research Database (Denmark)

    Thorslund, Tina; Ripplinger, Anita; Hoffmann, Saskia

    2015-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also...... known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked...

  11. Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

    OpenAIRE

    Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2012-01-01

    A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer e...

  12. NEK11: linking CHK1 and CDC25A in DNA damage checkpoint signaling

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Melixetian, Marina; Klein, Ditte Kjaersgaard

    2010-01-01

    The DNA damage induced G(2)/M checkpoint is an important guardian of the genome that prevents cell division when DNA lesions are present. The checkpoint prevents cells from entering mitosis by degrading CDC25A, a key CDK activator. CDC25A proteolysis is controlled by direct phosphorylation events...... is required for beta-TrCP mediated CDC25A polyubiquitylation and degradation. The activity of NEK11 is in turn controlled by CHK1 that activates NEK11 via phosphorylation on serine 273. Since inhibition of NEK11 activity forces checkpoint-arrested cells into mitosis and cell death, NEK11 is, like CHK1...

  13. The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

    DEFF Research Database (Denmark)

    Larsen, Dorthe Helena; Poinsignon, Catherine; Gudjonsson, Thorkell

    2010-01-01

    In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate...

  14. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Science.gov (United States)

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

  15. Controllable Shrinking of Glass Capillary Nanopores Down to sub-10 nm by Wet-Chemical Silanization for Signal-Enhanced DNA Translocation.

    Science.gov (United States)

    Xu, Xiaolong; Li, Chuanping; Zhou, Ya; Jin, Yongdong

    2017-10-27

    Diameter is a major concern for nanopore based sensing. However, directly pulling glass capillary nanopore with diameter down to sub-10 nm is very difficult. So, post treatment is sometimes necessary. Herein, we demonstrate a facile and effective wet-chemical method to shrink the diameter of glass capillary nanopore from several tens of nanometers to sub-10 nm by disodium silicate hydrolysis. Its benefits for DNA translocation are investigated. The shrinking of glass capillary nanopore not only slows down DNA translocation, but also enhances DNA translocation signal and signal-to-noise ratio significantly (102.9 for 6.4 nm glass nanopore, superior than 15 for a 3 nm silicon nitride nanopore). It also affects DNA translocation behaviors, making the approach and glass capillary nanopore platform promising for DNA translocation studies.

  16. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    Energy Technology Data Exchange (ETDEWEB)

    Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

    2010-01-05

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  17. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    International Nuclear Information System (INIS)

    Cogan, Nicola; Baird, Duncan M.; Phillips, Ryan; Crompton, Lucy A.; Caldwell, Maeve A.; Rubio, Miguel A.; Newson, Roger; Lyng, Fiona; Case, C. Patrick

    2010-01-01

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  18. Wavelet analysis of frequency chaos game signal: a time-frequency signature of the C. elegans DNA.

    Science.gov (United States)

    Messaoudi, Imen; Oueslati, Afef Elloumi; Lachiri, Zied

    2014-12-01

    Challenging tasks are encountered in the field of bioinformatics. The choice of the genomic sequence's mapping technique is one the most fastidious tasks. It shows that a judicious choice would serve in examining periodic patterns distribution that concord with the underlying structure of genomes. Despite that, searching for a coding technique that can highlight all the information contained in the DNA has not yet attracted the attention it deserves. In this paper, we propose a new mapping technique based on the chaos game theory that we call the frequency chaos game signal (FCGS). The particularity of the FCGS coding resides in exploiting the statistical properties of the genomic sequence itself. This may reflect important structural and organizational features of DNA. To prove the usefulness of the FCGS approach in the detection of different local periodic patterns, we use the wavelet analysis because it provides access to information that can be obscured by other time-frequency methods such as the Fourier analysis. Thus, we apply the continuous wavelet transform (CWT) with the complex Morlet wavelet as a mother wavelet function. Scalograms that relate to the organism Caenorhabditis elegans (C. elegans) exhibit a multitude of periodic organization of specific DNA sequences.

  19. Contribution of gold nanoparticles to the catalytic DNA strand displacement in leakage reduction and signal amplification.

    Science.gov (United States)

    Wang, Bei; Zhou, Xiang; Yao, Dongbao; Sun, Xianbao; He, Miao; Wang, Xiaojing; Yin, Xue; Liang, Haojun

    2017-10-03

    A new model using a gold nanoparticle (AuNP)-DNA system to constrain leakage and improve efficiency of catalytic toehold-mediated strand displacement reactions was outlined. A 10-bp spacer on AuNPs and fourfold amount of fuels were determined for good performance of this model with an optimized toehold strategy. After the reaction at 25 °C for 10 h, a 258 pM target could be identified, which is a remarkable improvement compared with the traditional AuNP-DNA system without fuel. Moreover, this model was also studied to differentiate specific single nucleotide polymorphism on target with superior selection factors. This model may help by introducing a proposition of target detection to guide further investigation.

  20. Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

    Czech Academy of Sciences Publication Activity Database

    Blažková, Hana; Krejčíková, Kateřina; Moudrý, Pavel; Frisan, T.; Hodný, Zdeněk; Bartek, Jiří

    2009-01-01

    Roč. 14, 1-2 (2009), s. 357-367 ISSN 1582-1838 R&D Projects: GA AV ČR IAA500390501; GA ČR GA204/08/1418; GA ČR GA301/08/0353 Institutional research plan: CEZ:AV0Z50520514 Keywords : cellular senescence * DNA damage response * bacterial toxins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.228, year: 2009

  1. Omni-PolyA: a method and tool for accurate recognition of Poly(A) signals in human genomic DNA

    KAUST Repository

    Magana-Mora, Arturo

    2017-08-15

    BackgroundPolyadenylation is a critical stage of RNA processing during the formation of mature mRNA, and is present in most of the known eukaryote protein-coding transcripts and many long non-coding RNAs. The correct identification of poly(A) signals (PAS) not only helps to elucidate the 3′-end genomic boundaries of a transcribed DNA region and gene regulatory mechanisms but also gives insight into the multiple transcript isoforms resulting from alternative PAS. Although progress has been made in the in-silico prediction of genomic signals, the recognition of PAS in DNA genomic sequences remains a challenge.ResultsIn this study, we analyzed human genomic DNA sequences for the 12 most common PAS variants. Our analysis has identified a set of features that helps in the recognition of true PAS, which may be involved in the regulation of the polyadenylation process. The proposed features, in combination with a recognition model, resulted in a novel method and tool, Omni-PolyA. Omni-PolyA combines several machine learning techniques such as different classifiers in a tree-like decision structure and genetic algorithms for deriving a robust classification model. We performed a comparison between results obtained by state-of-the-art methods, deep neural networks, and Omni-PolyA. Results show that Omni-PolyA significantly reduced the average classification error rate by 35.37% in the prediction of the 12 considered PAS variants relative to the state-of-the-art results.ConclusionsThe results of our study demonstrate that Omni-PolyA is currently the most accurate model for the prediction of PAS in human and can serve as a useful complement to other PAS recognition methods. Omni-PolyA is publicly available as an online tool accessible at www.cbrc.kaust.edu.sa/omnipolya/.

  2. Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for Staphylococcus aureus cutaneous host defense.

    Directory of Open Access Journals (Sweden)

    Philip O Scumpia

    2017-07-01

    Full Text Available Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs by pathogen recognition receptors (PRRs to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways-the Toll-like receptor (TLR and Stimulator of Interferon Gene (STING pathways-were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed S. aureus. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS. Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus. TLR signaling was required for host defense, with its absence reducing interleukin (IL-1β production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by S. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus.

  3. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang, E-mail: songyangwenrong@hotmail.com

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

  4. Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia

    NARCIS (Netherlands)

    Thijssen, Rachel; ter Burg, Johanna; Garrick, Brett; van Bochove, Gregor G. W.; Brown, Jennifer R.; Fernandes, Stacey M.; Rodríguez, María Solé; Michot, Jean-Marie; Hallek, Michael; Eichhorst, Barbara; Reinhardt, Hans Christian; Bendell, Johanna; Derks, Ingrid A. M.; van Kampen, Roel J. W.; Hege, Kristen; Kersten, Marie José; Trowe, Torsten; Filvaroff, Ellen H.; Eldering, Eric; Kater, Arnon P.

    2016-01-01

    Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity

  5. Monitoring of noble, signal and narrow-clawed crayfish using environmental DNA from freshwater samples

    DEFF Research Database (Denmark)

    Agersnap, Sune; Larsen, William Brenner; Knudsen, Steen Wilhelm

    2017-01-01

    human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution...

  6. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Czech Academy of Sciences Publication Activity Database

    Macůrek, Libor; Benada, Jan; Müllers, E.; Halim, V.A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Soňa; Hodný, Zdeněk; Lindqvist, A.; Medema, R.H.; Bartek, Jiří

    2013-01-01

    Roč. 12, č. 2 (2013), s. 251-262 ISSN 1538-4101 R&D Projects: GA ČR GPP305/10/P420; GA ČR GAP301/10/1525 Grant - others:Netherlands Genomic Initiative of NWO(NL) CGC; EK(XE) 259893 Institutional support: RVO:68378050 Keywords : DNA damage response * Wip1 phosphatase * cell cycle * mitotic progression * γH2AX Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.006, year: 2013

  7. Genetic imprint of the Mongol: signal from phylogeographic analysis of mitochondrial DNA.

    Science.gov (United States)

    Cheng, Baoweng; Tang, Wenru; He, Li; Dong, Yongli; Lu, Jing; Lei, Yunping; Yu, Haijing; Zhang, Jiali; Xiao, Chunjie

    2008-01-01

    Mitochondrial deoxyribonucleic acid (DNA) from 201 unrelated Mongolian individuals in the three different regions was analyzed. The Mongolians took the dominant East Asian-specific haplogroups, and some European-prevalent haplogroups were detected. The East Asians-specific haplogroups distributed from east to west in decreasing frequencies, and the European-specific haplogroups distributed conversely. These genetic data suggest that the Mongolian empire played an important role in the maternal genetic admixture across Mongolians and even Central Asian populations, whereas the Silk Road might have contributed little in the admixture between the East Asians and the Europeans.

  8. A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation

    Directory of Open Access Journals (Sweden)

    Eilis Hannon

    2015-07-01

    Full Text Available Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm, although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506 to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.

  9. PARP-1 modulation of mTOR signaling in response to a DNA alkylating agent.

    Directory of Open Access Journals (Sweden)

    Chantal Ethier

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N'-nitro-N'-nitrosoguanine (MNNG, we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose (PAR synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD⁺ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.

  10. Modeling DNA?damage-induced pneumopathy in mice: insight from danger signaling cascades

    OpenAIRE

    Wirsd?rfer, Florian; Jendrossek, Verena

    2017-01-01

    Radiation-induced pneumonitis and fibrosis represent severe and dose-limiting side effects in the radiotherapy of thorax-associated neoplasms leading to decreased quality of life or - as a consequence of treatment with suboptimal radiation doses - to fatal outcomes by local recurrence or metastatic disease. It is assumed that the initial radiation-induced damage to the resident cells triggers a multifaceted damage-signalling cascade in irradiated normal tissues including a multifactorial secr...

  11. Herpes Simplex Virus 1 UL24 Abrogates the DNA Sensing Signal Pathway by Inhibiting NF-κB Activation.

    Science.gov (United States)

    Xu, Haiyan; Su, Chenhe; Pearson, Angela; Mody, Christopher H; Zheng, Chunfu

    2017-04-01

    -κB in the DNA sensing signal pathway via binding to the RHDs of the NF-κB subunits p65 and p50 and abolishing their nuclear translocation. Copyright © 2017 American Society for Microbiology.

  12. A Signal, from Human mtDNA, of Postglacial Recolonization in Europe

    Science.gov (United States)

    Torroni, Antonio; Bandelt, Hans-Jürgen; Macaulay, Vincent; Richards, Martin; Cruciani, Fulvio; Rengo, Chiara; Martinez-Cabrera, Vicente; Villems, Richard; Kivisild, Toomas; Metspalu, Ene; Parik, Jüri; Tolk, Helle-Viivi; Tambets, Kristiina; Forster, Peter; Karger, Bernd; Francalacci, Paolo; Rudan, Pavao; Janicijevic, Branka; Rickards, Olga; Savontaus, Marja-Liisa; Huoponen, Kirsi; Laitinen, Virpi; Koivumäki, Satu; Sykes, Bryan; Hickey, Eileen; Novelletto, Andrea; Moral, Pedro; Sellitto, Daniele; Coppa, Alfredo; Al-Zaheri, Nadia; Santachiara-Benerecetti, A. Silvana; Semino, Ornella; Scozzari, Rosaria

    2001-01-01

    Mitochondrial HVS-I sequences from 10,365 subjects belonging to 56 populations/geographical regions of western Eurasia and northern Africa were first surveyed for the presence of the T→C transition at nucleotide position 16298, a mutation which has previously been shown to characterize haplogroup V mtDNAs. All mtDNAs with this mutation were then screened for a number of diagnostic RFLP sites, revealing two major subsets of mtDNAs. One is haplogroup V proper, and the other has been termed “pre*V,” since it predates V phylogenetically. The rather uncommon pre*V tends to be scattered throughout Europe (and northwestern Africa), whereas V attains two peaks of frequency: one situated in southwestern Europe and one in the Saami of northern Scandinavia. Geographical distributions and ages support the scenario that pre*V originated in Europe before the Last Glacial Maximum (LGM), whereas the more recently derived haplogroup V arose in a southwestern European refugium soon after the LGM. The arrival of V in eastern/central Europe, however, occurred much later, possibly with (post-)Neolithic contacts. The distribution of haplogroup V mtDNAs in modern European populations would thus, at least in part, reflect the pattern of postglacial human recolonization from that refugium, affecting even the Saami. Overall, the present study shows that the dissection of mtDNA variation into small and well-defined evolutionary units is an essential step in the identification of spatial frequency patterns. Mass screening of a few markers identified using complete mtDNA sequences promises to be an efficient strategy for inferring features of human prehistory. PMID:11517423

  13. The roles of DNA damage-dependent signals and MAPK cascades in tributyltin-induced germline apoptosis in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Yun; Wang, Shunchang; Luo, Xun; Yang, Yanan; Jian, Fenglei; Wang, Xuemin; Xie, Lucheng

    2014-08-01

    The induction of apoptosis is recognized to be a major mechanism of tributyltin (TBT) toxicity. However, the underlying signaling pathways for TBT-induced apoptosis remain unclear. In this study, using the nematode Caenorhabditis elegans, we examined whether DNA damage response (DDR) pathway and mitogen-activated protein kinase (MAPK) signaling cascades are involved in TBT-induced germline apoptosis and cell cycle arrest. Our results demonstrated that exposing worms to TBT at the dose of 10nM for 6h significantly increased germline apoptosis in N2 strain. Germline apoptosis was absent in strains that carried ced-3 or ced-4 loss-of-function alleles, indicating that both caspase protein CED-3 and Apaf-1 protein CED-4 were required for TBT-induced apoptosis. TBT-induced apoptosis was blocked in the Bcl-2 gain-of-function strain ced-9(n1950), whereas TBT induced a minor increase in the BH3-only protein EGL-1 mutated strain egl-1(n1084n3082). Checkpoint proteins HUS-1 and CLK-2 exerted proapoptotic effects, and the null mutation of cep-1, the homologue of tumor suppressor gene p53, significantly inhibited TBT-induced apoptosis. Apoptosis in the loss-of-function strains of ERK, JNK and p38 MAPK signaling pathways were completely or mildly suppressed under TBT stress. These results were supported by the results of mRNA expression levels of corresponding genes. The present study indicated that TBT-induced apoptosis required the core apoptotic machinery, and that DDR genes and MAPK pathways played essential roles in signaling the processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Common genomic signaling among initial DNA damage and radiation-induced apoptosis in peripheral blood lymphocytes from locally advanced breast cancer patients

    DEFF Research Database (Denmark)

    Henríquez-Hernández, Luis Alberto; Pinar, Beatriz; Carmona-Vigo, Ruth

    2013-01-01

    PURPOSE: To investigate the genomic signaling that defines sensitive lymphocytes to radiation and if such molecular profiles are consistent with clinical toxicity; trying to disclose the radiobiology mechanisms behind these cellular processes. PATIENTS AND METHODS: Twelve consecutive patients...... suffering from locally advanced breast cancer and treated with high-dose hyperfractionated radiotherapy were recruited. Initial DNA damage was measured by pulsed-field gel electrophoresis and radiation-induced apoptosis was measured by flow cytometry. Gene expression was assessed by DNA microarray. RESULTS...

  15. Structural basis for IL-1α recognition by a modified DNA aptamer that specifically inhibits IL-1α signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Xiaoming; Gelinas, Amy D.; von Carlowitz, Ira; Janjic, Nebojsa; Pyle, Anna Marie (Yale); (SomaLogic)

    2017-10-09

    IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.

  16. Human cDNA clones for an α subunit of G/sub i/ signal-transduction protein

    International Nuclear Information System (INIS)

    Bray, P.; Carter, A.; Guo, V.; Puckett, C.; Kamholz, J.; Spiegel, A.; Nirenberg, M.

    1987-01-01

    Two cDNA clones were obtained from a λgt11 cDNA human brain library that correspond to α/sub i/ subunits of G signal-transduction proteins (where α/sub i/ subunits refer to the α subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain α/sub i/ is highly homologous to that of bovine brain α/sub i/ and the predicted amino acid sequences are identical. However, human and bovine brain α/sub i/ cDNAs differ significantly from α/sub i/ cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homology) and nucleotide (71-75% homology) sequences. In addition, the nucleotide sequences of the 3' untranslated regions of human and bovine brain α/sub i/ cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macrophage α/sub i/ cDNAs. These results suggest there are at least two classes of α/sub i/ mRNA

  17. ENHANCING NETWORK SECURITY USING 'LEARNING-FROM-SIGNALS' AND FRACTIONAL FOURIER TRANSFORM BASED RF-DNA FINGERPRINTS

    Energy Technology Data Exchange (ETDEWEB)

    Buckner, Mark A [ORNL; Bobrek, Miljko [ORNL; Farquhar, Ethan [ORNL; Harmer, Paul K [Air Force Institute of Technology; Temple, Michael A [Air Force Institute of Technology

    2011-01-01

    Wireless Access Points (WAP) remain one of the top 10 network security threats. This research is part of an effort to develop a physical (PHY) layer aware Radio Frequency (RF) air monitoring system with multi-factor authentication to provide a first-line of defense for network security--stopping attackers before they can gain access to critical infrastructure networks through vulnerable WAPs. This paper presents early results on the identification of OFDM-based 802.11a WiFi devices using RF Distinct Native Attribute (RF-DNA) fingerprints produced by the Fractional Fourier Transform (FRFT). These fingerprints are input to a "Learning from Signals" (LFS) classifier which uses hybrid Differential Evolution/Conjugate Gradient (DECG) optimization to determine the optimal features for a low-rank model to be used for future predictions. Results are presented for devices under the most challenging conditions of intra-manufacturer classification, i.e., same-manufacturer, same-model, differing only in serial number. The results of Fractional Fourier Domain (FRFD) RF-DNA fingerprints demonstrate significant improvement over results based on Time Domain (TD), Spectral Domain (SD) and even Wavelet Domain (WD) fingerprints.

  18. A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

    Directory of Open Access Journals (Sweden)

    Hammond Richard

    2010-02-01

    Full Text Available Abstract Background Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor. Results In the study the performance of semi-homogeneous and homogeneous assay formats (suited to rapid, single step tests were evaluated utilising different diameter gold nanoparticles at varying DNA concentrations. Mathematical models were built to understand the effects of mass transport in the flow cell, the binding kinetics of targets to nanoparticles in solution, the packing geometries of targets on the nanoparticle, the packing of nanoparticles on the sensor surface and the effect of surface shear stiffness on the response of the acoustic sensor. This lead to the selection of optimised 15 nm nanoparticles that could be used with a 6 minute total assay time to achieve a limit of detection sensitivity of 5.2 × 10-12 M. Larger diameter nanoparticles gave poorer limits of detection than smaller particles. The limit of detection was three orders of magnitude lower than that observed using a hybridisation assay without nanoparticle signal amplification. Conclusions An analytical model was developed to determine optimal nanoparticle diameter, concentration and probe density, which allowed efficient and rapid optimisation of assay parameters

  19. DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    Directory of Open Access Journals (Sweden)

    Yaofeng Li

    2015-03-01

    Full Text Available Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K/Akt, the target of rapamycin (TOR and the extracellular signal-regulated kinase (ERK pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells.

  20. Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

    Science.gov (United States)

    Leaché, Adam D.; Chavez, Andreas S.; Jones, Leonard N.; Grummer, Jared A.; Gottscho, Andrew D.; Linkem, Charles W.

    2015-01-01

    Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

  1. DMPD: Activation of lymphokine genes in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available thatrespond to T cell activation signals. Arai N, Naito Y, Watanabe M, Masuda ES, Yamaguchi-Iwai Y, Tsuboi A, Heike T,Matsud... in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. Authors Arai N, Naito Y, Watanabe M, Masud...a ES, Yamaguchi-Iwai Y, Tsuboi A, Heike T,Matsuda I, Yokota

  2. Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

    Science.gov (United States)

    Hinrichsen, Inga; Weßbecher, Isabel M; Huhn, Meik; Passmann, Sandra; Zeuzem, Stefan; Plotz, Guido; Biondi, Ricardo M; Brieger, Angela

    2017-12-01

    MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1 L749P and MLH1 Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα. © 2017 Wiley Periodicals, Inc.

  3. DNA damage signaling and apoptosis in preinvasive tubal lesions of ovarian carcinoma.

    Science.gov (United States)

    Chene, Gautier; Ouellet, Veronique; Rahimi, Kurosh; Barres, Veronique; Caceres, Katia; Meunier, Liliane; Cyr, Louis; De Ladurantaye, Manon; Provencher, Diane; Mes Masson, Anne Marie

    2015-06-01

    High-grade serous ovarian cancer (HGSC) is the most life-threatening gynecological malignancy despite surgery and chemotherapy. A better understanding of the molecular basis of the preinvasive stages might be helpful in early detection and diagnosis. Genetic instability is 1 of the characteristics shared by most human cancers, and its level is variable through precancerous lesions to advanced cancer. Because DNA damage response (DDR) has been described as 1 of the first phases in genomic instability, we investigated the level of DDR activation and the apoptosis pathway in serous tubal intraepithelial carcinoma (STIC), the potential precursor of HGSC. A tissue microarray including 21 benign fallopian tubes, 21 STICs, 17 HGSCs from patients with STICs (associated ovarian cancer [AOC]) from the same individuals, and 30 HGSCs without STICs (non-AOC) was used in this study.Immunohistochemistry was performed to evaluate the level of DDR proteins (pATM, pChk2, γH2AX, 53BP1, and TRF2), apoptosis proteins (Bcl2, BAX, and BIM), and cyclin E. The expression of all DDR proteins increased from benign fallopian tubes to STICs. The level of expression of pATM, pChk2, γH2AX, and TRF2 was also increased in STICs in comparison with AOC. BAX, BIM, and cyclin E expressions were high in STICs, whereas Bcl2 expression was low. Immunohistochemical profiles of AOC and non-AOC were also different. These results suggest an activation of the DDR and apoptosis pathways in STICs, indicating that genomic instability may occur early in the precancerous lesions of HGSC.

  4. DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation.

    Science.gov (United States)

    Ghosh, Somnath; Narang, Himanshi; Sarma, Asitikantha; Krishna, Malini

    2011-11-01

    Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Gene expression analysis by cDNA-AFLP highlights a set of new signaling networks and translational control during seed dormancy breaking in Nicotiana plumbaginifolia.

    Science.gov (United States)

    Bove, Jérôme; Lucas, Philippe; Godin, Béatrice; Ogé, Laurent; Jullien, Marc; Grappin, Philippe

    2005-03-01

    Seed dormancy in Nicotiana plumbaginifolia is characterized by an abscisic acid accumulation linked to a pronounced germination delay. Dormancy can be released by 1 year after-ripening treatment. Using a cDNA-amplified fragment length polymorphism (cDNA-AFLP) approach we compared the gene expression patterns of dormant and after-ripened seeds, air-dry or during one day imbibition and analyzed 15,000 cDNA fragments. Among them 1020 were found to be differentially regulated by dormancy. Of 412 sequenced cDNA fragments, 83 were assigned to a known function by search similarities to public databases. The functional categories of the identified dormancy maintenance and breaking responsive genes, give evidence that after-ripening turns in the air-dry seed to a new developmental program that modulates, at the RNA level, components of translational control, signaling networks, transcriptional control and regulated proteolysis.

  6. Self-catalytic growth of unmodified gold nanoparticles as conductive bridges mediated gap-electrical signal transduction for DNA hybridization detection.

    Science.gov (United States)

    Zhang, Jing; Nie, Huagui; Wu, Zhan; Yang, Zhi; Zhang, Lijie; Xu, Xiangju; Huang, Shaoming

    2014-01-21

    A simple and sensitive gap-electrical biosensor based on self-catalytic growth of unmodified gold nanoparticles (AuNPs) as conductive bridges has been developed for amplifying DNA hybridization events. In this strategy, the signal amplification degree of such conductive bridges is closely related to the variation of the glucose oxidase (GOx)-like catalytic activity of AuNPs upon interaction with single- and double-stranded DNA (ssDNA and dsDNA), respectively. In the presence of target DNA, the obtained dsDNA product cannot adsorb onto the surface of AuNPs due to electrostatic interaction, which makes the unmodified AuNPs exhibit excellent GOx-like catalytic activity. Such catalytic activity can enlarge the diameters of AuNPs in the glucose and HAuCl4 solution and result in a connection between most of the AuNPs and a conductive gold film formation with a dramatically increased conductance. For the control sample, the catalytic activity sites of AuNPs are fully blocked by ssDNA due to the noncovalent interaction between nucleotide bases and AuNPs. Thus, the growth of the assembled AuNPs will not happen and the conductance between microelectrodes will be not changed. Under the optimal experimental conditions, the developed strategy exhibited a sensitive response to target DNA with a high signal-to-noise ratio. Moreover, this strategy was also demonstrated to provide excellent differentiation ability for single-nucleotide polymorphism. Such performances indicated the great potential of this label-free electrical strategy for clinical diagnostics and genetic analysis under real biological sample separation.

  7. Molecular docking and 3D-QSAR studies on inhibitors of DNA damage signaling enzyme human PARP-1.

    Science.gov (United States)

    Fatima, Sabiha; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

    2012-08-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) operates in a DNA damage signaling network. Molecular docking and three dimensional-quantitative structure activity relationship (3D-QSAR) studies were performed on human PARP-1 inhibitors. Docked conformation obtained for each molecule was used as such for 3D-QSAR analysis. Molecules were divided into a training set and a test set randomly in four different ways, partial least square analysis was performed to obtain QSAR models using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Derived models showed good statistical reliability that is evident from their r², q²(loo) and r²(pred) values. To obtain a consensus for predictive ability from all the models, average regression coefficient r²(avg) was calculated. CoMFA and CoMSIA models showed a value of 0.930 and 0.936, respectively. Information obtained from the best 3D-QSAR model was applied for optimization of lead molecule and design of novel potential inhibitors.

  8. A Cross-Cancer Genetic Association Analysis of the DNA Repair and DNA Damage Signaling Pathways for Lung, Ovary, Prostate, Breast, and Colorectal Cancer.

    Science.gov (United States)

    Scarbrough, Peter M; Weber, Rachel Palmieri; Iversen, Edwin S; Brhane, Yonathan; Amos, Christopher I; Kraft, Peter; Hung, Rayjean J; Sellers, Thomas A; Witte, John S; Pharoah, Paul; Henderson, Brian E; Gruber, Stephen B; Hunter, David J; Garber, Judy E; Joshi, Amit D; McDonnell, Kevin; Easton, Doug F; Eeles, Ros; Kote-Jarai, Zsofia; Muir, Kenneth; Doherty, Jennifer A; Schildkraut, Joellen M

    2016-01-01

    DNA damage is an established mediator of carcinogenesis, although genome-wide association studies (GWAS) have identified few significant loci. This cross-cancer site, pooled analysis was performed to increase the power to detect common variants of DNA repair genes associated with cancer susceptibility. We conducted a cross-cancer analysis of 60,297 single nucleotide polymorphisms, at 229 DNA repair gene regions, using data from the NCI Genetic Associations and Mechanisms in Oncology (GAME-ON) Network. Our analysis included data from 32 GWAS and 48,734 controls and 51,537 cases across five cancer sites (breast, colon, lung, ovary, and prostate). Because of the unavailability of individual data, data were analyzed at the aggregate level. Meta-analysis was performed using the Association analysis for SubSETs (ASSET) software. To test for genetic associations that might escape individual variant testing due to small effect sizes, pathway analysis of eight DNA repair pathways was performed using hierarchical modeling. We identified three susceptibility DNA repair genes, RAD51B (P cancer risk in the base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination pathways. Only three susceptibility loci were identified, which had all been previously reported. In contrast, hierarchical modeling identified several pleiotropic cancer risk associations in key DNA repair pathways. Results suggest that many common variants in DNA repair genes are likely associated with cancer susceptibility through small effect sizes that do not meet stringent significance testing criteria. ©2015 American Association for Cancer Research.

  9. Electrochemical gene sensor for Mycoplasma pneumoniae DNA using dual signal amplification via a Pt-Pd nanowire and horse radish peroxidase

    International Nuclear Information System (INIS)

    Liu, Linlin; Xiang, Guiming; Jiang, Dongneng; Du, Chunlan; Liu, Chang; Huang, Weiwei; Pu, Xiaoyun

    2016-01-01

    A dually amplified DNA biosensor was constructed for the determination of the DNA of Mycoplasma pneumoniae (M. pneu). A gold electrode was modified with 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA; a π-stacking perylene semiconductor dye with outstanding electronic and optical properties), a layer of gold nanoparticles (nano-Au), and capture DNA. Pt-Pd nanowires served as carriers for the co immobilization of complementary probe (CP2) and the mediator thionine (Thi). Horseradish peroxidase (HRP) acted as a blocking reagent and signal enhancer. Following base pairing, the modified Pt-Pd nanowires were captured on the surface of the gold electrode. After addition of H 2 O 2 , the Pt-Pd nanowires and HRP both catalyzed the reduction of H 2 O 2 and promoted the electron transfer via the mediator Thi, resulting in an amplified electrochemical signal. The electrical signal, best measured at a working voltage of −200 mV (vs a SCE), is logarithmically related to the concentration of the M. pneu DNA in the 0.1 pM to 20 nM concentration range, and the detection limit (at an S/N ratio of 3) is 0.03 pM. The assay is robust, sensitive and specific. Conceivably, it is a cost-effective alternative to the established PCR method for the detection of M. pneu in clinical samples. (author)

  10. Immunoglobulin heavy-chain fluorescence in situ hybridization-chromogenic in situ hybridization DNA probe split signal in the clonality assessment of lymphoproliferative processes on cytological samples.

    Science.gov (United States)

    Zeppa, Pio; Sosa Fernandez, Laura Virginia; Cozzolino, Immacolata; Ronga, Valentina; Genesio, Rita; Salatiello, Maria; Picardi, Marco; Malapelle, Umberto; Troncone, Giancarlo; Vigliar, Elena

    2012-12-25

    The human immunoglobulin heavy-chain (IGH) locus at chromosome 14q32 is frequently involved in different translocations of non-Hodgkin lymphoma (NHL), and the detection of any breakage involving the IGH locus should identify a B-cell NHL. The split-signal IGH fluorescence in situ hybridization-chromogenic in situ hybridization (FISH-CISH) DNA probe is a mixture of 2 fluorochrome-labeled DNAs: a green one that binds the telomeric segment and a red one that binds the centromeric segment, both on the IGH breakpoint. In the current study, the authors tested the capability of the IGH FISH-CISH DNA probe to detect IGH translocations and diagnose B-cell lymphoproliferative processes on cytological samples. Fifty cytological specimens from cases of lymphoproliferative processes were tested using the split-signal IGH FISH-CISH DNA probe and the results were compared with light-chain assessment by flow cytometry (FC), IGH status was tested by polymerase chain reaction (PCR), and clinicohistological data. The signal score produced comparable results on FISH and CISH analysis and detected 29 positive, 15 negative, and 6 inadequate cases; there were 29 true-positive cases (66%), 9 true-negative cases (20%), 6 false-negative cases (14%), and no false-positive cases (0%). Comparing the sensitivity of the IGH FISH-CISH DNA split probe with FC and PCR, the highest sensitivity was obtained by FC, followed by FISH-CISH and PCR. The split-signal IGH FISH-CISH DNA probe is effective in detecting any translocation involving the IGH locus. This probe can be used on different samples from different B-cell lymphoproliferative processes, although it is not useful for classifying specific entities. Cancer (Cancer Cytopathol) 2012;. © 2012 American Cancer Society. Copyright © 2012 American Cancer Society.

  11. Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

    Science.gov (United States)

    Hu, Yufang; Zhang, Qingqing; Xu, Lihua; Wang, Jiao; Rao, Jiajia; Guo, Zhiyong; Wang, Sui

    2017-11-01

    Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H 2 O 2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

  12. Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

    Science.gov (United States)

    Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

    2009-09-01

    DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

  13. Target-induced formation of gold amalgamation on DNA-based sensing platform for electrochemical monitoring of mercury ion coupling with cycling signal amplification strategy

    International Nuclear Information System (INIS)

    Chen, Jinfeng; Tang, Juan; Zhou, Jun; Zhang, Lan; Chen, Guonan; Tang, Dianping

    2014-01-01

    Graphical abstract: -- Highlights: •We report a new electrochemical sensing protocol for the detection of mercury ion. •Gold amalgamation on DNA-based sensing platform was used as nanocatalyst. •The signal was amplified by cycling signal amplification strategy. -- Abstract: Heavy metal ion pollution poses severe risks in human health and environmental pollutant, because of the likelihood of bioaccumulation and toxicity. Driven by the requirement to monitor trace-level mercury ion (Hg 2+ ), herein we construct a new DNA-based sensor for sensitive electrochemical monitoring of Hg 2+ by coupling target-induced formation of gold amalgamation on DNA-based sensing platform with gold amalgamation-catalyzed cycling signal amplification strategy. The sensor was simply prepared by covalent conjugation of aminated poly-T (25) oligonucleotide onto the glassy carbon electrode by typical carbodiimide coupling. Upon introduction of target analyte, Hg 2+ ion was intercalated into the DNA polyion complex membrane based on T–Hg 2+ –T coordination chemistry. The chelated Hg 2+ ion could induce the formation of gold amalgamation, which could catalyze the p-nitrophenol with the aid of NaBH 4 and Ru(NH 3 ) 6 3+ for cycling signal amplification. Experimental results indicated that the electronic signal of our system increased with the increasing Hg 2+ level in the sample, and has a detection limit of 0.02 nM with a dynamic range of up to 1000 nM Hg 2+ . The strategy afforded exquisite selectivity for Hg 2+ against other environmentally related metal ions. In addition, the methodology was evaluated for the analysis of Hg 2+ in spiked tap-water samples, and the recovery was 87.9–113.8%

  14. Target-induced formation of gold amalgamation on DNA-based sensing platform for electrochemical monitoring of mercury ion coupling with cycling signal amplification strategy

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Jinfeng; Tang, Juan; Zhou, Jun; Zhang, Lan; Chen, Guonan; Tang, Dianping, E-mail: dianping.tang@fzu.edu.cn

    2014-01-31

    Graphical abstract: -- Highlights: •We report a new electrochemical sensing protocol for the detection of mercury ion. •Gold amalgamation on DNA-based sensing platform was used as nanocatalyst. •The signal was amplified by cycling signal amplification strategy. -- Abstract: Heavy metal ion pollution poses severe risks in human health and environmental pollutant, because of the likelihood of bioaccumulation and toxicity. Driven by the requirement to monitor trace-level mercury ion (Hg{sup 2+}), herein we construct a new DNA-based sensor for sensitive electrochemical monitoring of Hg{sup 2+} by coupling target-induced formation of gold amalgamation on DNA-based sensing platform with gold amalgamation-catalyzed cycling signal amplification strategy. The sensor was simply prepared by covalent conjugation of aminated poly-T{sub (25)} oligonucleotide onto the glassy carbon electrode by typical carbodiimide coupling. Upon introduction of target analyte, Hg{sup 2+} ion was intercalated into the DNA polyion complex membrane based on T–Hg{sup 2+}–T coordination chemistry. The chelated Hg{sup 2+} ion could induce the formation of gold amalgamation, which could catalyze the p-nitrophenol with the aid of NaBH{sub 4} and Ru(NH{sub 3}){sub 6}{sup 3+} for cycling signal amplification. Experimental results indicated that the electronic signal of our system increased with the increasing Hg{sup 2+} level in the sample, and has a detection limit of 0.02 nM with a dynamic range of up to 1000 nM Hg{sup 2+}. The strategy afforded exquisite selectivity for Hg{sup 2+} against other environmentally related metal ions. In addition, the methodology was evaluated for the analysis of Hg{sup 2+} in spiked tap-water samples, and the recovery was 87.9–113.8%.

  15. Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

    Science.gov (United States)

    Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

    2014-09-02

    A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

  16. The effect of microscopic attractive interactions on piezoelectric coefficients of nanoscale DNA films and its resultant mirocantilever-based biosensor signals

    International Nuclear Information System (INIS)

    Wu, Jun-Zheng; Zhang, Neng-Hui; Zhou, Mei-Hong

    2017-01-01

    The adsorption of charged biomolecules on a substrate will trigger a self-induced electric potential field that could deflect microcantilever biosensors in the nanometer regime. The paper is devoted to a multiscale characterization of the piezoelectric coefficient of double-stranded DNA (dsDNA) films with microscopic attractive interactions in multivalence salt solutions, which has a close relationship with biosensor signals. First, two different analytical models of cantilever deflections based on macroscopic piezoelectric theories or mesoscopic liquid crystal theories were combined in the sense of equivalent deformation in order to bridge the relation between the macroscopic piezoelectric coefficient of an adsorbate film and the sensitivity of its microstructure to surrounding conditions. Second, two interaction potentials of the free energy for repulsion-dominated DNA films in NaCl solution or attraction-repulsion-coexisted DNA films in multivalent salt solutions were used to compare the piezoelectric effect and the resultant cantilever deformation at various packing conditions, such as different packing density, various nucleotide numbers and two packing technologies, i.e. nano-grafting or self-assembling technology. The variational tendency of microcantilever deflections predicted by the present multiscale analytical model agrees well with the related DNA-mirocantilever experiments. Negative piezoelectric coefficient of dsDNA film exists in multivalent salt solutions, and its distinctive size effect with different packing densities and nucleotide numbers provides us with an opportunity to obtain a more sensitive microcantilever sensor by careful control of packing conditions. (paper)

  17. IFNγ induces oxidative stress, DNA damage and tumor cell senescence via TGFβ/SMAD signaling-dependent induction of Nox4 and suppression of ANT2

    Czech Academy of Sciences Publication Activity Database

    Hubáčková, Soňa; Kučerová, Alena; Michlits, Georg; Kyjacová, Lenka; Reiniš, Milan; Korolov, Oleksandr; Bartek, Jiří; Hodný, Zdeněk

    2016-01-01

    Roč. 35, č. 10 (2016), s. 1236-1249 ISSN 0950-9232 R&D Projects: GA ČR GA13-17658S; GA MZd NT14461; GA AV ČR(CZ) L200521301 Institutional support: RVO:68378050 Keywords : IFNγ * DNA damage * TGFβ/SMAD signaling * Nox4 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.519, year: 2016

  18. Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

    Directory of Open Access Journals (Sweden)

    Justin W Leung

    2014-03-01

    Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

  19. Suppressor of cytokine signaling (SOCS genes are silenced by DNA hypermethylation and histone deacetylation and regulate response to radiotherapy in cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Moon-Hong Kim

    Full Text Available Suppressor of cytokine signaling (SOCS family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.

  20. Mechanisms of mtDNA segregation and mitochondrial signalling in cells with the pathogenic A3243G mutation

    NARCIS (Netherlands)

    Jahangir Tafrechi, Roshan Sakineh

    2008-01-01

    Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene

  1. Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

    Energy Technology Data Exchange (ETDEWEB)

    Abdelali, Ala [Department of Anatomy, Faculty of Medicine, Kuwait University (Kuwait); Al-Bader, Maie [Department of Physiology, Faculty of Medicine, Kuwait University (Kuwait); Kilarkaje, Narayana, E-mail: knarayana@hsc.edu.kw [Department of Anatomy, Faculty of Medicine, Kuwait University (Kuwait)

    2016-11-15

    Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

  2. Effects of Trans-Resveratrol on hyperglycemia-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase signaling in rat testis

    International Nuclear Information System (INIS)

    Abdelali, Ala; Al-Bader, Maie; Kilarkaje, Narayana

    2016-01-01

    Diabetes induces oxidative stress, DNA damage and alters several intracellular signaling pathways in organ systems. This study investigated modulatory effects of Trans-Resveratrol on type 1 diabetes mellitus (T1DM)-induced abnormal spermatogenesis, DNA damage and alterations in poly (ADP-ribose) polymerase (PARP) signaling in rat testis. Trans-Resveratrol administration (5mg/kg/day, ip) to Streptozotocin-induced T1DM adult male Wistar rats from day 22–42 resulted in recovery of induced oxidative stress, abnormal spermatogenesis and inhibited DNA synthesis, and led to mitigation of 8-hydroxy-2'-deoxyguanosine formation in the testis and spermatozoa, and DNA double-strand breaks in the testis. Trans-Resveratrol aggravated T1DM-induced up-regulation of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 expression; however, it did not modify the up-regulated total PARP and down-regulated PARP1 expressions, but recovered the decreased SirT1 (Sirtuin 1) levels in T1DM rat testis. Trans-Resveratrol, when given alone, reduced the poly (ADP-ribosyl)ation (pADPr) process in the testis due to an increase in PAR glycohydrolase activity, but when given to T1DM rats it did not affect the pADPr levels. T1DM with or without Trans-Resveratrol did not induce nuclear translocation of apoptosis-inducing factor and the formation of 50 kb DNA breaks, suggesting to the lack of caspase-3-independent cell death called parthanatos. T1DM with or without Trans-Resveratrol did not increase necrotic cell death in the testis. Primary spermatocytes, Sertoli cells, Leydig cells and intra-testicular vessels showed the expression of PARP pathway related proteins. In conclusion, Trans-Resveratrol mitigates T1DM-induced sperm abnormality and DNA damage, but does not significantly modulate PARP signaling pathway, except the SirT1 expression, in the rat testis. - Highlights: • Resveratrol inhibits diabetes-induced abnormal sperm morphogenesis • Resveratrol recovers

  3. Highly sensitive aptasensor based on synergetic catalysis activity of MoS2-Au-HE composite using cDNA-Au-GOD for signal amplification.

    Science.gov (United States)

    Song, Hai-Yan; Kang, Tian-Fang; Lu, Li-Ping; Cheng, Shui-Yuan

    2017-03-01

    Single or few-layer nanosheets of MoS 2 (MoS 2 nanosheets) and a composite composed of MoS 2 nanosheets, Au nanoparticles (AuNPs) and hemin (HE) (denoted as MoS 2 -Au-HE) were prepared. The composites possessed high synergetic catalysis activity towards the electroreduction of hydrogen peroxide. Furthermore, glucose oxidase (GOD) and AuNPs were used as marker of the complementary DNA (cDNA) strand of kanamycin aptamer to prepare a conjugate (reffered as cDNA-Au-GOD) that was designed as the signal probe. Both cDNA-Au-GOD and MoS 2 -Au-HE were applied to fabricate aptasensor for kanamycin. MoS 2 -Au-HE acted as solid platform for kanamycin aptamer and signal transmitters. AuNPs were employed as the supporter of cDNA and GOD which catalyze dissolved oxygen to produce hydrogen peroxide in the presence of glucose. Then cathodic peak current of H 2 O 2 was recorded by differential pulse voltammetry (DPV). The electrochemical reduction of H 2 O 2 was catalyzed by MoS 2 -Au-HE that was modified onto the surface of a glassy carbon electrode (GCE). The cathodic peak current of H 2 O 2 was highly linearly decreased with an increase of kanamycin concentrations from 1.0ng/L to 1.0×10 5 ng/L, with a detection limit of 0.8ng/L. This aptasensor can be used to detect kanamycin in milk with high specificity, sensitivity and selectivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. DNA methylation changes in genes frequently mutated in sporadic colorectal cancer and in the DNA repair and Wnt/β-catenin signaling pathway genes

    Czech Academy of Sciences Publication Activity Database

    Farkas, S. A.; Vymetálková, Veronika; Vodičková, Ludmila; Vodička, Pavel; Torbjörn, K. N.

    2014-01-01

    Roč. 6, č. 2 (2014), s. 179-191 ISSN 1750-1911 R&D Projects: GA ČR GPP304/11/P715; GA ČR(CZ) GAP304/12/1585; GA MZd NT14329 Institutional support: RVO:68378041 Keywords : CpG * DNA repair genes * sporadic colorectal cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.649, year: 2014

  5. Singapore grouper iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling.

    Science.gov (United States)

    Huang, Xiaohong; Huang, Youhua; Ouyang, Zhengliang; Xu, Lixiao; Yan, Yang; Cui, Huachun; Han, Xin; Qin, Qiwei

    2011-08-01

    Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (ΔΨm) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.

  6. Amplified amperometric aptasensor for selective detection of protein using catalase-functional DNA-PtNPs dendrimer as a synergetic signal amplification label.

    Science.gov (United States)

    Zhang, Juan; Yuan, Yali; biXie, Shun; Chai, Yaqin; Yuan, Ruo

    2014-10-15

    In this work, we present a new strategy to construct an electrochemical aptasensor for sensitive detection of platelet-derived growth factor BB (PDGF-BB) based on the synergetic amplification of a three-dimensional (3D) nanoscale catalase (CAT) enzyme-functional DNA-platinum nanoparticles (PtNPs) dendrimer through autonomous layer-by-layer assembly. Firstly, polyamidoaminedendrimer (PAMAM) with a hyper-branched and three-dimensional structure was served as nanocarriers to coimmobilize a large number of PDGF-BB binding aptamer (PBA II) and ssDNA 1 (S1) to form PBA II-PAMAM-S1 bioconjugate. In the presence of PDGF-BB, the bioconjugate was self-assembled on the electrode by sandwich assay. Following that, the carried S1 propagated a chain reaction of hybridization events between CAT-PtNPs-S1 and CAT-PtNPs-ssDNA 2 (S2) to form a 3D nanoscale CAT-functional PtNPs-DNA dendrimer, which successfully immobilized substantial CAT enzyme and PtNPs with superior catalysis activity. In this process, the formed negatively charged double-helix DNA could cause the intercalation of hexaammineruthenium(III) chloride (RuHex) into the groove via electrostatic interactions. Thus, numerous RuHex redox probes and CAT were decorated inside/outside of the dendrimer. In the presence of H2O2 in electrolytic cell, the synergistic reaction of CAT and PtNPs towards electrocatalysis could further amplify electrochemical signal. Under optimal condition, the CAT-PtNPs-DNA dendrimer-based sensing system presented a linear dependence between the reduction peak currents and logarithm of PDGF-BB concentrations in the range of 0.00005-35 nM with a relatively low detection limit of 0.02 pM. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma.

    Science.gov (United States)

    Li, Zhigang; Fan, Erica K; Liu, Jinghua; Scott, Melanie J; Li, Yuehua; Li, Song; Xie, Wen; Billiar, Timothy R; Wilson, Mark A; Jiang, Yong; Wang, Ping; Fan, Jie

    2017-05-11

    Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mφ) direct trauma-induced inflammation, and Mφ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mφ and the subsequent regulation of Mφ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)-TLR4-MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mφ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mφ. However, autophagy activation also suppressed Mφ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mφ homeostasis in response to trauma.

  8. Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

    Science.gov (United States)

    Bogacheva, Mariia; Egorova, Anna; Slita, Anna; Maretina, Marianna; Baranov, Vladislav; Kiselev, Anton

    2017-11-01

    The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    Energy Technology Data Exchange (ETDEWEB)

    Edelbrock, Michael A., E-mail: Edelbrock@findlay.edu [The University of Findlay, 1000 North Main Street, Findlay, OH 45840 (United States); Kaliyaperumal, Saravanan, E-mail: Saravanan.Kaliyaperumal@hms.harvard.edu [Division of Comparative Medicine and Pathology, New England Primate Research Center, One Pine Hill Drive, Southborough, MA 01772 (United States); Williams, Kandace J., E-mail: Kandace.williams@utoledo.edu [University of Toledo College of Medicine and Life Sciences, Department of Biochemistry and Cancer Biology, 3000 Transverse Dr., Toledo, OH 43614 (United States)

    2013-03-15

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα, and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O{sup 6}meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6.

  10. Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

    International Nuclear Information System (INIS)

    Edelbrock, Michael A.; Kaliyaperumal, Saravanan; Williams, Kandace J.

    2013-01-01

    The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα, and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O 6 meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6

  11. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

  12. Target-induced formation of gold amalgamation on DNA-based sensing platform for electrochemical monitoring of mercury ion coupling with cycling signal amplification strategy.

    Science.gov (United States)

    Chen, Jinfeng; Tang, Juan; Zhou, Jun; Zhang, Lan; Chen, Guonan; Tang, Dianping

    2014-01-31

    Heavy metal ion pollution poses severe risks in human health and environmental pollutant, because of the likelihood of bioaccumulation and toxicity. Driven by the requirement to monitor trace-level mercury ion (Hg(2+)), herein we construct a new DNA-based sensor for sensitive electrochemical monitoring of Hg(2+) by coupling target-induced formation of gold amalgamation on DNA-based sensing platform with gold amalgamation-catalyzed cycling signal amplification strategy. The sensor was simply prepared by covalent conjugation of aminated poly-T(25) oligonucleotide onto the glassy carbon electrode by typical carbodiimide coupling. Upon introduction of target analyte, Hg(2+) ion was intercalated into the DNA polyion complex membrane based on T-Hg(2+)-T coordination chemistry. The chelated Hg(2+) ion could induce the formation of gold amalgamation, which could catalyze the p-nitrophenol with the aid of NaBH4 and Ru(NH3)6(3+) for cycling signal amplification. Experimental results indicated that the electronic signal of our system increased with the increasing Hg(2+) level in the sample, and has a detection limit of 0.02nM with a dynamic range of up to 1000nM Hg(2+). The strategy afforded exquisite selectivity for Hg(2+) against other environmentally related metal ions. In addition, the methodology was evaluated for the analysis of Hg(2+) in spiked tap-water samples, and the recovery was 87.9-113.8%. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

    International Nuclear Information System (INIS)

    Elsen, S.; Collin-Faure, V.; Gidrol, X.; Lemercier, C.

    2013-01-01

    Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design. (authors)

  14. Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

    International Nuclear Information System (INIS)

    Cucinotta, Francis A

    2016-01-01

    The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

  15. Systems Biology Model of Interactions Between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFbeta and ATM Signaling

    Energy Technology Data Exchange (ETDEWEB)

    O' Neill, Peter [University of Oxford; Anderson, Jennifer [University of Oxford

    2014-10-02

    The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low dose responses and cross-talk between the ATM and TGFβ pathways initiated by low and high LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to cross- talk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

  16. Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Cucinotta, Francis A [Univ. of Nevada, Las Vegas, NV (United States)

    2016-09-01

    The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental

  17. Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes

    Czech Academy of Sciences Publication Activity Database

    Vlková, Veronika; Štěpánek, Ivan; Hrušková, Veronika; Šenigl, Filip; Mayerová, Veronika; Šrámek, Martin; Šímová, Jana; Bieblová, Jana; Indrová, Marie; Hejhal, Tomáš; Dérian, N.; Klatzmann, D.; Six, A.; Reiniš, Milan

    2014-01-01

    Roč. 5, č. 16 (2014), s. 6923-35 ISSN 1949-2553 R&D Projects: GA ČR GAP301/10/2174; GA MZd NT14461 EU Projects: European Commission(XE) 18933 - CLINIGENE Grant - others:French state funds within the Investissements d’Avenir program(FR) ANR-11-IDEX-0004-02 Institutional support: RVO:68378050 Keywords : IFNγ signalling pathway * DNA demethylation * tumour Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.359, year: 2014

  18. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

  19. Cytogenetic responses to ionizing radiation exposure of human fibroblasts with knocked-down expressions of various DNA damage signaling genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry; Wu, Honglu

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with up-regulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. Here, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yields of MN and/or CA formation were significantly increased by suppressed expression of some of the selected genes in DSB and other DNA repair pathways. Knocked-down expression of other genes showed significant impact on cell cycle progression, possibly because of severe impairment of DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  20. Epigenetic Alteration by DNA Promoter Hypermethylation of Genes Related to Transforming Growth Factor-β (TGF-β) Signaling in Cancer

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    Khin, Sann Sanda [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Pathology Research Unit, Department of Medical Research (Central Myanmar), Naypyitaw, Union of (Myanmar); Kitazawa, Riko [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Ehime University Graduate School of Medicine, Toon 791-0295, Ehime (Japan); Kondo, Takeshi; Idei, Yuka; Fujimoto, Masayo [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Haraguchi, Ryuma [Ehime University Graduate School of Medicine, Toon 791-0295, Ehime (Japan); Mori, Kiyoshi [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Kitazawa, Sohei, E-mail: kitazawa@m.ehime-u.ac.jp [Kobe University Graduate School of Medicine, Division of Diagnostic Molecular Pathology, Kobe 650-0017 (Japan); Ehime University Graduate School of Medicine, Toon 791-0295, Ehime (Japan)

    2011-03-03

    Epigenetic alterations in cancer, especially DNA methylation and histone modification, exert a significant effect on the deregulated expression of cancer-related genes and lay an epigenetic pathway to carcinogenesis and tumor progression. Global hypomethylation and local hypermethylation of CpG islands in the promoter region, which result in silencing tumor suppressor genes, constitute general and major epigenetic modification, the hallmark of the neoplastic epigenome. Additionally, methylation-induced gene silencing commonly affects a number of genes and increases with cancer progression. Indeed, cancers with a high degree of methylation (CpG island methylator phenotype/CIMP) do exist and represent a distinct subset of certain cancers including colorectal, bladder and kidney. On the other hand, signals from the microenvironment, especially those from transforming growth factor-β (TGF-β), induce targeted de novo epigenetic alterations of cancer-related genes. While TGF-β signaling has been implicated in two opposite roles in cancer, namely tumor suppression and tumor promotion, its deregulation is also partly induced by epigenetic alteration itself. Although the epigenetic pathway to carcinogenesis and cancer progression has such reciprocal complexity, the important issue is to identify genes or signaling pathways that are commonly silenced in various cancers in order to find early diagnostic and therapeutic targets. In this review, we focus on the epigenetic alteration by DNA methylation and its role in molecular modulations of the TGF-β signaling pathway that cause or underlie altered cancer-related gene expression in both phases of early carcinogenesis and late cancer progression.

  1. Epigenetic Alteration by DNA Promoter Hypermethylation of Genes Related to Transforming Growth Factor-β (TGF-β) Signaling in Cancer

    International Nuclear Information System (INIS)

    Khin, Sann Sanda; Kitazawa, Riko; Kondo, Takeshi; Idei, Yuka; Fujimoto, Masayo; Haraguchi, Ryuma; Mori, Kiyoshi; Kitazawa, Sohei

    2011-01-01

    Epigenetic alterations in cancer, especially DNA methylation and histone modification, exert a significant effect on the deregulated expression of cancer-related genes and lay an epigenetic pathway to carcinogenesis and tumor progression. Global hypomethylation and local hypermethylation of CpG islands in the promoter region, which result in silencing tumor suppressor genes, constitute general and major epigenetic modification, the hallmark of the neoplastic epigenome. Additionally, methylation-induced gene silencing commonly affects a number of genes and increases with cancer progression. Indeed, cancers with a high degree of methylation (CpG island methylator phenotype/CIMP) do exist and represent a distinct subset of certain cancers including colorectal, bladder and kidney. On the other hand, signals from the microenvironment, especially those from transforming growth factor-β (TGF-β), induce targeted de novo epigenetic alterations of cancer-related genes. While TGF-β signaling has been implicated in two opposite roles in cancer, namely tumor suppression and tumor promotion, its deregulation is also partly induced by epigenetic alteration itself. Although the epigenetic pathway to carcinogenesis and cancer progression has such reciprocal complexity, the important issue is to identify genes or signaling pathways that are commonly silenced in various cancers in order to find early diagnostic and therapeutic targets. In this review, we focus on the epigenetic alteration by DNA methylation and its role in molecular modulations of the TGF-β signaling pathway that cause or underlie altered cancer-related gene expression in both phases of early carcinogenesis and late cancer progression

  2. Epigenetic Alteration by DNA Promoter Hypermethylation of Genes Related to Transforming Growth Factor-β (TGF-β Signaling in Cancer

    Directory of Open Access Journals (Sweden)

    Kiyoshi Mori

    2011-03-01

    Full Text Available Epigenetic alterations in cancer, especially DNA methylation and histone modification, exert a significant effect on the deregulated expression of cancer-related genes and lay an epigenetic pathway to carcinogenesis and tumor progression. Global hypomethylation and local hypermethylation of CpG islands in the promoter region, which result in silencing tumor suppressor genes, constitute general and major epigenetic modification, the hallmark of the neoplastic epigenome. Additionally, methylation-induced gene silencing commonly affects a number of genes and increases with cancer progression. Indeed, cancers with a high degree of methylation (CpG island methylator phenotype/CIMP do exist and represent a distinct subset of certain cancers including colorectal, bladder and kidney. On the other hand, signals from the microenvironment, especially those from transforming growth factor-β (TGF-β, induce targeted de novo epigenetic alterations of cancer-related genes. While TGF-β signaling has been implicated in two opposite roles in cancer, namely tumor suppression and tumor promotion, its deregulation is also partly induced by epigenetic alteration itself. Although the epigenetic pathway to carcinogenesis and cancer progression has such reciprocal complexity, the important issue is to identify genes or signaling pathways that are commonly silenced in various cancers in order to find early diagnostic and therapeutic targets. In this review, we focus on the epigenetic alteration by DNA methylation and its role in molecular modulations of the TGF-β signaling pathway that cause or underlie altered cancer-related gene expression in both phases of early carcinogenesis and late cancer progression.

  3. KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1

    Science.gov (United States)

    Borgel, Julie; Tyl, Marek; Schiller, Karin; Pusztai, Zsofia; Dooley, Christopher M.; Deng, Wen; Wooding, Carol; White, Richard J.; Warnecke, Tobias; Leonhardt, Heinrich; Busch-Nentwich, Elisabeth M.

    2017-01-01

    Abstract Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing. PMID:28180290

  4. The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals.

    OpenAIRE

    Ding, W V; Johnston, S A

    1997-01-01

    The transcriptional activation function of the Saccharomyces cerevisiae activator Gal4p is known to rely on a DNA binding activity at its amino terminus and an activation domain at its carboxy terminus. Although both domains are required for activation, truncated forms of Gal4p containing only these domains activate poorly in vivo. Also, mutations in an internal conserved region of Gal4p inactivate the protein, suggesting that this internal region has some function critical to the activity of...

  5. Bipartite recognition of DNA by TCF/Pangolin is remarkably flexible and contributes to transcriptional responsiveness and tissue specificity of wingless signaling.

    Directory of Open Access Journals (Sweden)

    Hilary C Archbold

    2014-09-01

    Full Text Available The T-cell factor (TCF family of transcription factors are major mediators of Wnt/β-catenin signaling in metazoans. All TCFs contain a High Mobility Group (HMG domain that possesses specific DNA binding activity. In addition, many TCFs contain a second DNA binding domain, the C-clamp, which binds to DNA motifs referred to as Helper sites. While HMG and Helper sites are both important for the activation of several Wnt dependent cis-regulatory modules (W-CRMs, the rules of what constitutes a functional HMG-Helper site pair are unknown. In this report, we employed a combination of in vitro binding, reporter gene analysis and bioinformatics to address this question, using the Drosophila family member TCF/Pangolin (TCF/Pan as a model. We found that while there were constraints for the orientation and spacing of HMG-Helper pairs, the presence of a Helper site near a HMG site in any orientation increased binding and transcriptional response, with some orientations displaying tissue-specific patterns. We found that altering an HMG-Helper site pair from a sub-optimal to optimal orientation/spacing dramatically increased the responsiveness of a W-CRM in several fly tissues. In addition, we used the knowledge gained to bioinformatically identify two novel W-CRMs, one that was activated by Wnt/β-catenin signaling in the prothoracic gland, a tissue not previously connected to this pathway. In sum, this work extends the importance of Helper sites in fly W-CRMs and suggests that the type of HMG-Helper pair is a major factor in setting the threshold for Wnt activation and tissue-responsiveness.

  6. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

    Science.gov (United States)

    Thompson, Larry H

    2012-01-01

    The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Chung Jae

    2009-06-01

    Full Text Available Abstract Background Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition. Methods To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis. Results Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99 between the total number of hypermethylated CGIs and GI50 values (i.e., increased drug resistance following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells. Conclusion Selective epigenetic disruption of distinct biological

  8. Cobalt oxide nanoparticles aggravate DNA damage and cell death in eggplant via mitochondrial swelling and NO signaling pathway.

    Science.gov (United States)

    Faisal, Mohammad; Saquib, Quaiser; Alatar, Abdulrahman A; Al-Khedhairy, Abdulaziz A; Ahmed, Mukhtar; Ansari, Sabiha M; Alwathnani, Hend A; Dwivedi, Sourabh; Musarrat, Javed; Praveen, Shelly

    2016-03-18

    Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant. The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (ΔΨm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis. This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.

  9. Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

    Directory of Open Access Journals (Sweden)

    Shamim H Rahman

    2015-05-01

    Full Text Available In vitro disease modeling based on induced pluripotent stem cells (iPSCs provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID, a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(DJ recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.

  10. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  11. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    International Nuclear Information System (INIS)

    Railo, Antti; Pajunen, Antti; Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka; Vainio, Seppo

    2009-01-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-β-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of β-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  12. The innate immunity adaptor SARM translocates to the nucleus to stabilize lamins and prevent DNA fragmentation in response to pro-apoptotic signaling.

    Directory of Open Access Journals (Sweden)

    Chad R Sethman

    Full Text Available Sterile alpha and armadillo-motif containing protein (SARM, a highly conserved and structurally unique member of the MyD88 family of Toll-like receptor adaptors, plays an important role in innate immunity signaling and apoptosis. Its exact mechanism of intracellular action remains unclear. Apoptosis is an ancient and ubiquitous process of programmed cell death that results in disruption of the nuclear lamina and, ultimately, dismantling of the nucleus. In addition to supporting the nuclear membrane, lamins serve important roles in chromatin organization, epigenetic regulation, transcription, nuclear transport, and mitosis. Mutations and other damage that destabilize nuclear lamins (laminopathies underlie a number of intractable human diseases. Here, we report that SARM translocates to the nucleus of human embryonic kidney cells by using its amino-terminal Armadillo repeat region. Within the nucleus, SARM forms a previously unreported lattice akin to the nuclear lamina scaffold. Moreover, we show that SARM protects lamins from apoptotic degradation and reduces internucleosomal DNA fragmentation in response to signaling induced by the proinflammatory cytokine Tumor Necrosis Factor alpha. These findings indicate an important link between the innate immunity adaptor SARM and stabilization of nuclear lamins during inflammation-driven apoptosis in human cells.

  13. Signal transduction and HIV transcriptional activation after exposure to ultraviolet light and other DNA-damaging agents

    International Nuclear Information System (INIS)

    Valerie, K.; Laster, W.S.; Luhua Cheng; Kirkham, J.C.; Reavey, Peter; Kuemmerle, N.B.

    1996-01-01

    Short wavelength (254 nm) ultraviolet light (UVC) radiation was much more potent in activating transcription of human immunodeficiency virus 1 (HIV) reporter genes stably integrated into the genomes of human and monkey cells than ionizing radiation (IR) from a 137 Cs source at similarly cytotoxic doses. A similar differential was also observed when c-jun transcription levels were examined. However, these transcription levels do not correlate with activation of nuclear factor (NF)-kB and AP-1 measured by band-shift assays, i.e. both types of radiation produce similar increases in NF-kB and AP-1 activity, suggesting existence of additional levels of regulation during these responses. Because of the well-established involvement of cytoplasmic signaling pathways in the cellular response to tumor necrosis factor-α (TNF-α), UVC, and IR using other types of assays, the role of TNF-α in the UVC response of HIV and c-jun was investigated in our cell system. We demonstrate that UVC and TNF-α activate HIV gene expression in a synergistic fashion, suggesting that it is unlikely that TNF-α is involved in UVC activation of HIV transcription in stably transfected HeLa cells. Moreover, maximum TNF-α stimulation resulted in one order of magnitude lower levels of HIV expression than that observed after UVC exposure. We also observed an additive effect of UVC and TNF-α on c-jun steady-state mRNA levels, suggestive of a partial overlap in activation mechanism of c-jun by UVC and TNF-α; yet these responses are distinct to some extent. Our results indicate that the HIV, and to some extent also the c-jun, transcriptional responses to UVC are not the result of TNF-α stimulation and subsequent downstream cytoplasmic signaling events in HeLa cells. In addition to the new data, this report also summarizes our current views regarding UVC-induced activations of HIV gene expression in stably transfected cells. (Author)

  14. Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA.

    Science.gov (United States)

    Zhang, Wenjun; Hubbard, Antony; Baca-Parkinson, Leslie; Stanislaw, Stacey; Vladich, Frank; Robida, Mark D; Grille, James G; Maxwell, Daniel; Tsao, Tsu-Shuen; Carroll, William; Gardner, Tracie; Clements, June; Singh, Shalini; Tang, Lei

    2015-09-01

    The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA.

    Science.gov (United States)

    Tu, Haijian; Lin, Kun; Lun, Yongzhi; Yu, Liuming

    2018-06-01

    A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5' end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3' end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5'-GGATCC-3' between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 μM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas ® Amplicor HCV Test Analyzer. Graphical abstract A digital detection strategy utilizing a personal glucometer was developed for the detection of hepatitis C virus RNA. The strategy involved the use of the

  16. Effects of signal transducer and activator of transcription 3 RNAi on content of reactive oxygen species and DNA damage in glioma cell

    International Nuclear Information System (INIS)

    Gao Ling; Li Fengsheng; Dong Bo; Liu Lihui; Liu Qingjie; Chen Xiaohua; Mao Bingzhi

    2011-01-01

    Objective: To investigate the effects of signal transducer and activator of transcription 3 (STAT3) RNAi on the content of reactive oxygen species (ROS) and the DNA damage in glioma cells. Methods: Glioma cells of the line U251 cells were cultured and transfected with STAT3 RNAi plasmid (pSilencer2.1-STAT3, STAT3 group) and pSilencer2.1-GFP (GFP control group) respectively. Part of the U251 cells were irradiated with γ-rays of 60 Co as positive control group of smear phenomenon. The levels of ROS and malondialdehyde (MDA) in the cells were detected 24, 48, and 72 h later by flow cytometry and fluorescence chamoluminescence analyzer, respectively. The DNA damage in the transfected U251 cells was examined by using single cell gel electrophoresis assay, and the cell cycle distribution was examined using FACS PI staining 12, 24, and 36 h later. Results: At 24 h after the transfection, the ROS level of the siSTAT3-transfected cells was 8.91 times that of the control group (F=89.296, P<0.05), and returned to the normal level 48 h later. There were not significant differences in the MDA level of the cells 24, 48, and 72 h later between the siSTAT3 group and siGFP group. Compared with the 8 Gy irradiation positive group with obvious smear phenomenon, smear phenomenon was shown in part of the cells in the siSTAT3 group 6 h later, became less 12 h later, and disappeared completely 24 h later. Compared with the control group,lag of S stage rate was 17.22% and the lag of G 2 /M stage rate was 6.4% 12 h later in the siSTAT-transfected group,and the G 0 /G 1 stage lag rate was 18.44% 24 h later, and the lag of S stage rate was 17.99% 36 h later. Conclusions: Inhibition of STAT3 results in the change of oxido reduction status in glioma cells, as well as damage and reparation of DNA. (authors)

  17. TFII-I regulates target genes in the PI-3K and TGF-β signaling pathways through a novel DNA binding motif.

    Science.gov (United States)

    Segura-Puimedon, Maria; Borralleras, Cristina; Pérez-Jurado, Luis A; Campuzano, Victoria

    2013-09-25

    General transcription factor (TFII-I) is a multi-functional protein involved in the transcriptional regulation of critical developmental genes, encoded by the GTF2I gene located on chromosome 7q11.23. Haploinsufficiency at GTF2I has been shown to play a major role in the neurodevelopmental features of Williams-Beuren syndrome (WBS). Identification of genes regulated by TFII-I is thus critical to detect molecular determinants of WBS as well as to identify potential new targets for specific pharmacological interventions, which are currently absent. We performed a microarray screening for transcriptional targets of TFII-I in cortex and embryonic cells from Gtf2i mutant and wild-type mice. Candidate genes with altered expression were verified using real-time PCR. A novel motif shared by deregulated genes was found and chromatin immunoprecipitation assays in embryonic fibroblasts were used to document in vitro TFII-I binding to this motif in the promoter regions of deregulated genes. Interestingly, the PI3K and TGFβ signaling pathways were over-represented among TFII-I-modulated genes. In this study we have found a highly conserved DNA element, common to a set of genes regulated by TFII-I, and identified and validated novel in vivo neuronal targets of this protein affecting the PI3K and TGFβ signaling pathways. Overall, our data further contribute to unravel the complexity and variability of the different genetic programs orchestrated by TFII-I. © 2013 Elsevier B.V. All rights reserved.

  18. Signalization and repair of the DNA double-strand breaks of in the cerebral tumors: modulation of the radiation response with the chemotherapy treatments

    International Nuclear Information System (INIS)

    Marcinkova-Bencokova, Z.

    2007-07-01

    There are about 6000 new cases of nervous system tumours each year in France. However, the current radio chemotherapeutic approaches against brain tumours remain still insufficient to produce a satisfactory therapeutic index. In parallel, the knowledge of the early radiobiological events has considerably progressed in the last few years. This thesis aims to provide new insights in the molecular and cellular response of brain tumours to radio chemotherapy. This thesis was divided into four stages. Stage 1: a novel DNA double-strand breaks repair pathway depending on the MRE11 protein but independent of the phosphorylation of H2AX emerged from the study of artefacts of the immunofluorescence technique and a systematic analysis of the radiosensitivity of human cells. Stage 2: the radiobiological features of 3 rodent models of glioma among the most used in preclinical trials and of 7 human glioma cell lines were investigated. Functional impairments of the BRCA1 protein in response to radiation and/or cisplatin were observed in the majority of the models tested, raising the question of the role of this protein in the anti-glioma treatments and in glioma genesis. Stage 3: in order to extend our approach to genetic syndromes associated with cerebral tumours predisposition, the radiobiological characteristics of the fibroblasts resulting from patients suffering from neurofibromatosis type 1 (NF1), a pathology associated with a strong incidence of peripheral nervous system tumours, were investigated. NF1 appeared to be a syndrome with moderated radiosensitivity, associated with a weak deficiency of DNA end-joining repair but with a strong activity of MRE11. These results enabled us to propose a preliminary model involving both proteins BRCA1 and NF1. Stage 4: considering the role of BRCA1 in the inhibition of some tyrosine kinase activity and in the response to cisplatin, we tested the radiobiological effects of treatments combining radiation, cisplatin and tyrosine kinase

  19. DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

    International Nuclear Information System (INIS)

    Chen, Rui; Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

    2016-01-01

    Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

  20. DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Rui [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Department of Forensic Medicine, Guangdong Medical University, Dongguan 523808 (China); Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Liu, Chao [Guangzhou Forensic Science Institute, Guangzhou 510030 (China); Lin, Zhoumeng [Institute of Computational Comparative Medicine and Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (United States); Xie, Wei-Bing, E-mail: xieweib@126.com [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Wang, Huijun, E-mail: hjwang711@yahoo.cn [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China)

    2016-03-15

    Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

  1. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  2. IL1-and TGF beta-Nox4 signaling, oxidative stress and DNA damage response are shared features of replicative, oncogene-induced, and drug-induced paracrine 'Bystander senescence'

    Czech Academy of Sciences Publication Activity Database

    Hubáčková, Soňa; Krejčíková, Kateřina; Bartek, Jiří; Hodný, Zdeněk

    2012-01-01

    Roč. 4, č. 12 (2012), 932-951 ISSN 1945-4589 R&D Projects: GA ČR GA204/08/1418; GA ČR GAP301/10/1525 Institutional support: RVO:68378050 Keywords : senescence-associated secretome * DNA damage response * cytokines * JAK/STAT3 * TGF beta * NF kappa B * IL6 * IL beta * Nox4 * autocrine and paracrine signaling * tumor Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.696, year: 2012

  3. Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

    Science.gov (United States)

    Kang, Jian; Kusnadi, Eric P.; Ogden, Allison J.; Hicks, Rodney J.; Bammert, Lukas; Kutay, Ulrike; Hung, Sandy; Sanij, Elaine; Hannan, Ross D.; Hannan, Katherine M.; Pearson, Richard B.

    2016-01-01

    Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer. PMID:27385002

  4. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  5. Human plasmacytoid dentritic cells elicit a Type I Interferon response by sensing DNA via the cGAS-STING signaling pathway.

    Science.gov (United States)

    Bode, Christian; Fox, Mario; Tewary, Poonam; Steinhagen, Almut; Ellerkmann, Richard K; Klinman, Dennis; Baumgarten, Georg; Hornung, Veit; Steinhagen, Folkert

    2016-07-01

    Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN-driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self-DNA and has been linked to TLR9-independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS-STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS-STING pathway and that targeting this pathway could be of therapeutic interest. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Copper exposure induces toxicity to the antioxidant system via the destruction of Nrf2/ARE signaling and caspase-3-regulated DNA damage in fish muscle: Amelioration by myo-inositol

    International Nuclear Information System (INIS)

    Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Feng, Lin; Zhou, Xiao-Qiu

    2015-01-01

    Highlights: • Cu stress decreased fish muscle CuZnSOD, GPx1a, GPx1b and PKCδ mRNA levels. • Cu stress caused fish muscle lower nuclear Nrf2 levels and poor ARE binding ability. • Cu stress induced caspase-3 signaling-modulated DNA fragmentation in fish muscle. • Pre-treatment with MI prevented fish muscle from Cu-induced oxidative damages. - Abstract: The muscle is the main portion of fish that is consumed by humans. Copper (Cu) can induce oxidative damage in fish muscle. However, the effects of Cu exposure on the muscle antioxidant system and molecular patterns and preventive measures against these effects remain unclear. In this study, ROS production, enzymatic and mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2) signaling-related molecules, antioxidant response element (ARE) binding ability, DNA fragmentation and caspase-3 activities were analyzed in fish muscle following Cu exposure or myo-inositol (MI) pre-administration. The results indicated that contamination due to copper exposure caused an approximately three-fold increase in ROS production, induced lipid peroxidation and protein oxidation, and resulted in depletion of the glutathione (GSH) content of fish muscle. Moreover, Cu exposure caused decreases in the activities of total superoxide dismutase (T-SOD), CuZnSOD, and glutathione peroxidase (GPx) that were accompanied by decreases in CuZnSOD, GPx1a, GPx1b and signaling factor protein kinase C delta mRNA levels. The decreases in the antioxidant enzyme gene mRNA levels were confirmed to be partly due to the reduced nuclear Nrf2 protein levels, poor ARE binding ability and increased caspase-3 signaling-modulated DNA fragmentation in the fish muscle. Interestingly, MI pre-treatment prevented fish muscle from Cu-induced oxidative damages mainly through increasing the GSH content, and increasing the CuZnSOD and GPx activities and corresponding mRNA levels and ARE binding ability. Taken together, our results show for the first

  7. Copper exposure induces toxicity to the antioxidant system via the destruction of Nrf2/ARE signaling and caspase-3-regulated DNA damage in fish muscle: Amelioration by myo-inositol

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Wei-Dan; Liu, Yang [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Jiang, Jun [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Wu, Pei [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Feng, Lin, E-mail: fenglin@sicau.edu.cn [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Zhou, Xiao-Qiu, E-mail: zhouxq@sicau.edu.cn [Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, Sichuan (China); Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, Sichuan (China)

    2015-02-15

    Highlights: • Cu stress decreased fish muscle CuZnSOD, GPx1a, GPx1b and PKCδ mRNA levels. • Cu stress caused fish muscle lower nuclear Nrf2 levels and poor ARE binding ability. • Cu stress induced caspase-3 signaling-modulated DNA fragmentation in fish muscle. • Pre-treatment with MI prevented fish muscle from Cu-induced oxidative damages. - Abstract: The muscle is the main portion of fish that is consumed by humans. Copper (Cu) can induce oxidative damage in fish muscle. However, the effects of Cu exposure on the muscle antioxidant system and molecular patterns and preventive measures against these effects remain unclear. In this study, ROS production, enzymatic and mRNA levels of antioxidant enzymes and NF-E2-related factor 2 (Nrf2) signaling-related molecules, antioxidant response element (ARE) binding ability, DNA fragmentation and caspase-3 activities were analyzed in fish muscle following Cu exposure or myo-inositol (MI) pre-administration. The results indicated that contamination due to copper exposure caused an approximately three-fold increase in ROS production, induced lipid peroxidation and protein oxidation, and resulted in depletion of the glutathione (GSH) content of fish muscle. Moreover, Cu exposure caused decreases in the activities of total superoxide dismutase (T-SOD), CuZnSOD, and glutathione peroxidase (GPx) that were accompanied by decreases in CuZnSOD, GPx1a, GPx1b and signaling factor protein kinase C delta mRNA levels. The decreases in the antioxidant enzyme gene mRNA levels were confirmed to be partly due to the reduced nuclear Nrf2 protein levels, poor ARE binding ability and increased caspase-3 signaling-modulated DNA fragmentation in the fish muscle. Interestingly, MI pre-treatment prevented fish muscle from Cu-induced oxidative damages mainly through increasing the GSH content, and increasing the CuZnSOD and GPx activities and corresponding mRNA levels and ARE binding ability. Taken together, our results show for the first

  8. Structural modelling and molecular dynamics of a multi-stress responsive WRKY TF-DNA complex towards elucidating its role in stress signalling mechanisms in chickpea.

    Science.gov (United States)

    Konda, Aravind Kumar; Farmer, Rohit; Soren, Khela Ram; P S, Shanmugavadivel; Setti, Aravind

    2017-07-28

    Chickpea is a premier food legume crop with high nutritional quality and attains prime importance in the current era of 795 million people being undernourished worldwide. Chickpea production encounters setbacks due to various stresses and understanding the role of key transcription factors (TFs) involved in multiple stresses becomes inevitable. We have recently identified a multi-stress responsive WRKY TF in chickpea. The present study was conducted to predict the structure of WRKY TF to identify the DNA-interacting residues and decipher DNA-protein interactions. Comparative modelling approach produced 3D model of the WRKY TF with good stereochemistry, local/global quality and further revealed W19, R20, K21, and Y22 motifs within a vicinity of 5 Å to the DNA amongst R18, G23, Q24, K25, Y36, Y37, R38 and K47 and these positions were equivalent to the 2LEX WRKY domain of Arabidopsis. Molecular simulations analysis of reference protein -PDB ID 2LEX, along with Car-WRKY TF modelled structure with the DNA coordinates derived from PDB ID 2LEX and docked using HADDOCK were executed. Root Mean Square (RMS) Deviation and RMS Fluctuation values yielded consistently stable trajectories over 50 ns simulation. Strengthening the obtained results, neither radius of gyration, distance and total energy showed any signs of DNA-WRKY complex falling apart nor any significant dissociation event over 50 ns run. Therefore, the study provides first insights into the structural properties of multi-stress responsive WRKY TF-DNA complex in chickpea, enabling genome wide identification of TF binding sites and thereby deciphers their gene regulatory networks.

  9. Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling

    NARCIS (Netherlands)

    G. Smeenk (Godelieve); W.W. Wiegant (Wouter); J.A. Marteijn (Jurgen); M.S. Luijsterburg (Martijn); N. Sroczynski (Nicholas); T. Costelloe (Thomas); R. Romeijn (Ron); A. Pastink (Albert); N. Mailand (Niels); W. Vermeulen (Wim); H. van Attikum (Haico)

    2013-01-01

    textabstractIonizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains

  10. Limited phylogeographic signal in sex-linked and autosomal loci despite geographically, ecologically, and phenotypically concordant structure of mtDNA variation in the Holarctic avian genus Eremophila.

    Directory of Open Access Journals (Sweden)

    Sergei V Drovetski

    Full Text Available Phylogeographic studies of Holarctic birds are challenging because they involve vast geographic scale, complex glacial history, extensive phenotypic variation, and heterogeneous taxonomic treatment across countries, all of which require large sample sizes. Knowledge about the quality of phylogeographic information provided by different loci is crucial for study design. We use sequences of one mtDNA gene, one sex-linked intron, and one autosomal intron to elucidate large scale phylogeographic patterns in the Holarctic lark genus Eremophila. The mtDNA ND2 gene identified six geographically, ecologically, and phenotypically concordant clades in the Palearctic that diverged in the Early-Middle Pleistocene and suggested paraphyly of the horned lark (E. alpestris with respect to the Temminck's lark (E. bilopha. In the Nearctic, ND2 identified five subclades which diverged in the Late Pleistocene. They overlapped geographically and were not concordant phenotypically or ecologically. Nuclear alleles provided little information on geographic structuring of genetic variation in horned larks beyond supporting the monophyly of Eremophila and paraphyly of the horned lark. Multilocus species trees based on two nuclear or all three loci provided poor support for haplogroups identified by mtDNA. The node ages calculated using mtDNA were consistent with the available paleontological data, whereas individual nuclear loci and multilocus species trees appeared to underestimate node ages. We argue that mtDNA is capable of discovering independent evolutionary units within avian taxa and can provide a reasonable phylogeographic hypothesis when geographic scale, geologic history, and phenotypic variation in the study system are too complex for proposing reasonable a priori hypotheses required for multilocus methods. Finally, we suggest splitting the currently recognized horned lark into five Palearctic and one Nearctic species.

  11. DNA topology and transcription

    Science.gov (United States)

    Kouzine, Fedor; Levens, David; Baranello, Laura

    2014-01-01

    Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

  12. Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling

    DEFF Research Database (Denmark)

    Smeenk, G.; Wiegant, W.W.; Luijsterburg, M.S.

    2013-01-01

    Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely...... unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of νH2AX into damaged chromatin......, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA...

  13. Genetic Variability as a Regulator of TLR4 and NOD Signaling in Response to Bacterial Driven DNA Damage Response (DDR and Inflammation: Focus on the Gastrointestinal (GI Tract

    Directory of Open Access Journals (Sweden)

    Evagelia Spanou

    2017-05-01

    Full Text Available The fundamental role of human Toll-like receptors (TLRs and NOD-like receptors (NLRs, the two most studied pathogen recognition receptors (PRRs, is the protection against pathogens and excessive tissue injury. Recent evidence supports the association between TLR/NLR gene mutations and susceptibility to inflammatory, autoimmune, and malignant diseases. PRRs also interfere with several cellular processes, such as cell growth, apoptosis, cell proliferation, differentiation, autophagy, angiogenesis, cell motility and migration, and DNA repair mechanisms. We briefly review the impact of TLR4 and NOD1/NOD2 and their genetic variability in the process of inflammation, tumorigenesis and DNA repair, focusing in the gastrointestinal tract. We also review the available data on new therapeutic strategies utilizing TLR/NLR agonists and antagonists for cancer, allergic diseases, viral infections and vaccine development against both infectious diseases and cancer.

  14. The DNA damage response during mitosis

    NARCIS (Netherlands)

    Heijink, Anne Margriet; Krajewska, Malgorzata; van Vugt, Marcel A. T. M.

    2013-01-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance

  15. HPV18 Persistence Impairs Basal and DNA Ligand-Mediated IFN-β and IFN-λ1 Production through Transcriptional Repression of Multiple Downstream Effectors of Pattern Recognition Receptor Signaling.

    Science.gov (United States)

    Albertini, Silvia; Lo Cigno, Irene; Calati, Federica; De Andrea, Marco; Borgogna, Cinzia; Dell'Oste, Valentina; Landolfo, Santo; Gariglio, Marisa

    2018-03-15

    Although it is clear that high-risk human papillomaviruses (HPVs) can selectively infect keratinocytes and persist in the host, it still remains to be unequivocally determined whether they can escape antiviral innate immunity by interfering with pattern recognition receptor (PRR) signaling. In this study, we have assessed the innate immune response in monolayer and organotypic raft cultures of NIKS cells harboring multiple copies of episomal HPV18 (NIKSmcHPV18), which fully recapitulates the persistent state of infection. We show for the first time, to our knowledge, that NIKSmcHPV18, as well as HeLa cells (a cervical carcinoma-derived cell line harboring integrated HPV18 DNA), display marked downregulation of several PRRs, as well as other PRR downstream effectors, such as the adaptor protein stimulator of IFN genes and the transcription factors IRF1 and 7. Importantly, we provide evidence that downregulation of stimulator of IFN genes, cyclic GMP-AMP synthase, and retinoic acid-inducible gene I mRNA levels occurs at the transcriptional level through a novel epigenetic silencing mechanism, as documented by the accumulation of repressive heterochromatin markers seen at the promoter region of these genes. Furthermore, stimulation of NIKSmcHPV18 cells with salmon sperm DNA or poly(deoxyadenylic-deoxythymidylic) acid, two potent inducers of PRR signaling, only partially restored PRR protein expression. Accordingly, the production of IFN-β and IFN-λ 1 was significantly reduced in comparison with the parental NIKS cells, indicating that HPV18 exerts its immunosuppressive activity through downregulation of PRR signaling. Altogether, our findings indicate that high-risk human papillomaviruses have evolved broad-spectrum mechanisms that allow simultaneous depletion of multiple effectors of the innate immunity network, thereby creating an unreactive cellular milieu suitable for viral persistence. Copyright © 2018 by The American Association of Immunologists, Inc.

  16. Depressive-like effect of prenatal exposure to DDT involves global DNA hypomethylation and impairment of GPER1/ESR1 protein levels but not ESR2 and AHR/ARNT signaling.

    Science.gov (United States)

    Kajta, Malgorzata; Wnuk, Agnieszka; Rzemieniec, Joanna; Litwa, Ewa; Lason, Wladyslaw; Zelek-Molik, Agnieszka; Nalepa, Irena; Rogóż, Zofia; Grochowalski, Adam; Wojtowicz, Anna K

    2017-07-01

    Several lines of evidence suggest that exposures to Endocrine Disrupting Chemicals (EDCs) such as pesticides increase the risks of neuropsychiatric disorders. Despite extended residual persistence of dichlorodiphenyltrichloroethane (DDT) in the environment, the mechanisms of perinatal actions of DDT that could account for adult-onset of depression are largely unknown. This study demonstrated the isomer-specific induction of depressive-like behavior and impairment of Htr1a/serotonin signaling in one-month-old mice that were prenatally exposed to DDT. The effects were reversed by the antidepressant citalopram as evidenced in the forced swimming (FST) and tail suspension (TST) tests in the male and female mice. Prenatally administered DDT accumulated in mouse brain as determined with gas chromatography and tandem mass spectrometry, led to global DNA hypomethylation, and altered the levels of methylated DNA in specific genes. The induction of depressive-like behavior and impairment of Htr1a/serotonin signaling were accompanied by p,p'-DDT-specific decrease in the levels of estrogen receptors i.e. ESR1 and/or GPER1 depending on sex. In contrast, o,p'-DDT did not induce depressive-like effects and exhibited quite distinct pattern of biochemical alterations that was related to aryl hydrocarbon receptor (AHR), its nuclear translocator ARNT, and ESR2. Exposure to o,p'-DDT increased AHR expression in male and female brains, and reduced expression levels of ARNT and ESR2 in the female brains. The evolution of p,p'-DDT-induced depressive-like behavior was preceded by attenuation of Htr1a and Gper1/GPER1 expression as observed in the 7-day-old mouse pups. Because p,p'-DDT caused sex- and age-independent attenuation of GPER1, we suggest that impairment of GPER1 signaling plays a key role in the propagation of DDT-induced depressive-like symptoms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Population History of the Red Sea. Genetic Exchanges Between the Arabian Peninsula and East Africa Signaled in the Mitochondrial DNA HV1 Haplogroup

    Czech Academy of Sciences Publication Activity Database

    Musilová, E.; Fernandes, V.; Silva, N. M.; Soares, P.; Alshamali, F.; Harich, N.; Cherni, L.; El Gaaied, A. B. A.; Al-Meeri, A.; Pereira, L.; Černý, Viktor

    2011-01-01

    Roč. 145, č. 4 (2011), s. 592-598 ISSN 0002-9483 R&D Projects: GA MŠk ME 917 Institutional research plan: CEZ:AV0Z80020508 Keywords : mtDNA genomes * HV1 haplogroup * Arabian Peninsula * East Africa Subject RIV: AC - Archeology, Anthropology, Ethnology Impact factor: 2.824, year: 2011 http://onlinelibrary.wiley.com/doi/10.1002/ajpa.21522/abstract;jsessionid=06420E45513D054B63DFDE068F111692.d03t01

  18. Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

    Science.gov (United States)

    Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

    2005-01-01

    We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

  19. Effect of low dose of gamma radiation on the induction and signalling of complex damages of DNA in the mammals cells

    International Nuclear Information System (INIS)

    Boucher, D.

    2006-09-01

    The work presented in the frame of this thesis are in line with the studies on the radioinduced damages of DNA in order to better understand their genotoxic effects and the associated risks. The different radioinduced damages of DNA are given, the complex damages, double strand-break and multiple damages are particularly detailed as well as their methods of detection. Are presented the systems implemented in the cell in order to point the radioinduced damages and to implement the repair systems of these lesions. Then, are presented the knowledge on the effects of low doses and low dose rates. The whole of this work has for objective to highlight the mechanisms by which the reduction of dose rate allows an increase of cell survival. By a better knowledge of the system implemented in response to a low dose rate irradiation, it is possible to valid or not the linear without threshold model of low doses effects and consequently to better understand the eventual risks of cancer linked to exposures of low doses of ionizing radiation. (N.C.)

  20. Low concentrations of antimony impair DNA damage signaling and the repair of radiation-induced DSB in HeLa S3 cells.

    Science.gov (United States)

    Koch, Barbara; Maser, Elena; Hartwig, Andrea

    2017-12-01

    Antimony is utilized in a large variety of industrial applications, leading to significant environmental and occupational exposure. Mainly based on animal experiments, the IARC and MAK Commission have classified antimony and its inorganic compounds as Group 2B or 2 carcinogens, respectively. However, the underlying mode(s) of action are still largely unknown. In the present study, we investigated the impact of non-cytotoxic up to cytotoxic concentrations of SbCl 3 on DNA DSB repair and cell cycle control in HeLa S3 cells. We induced DSB by γ-irradiation and analyzed inhibitory actions of antimony on potential molecular targets of the DSB repair machinery. Antimony disturbed cell cycle control, affecting phosphorylation of Chk1. Furthermore, the repair of DSB was impaired in the presence of antimony, as monitored by pulsed-field gel electrophoresis and γH2AX foci formation of cells in G1 and G2 phase. Specifically, BRCA1 and RAD51 were identified as molecular targets. Our results point towards an interference with both non-homologous end-joining (NHEJ) and homologous recombination (HR), and inhibitory effects may be explained by interactions with critical cysteine groups; this needs to be further investigated. Altogether, the results provide further evidence for the impairment of DNA repair processes as one underlying mechanism in antimony-induced carcinogenicity.

  1. Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Karanam, Narasimha Kumar; Srinivasan, Kalayarasan; Ding, Lianghao; Sishc, Brock; Saha, Debabrata; Story, Michael D

    2017-03-30

    The use of tumor-treating fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma (GBM). TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis. Using five non-small cell lung cancer (NSCLC) cell lines we found that there is a variable response in cell proliferation and cell killing between these NSCLC cell lines that was independent of p53 status. TTFields treatment increased the G2/M population, with a concomitant reduction in S-phase cells followed by the appearance of a sub-G1 population indicative of apoptosis. Temporal changes in gene expression during TTFields exposure was evaluated to identify molecular signaling changes underlying the differential TTFields response. The most differentially expressed genes were associated with the cell cycle and cell proliferation pathways. However, the expression of genes found within the BRCA1 DNA-damage response were significantly downregulated (Pionizing radiation resulted in increased chromatid aberrations and a reduced capacity to repair DNA DSBs, which were likely responsible for at least a portion of the enhanced cell killing seen with the combination. These findings suggest that TTFields induce a state of 'BRCAness' leading to a conditional susceptibility resulting in enhanced sensitivity to ionizing radiation and provides a strong rationale for the use of TTFields as a combined modality therapy with radiation or other DNA-damaging agents.

  2. DNA nanotechnology: On-command molecular Trojans

    Science.gov (United States)

    Niemeyer, Christof M.

    2017-12-01

    Lipid-motif-decorated DNA nanocapsules filled with photoresponsive polymers are capable of delivering signalling molecules into target organisms for biological perturbations at high spatiotemporal resolution.

  3. Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

    DEFF Research Database (Denmark)

    Bennetzen, Martin; Andersen, J.S.; Lasen, D.H.

    2013-01-01

    Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time...

  4. Distinct roles of FANCO/RAD51C in DNA damage signaling and repair: implications for fanconi anemia and breast cancer susceptibility

    International Nuclear Information System (INIS)

    Nagaraju, G.; Somyajit, K.; Subramanya, S.

    2012-01-01

    Unrepaired or misrepaired chromosomal double-strand breaks (DSBs) can cause gross chromosomal rearrangements which eventually can lead to tumorigenesis through inactivation of tumor suppressor genes or activation of oncogenes. There are two major mechanisms of DSB repair: non-homologous end joining (NHEJ) and homologous recombination (HR). DSBs that are generated during S and G2 phase of the cell are preferentially repaired by sister chromatid recombination (SCR), an HR pathway that utilizes neighboring sister chromatid as a template. Since the copied information is accurate, SCR is potentially an error-free pathway. HR also plays a critical role in the repair of daughter strand gaps (DSGs) that arise as a result of replication fork stalling and facilitates replication fork recovery. Furthermore, in collaboration with nucleotide excision repair and translesion synthesis, HR is involved in the repair of DNA interstrand cross-links (ICLs). Thus, HR is important for the maintenance of genome integrity and its dysfunction can lead to various genetic disorders and cancer

  5. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  6. Modeling DNA

    Science.gov (United States)

    Robertson, Carol

    2016-01-01

    Deoxyribonucleic acid (DNA) is life's most amazing molecule. It carries the genetic instructions that almost every organism needs to develop and reproduce. In the human genome alone, there are some three billion DNA base pairs. The most difficult part of teaching DNA structure, however, may be getting students to visualize something as small as a…

  7. Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

    DEFF Research Database (Denmark)

    Gilroy, Emma L.; Hoffmann, Søren Vrønning; Jones, Nykola C.

    2011-01-01

    ) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)2], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed...

  8. DNA Camouflage

    Science.gov (United States)

    2016-01-08

    1 DNA Camouflage Supplementary Information Bijan Zakeri1,2*, Timothy K. Lu1,2*, Peter A. Carr2,3* 1Department of Electrical Engineering and...ll.mit.edu). Distribution A: Public Release   2 Supplementary Figure 1 DNA camouflage with the 2-state device. (a) In the presence of Cre, DSD-2[α...10 1 + Cre 1 500 1,000 length (bp) chromatogram alignment template − Cre   4 Supplementary Figure 3 DNA camouflage with a switchable

  9. Signal Words

    Science.gov (United States)

    SIGNAL WORDS TOPIC FACT SHEET NPIC fact sheets are designed to answer questions that are commonly asked by the ... making decisions about pesticide use. What are Signal Words? Signal words are found on pesticide product labels, ...

  10. ATM signaling and 53BP1

    International Nuclear Information System (INIS)

    Zgheib, Omar; Huyen, Yentram; DiTullio, Richard A.; Snyder, Andrew; Venere, Monica; Stavridi, Elena S.; Halazonetis, Thanos D.

    2005-01-01

    The ATM (mutated in Ataxia-Telangiectasia) protein kinase is an important player in signaling the presence of DNA double strand breaks (DSBs) in higher eukaryotes. Recent studies suggest that ATM monitors the presence of DNA DSBs indirectly, through DNA DSB-induced changes in chromatin structure. One of the proteins that sense these chromatin structure changes is 53BP1, a DNA damage checkpoint protein conserved in all eukaryotes and the putative ortholog of the S. cerevisiae RAD9 protein. We review here the mechanisms by which ATM is activated in response to DNA DSBs, as well as key ATM substrates that control cell cycle progression, apoptosis and DNA repair

  11. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.

    2008-01-01

    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  12. Hyperstretching DNA

    NARCIS (Netherlands)

    Schakenraad, Koen; Biebricher, Andreas S.; Sebregts, Maarten; Ten Bensel, Brian; Peterman, Erwin J.G.; Wuite, Gijs J L; Heller, Iddo; Storm, Cornelis; Van Der Schoot, Paul

    2017-01-01

    The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely

  13. RAD51 interconnects between DNA replication, DNA repair and immunity.

    Science.gov (United States)

    Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

    2017-05-05

    RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. DNA nanotechnology-enabled biosensors.

    Science.gov (United States)

    Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai

    2016-02-15

    Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.

    1992-01-01

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  16. DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Castelino, J

    1993-12-31

    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with {sup 32}P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism`s genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens 10 figs, 2 tabs

  17. Drug Addiction and DNA Modifications.

    Science.gov (United States)

    Brown, Amber N; Feng, Jian

    2017-01-01

    Drug addiction is a complex disorder which can be influenced by both genetic and environmental factors. Research has shown that epigenetic modifications can translate environmental signals into changes in gene expression, suggesting that epigenetic changes may underlie the causes and possibly treatment of substance use disorders. This chapter will focus on epigenetic modifications to DNA, which include DNA methylation and several recently defined additional DNA epigenetic changes. We will discuss the functions of DNA modifications and methods for detecting them, followed by a description of the research investigating the function and consequences of drug-induced changes in DNA methylation patterns. Understanding these epigenetic changes may provide us translational tools for the diagnosis and treatment of addiction in the future.

  18. The DNA damage response during mitosis

    International Nuclear Information System (INIS)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van

    2013-01-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed

  19. The DNA damage response during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van, E-mail: m.vugt@umcg.nl

    2013-10-15

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

  20. The DNA damage response during mitosis.

    Science.gov (United States)

    Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

    2013-10-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  2. DNA data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  3. DNA nanotechnology

    Science.gov (United States)

    Seeman, Nadrian C.; Sleiman, Hanadi F.

    2018-01-01

    DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

  4. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  5. DNA-PK dependent targeting of DNA-ends to a protein complex assembled on matrix attachment region DNA sequences

    International Nuclear Information System (INIS)

    Mauldin, S.K.; Getts, R.C.; Perez, M.L.; DiRienzo, S.; Stamato, T.D.

    2003-01-01

    Full text: We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end-binding was observed. Calculation of relative binding activities indicates that DNA-end binding activities to MAR sequences was 7 to 21 fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV, scaffold attachment factor A, topoisomerase II, and poly(ADP-ribose) polymerase preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends. After electroporation of a 32P-labeled DNA probe into human cells and cell fractionation, 87% of the total intercellular radioactivity remained in nuclei after a 0.5M NaCl extraction suggesting the probe was strongly bound in the nucleus. The above observations raise the possibility that DNA-PK targets DNA-ends to a repair and/or DNA damage signaling complex which is assembled on MAR sites in the nucleus

  6. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  7. ATP signals

    DEFF Research Database (Denmark)

    Novak, Ivana

    2016-01-01

    The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas......The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas...

  8. Indicator Based and Indicator - Free Electrochemical DNA Biosensors

    National Research Council Canada - National Science Library

    Kerman, Kagan

    2001-01-01

    The utility and advantages of an indicator free and MB based sequence specific DNA hybridization biosensor based on guanine and adenine oxidation signals and MB reduction signals have been demonstrated...

  9. Electrokinetic acceleration of DNA hybridization in microsystems.

    Science.gov (United States)

    Lei, Kin Fong; Wang, Yun-Hsiang; Chen, Huai-Yi; Sun, Jia-Hong; Cheng, Ji-Yen

    2015-06-01

    In this work, electrokinetic acceleration of DNA hybridization was investigated by different combinations of frequencies and amplitudes of actuating electric signals. Because the frequencies from low to high can induce different kinds of electrokinetic forces, i.e., electroosmotic to electrothermal forces, this work provides an in-depth investigation of electrokinetic enhanced hybridization. Concentric circular Cr/Au microelectrodes of 350 µm in diameter were fabricated on a glass substrate and probe DNA was immobilized on the electrode surface. Target DNA labeled with fluorescent dyes suspending in solution was then applied to the electrode. Different electrokinetic forces were induced by the application of different electric signals to the circular microelectrodes. Local microfluidic vortexes were generated to increase the collision efficiency between the target DNA suspending in solution and probe DNA immobilized on the electrode surface. DNA hybridization on the electrode surface could be accelerated by the electrokinetic forces. The level of hybridization was represented by the fluorescent signal intensity ratio. Results revealed that such 5-min dynamic hybridization increased 4.5 fold of signal intensity ratio as compared to a 1-h static hybridization. Moreover, dynamic hybridization was found to have better differentiation ability between specific and non-specific target DNA. This study provides a strategy to accelerate DNA hybridization in microsystems. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. DNA Electrochemistry with Tethered Methylene Blue

    Science.gov (United States)

    Pheeney, Catrina G.

    2012-01-01

    Methylene blue (MB′), covalently attached to DNA through a flexible C12 alkyl linker, provides a sensitive redox reporter in DNA electrochemistry measurements. Tethered, intercalated MB′ is reduced through DNA-mediated charge transport; the incorporation of a single base mismatch at position 3, 10, or 14 of a 17-mer causes an attenuation of the signal to 62 ± 3% of the well-matched DNA, irrespective of position in the duplex. The redox signal intensity for MB′–DNA is found to be least 3-fold larger than that of Nile blue (NB)–DNA, indicating that MB′ is even more strongly coupled to the π-stack. The signal attenuation due to an intervening mismatch does, however, depend on DNA film density and the backfilling agent used to passivate the surface. These results highlight two mechanisms for reduction of MB′ on the DNA-modified electrode: reduction mediated by the DNA base pair stack and direct surface reduction of MB′ at the electrode. These two mechanisms are distinguished by their rates of electron transfer that differ by 20-fold. The extent of direct reduction at the surface can be controlled by assembly and buffer conditions. PMID:22512327

  11. SIRT participates at DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi Yong; Joeng, Jae Min; Lee, Kee Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Park, Gil Hong [College of Medicine, Korea University, Seoul (Korea, Republic of)

    2009-05-15

    Sir2 maintains genomic stability in multiple ways in yeast. As a NAD{sup +}-dependent histone deacetylase, Sir2 has been reported to control chromatin silencing. In both budding yeast and Drosophila, overexpression of Sir2 extends life span. Previous reports have also demonstrated that Sir2 participate at DNA damage repair. A protein complex containing Sir2 has been reported to translocate to DNA double-strand breaks. Following DNA damage response, SIRT1 deacetylates p53 protein and attenuates its ability as a transcription factor. Consequently, SIRT1 over-expression increases cell survival under DNA damage inducing conditions. These previous observations mean a possibility that signals generated during the process of DNA repair are delivered through SIRT1 to acetylated p53. We present herein functional evidence for the involvement of SIRT1 in DNA repair response to radiation. In addition, this modulation of DNA repair activity may be connected to deacetylation of MRN proteins.

  12. DNA Vaccines

    Indian Academy of Sciences (India)

    diseases. Keywords. DNA vaccine, immune response, antibodies, infectious diseases. GENERAL .... tein vaccines require expensive virus/protein purification tech- niques as ... sphere continue to remain major health hazards in developing nations. ... significance since it can be produced at a very low cost and can be stored ...

  13. DNA Investigations.

    Science.gov (United States)

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  14. Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

    Science.gov (United States)

    Tymoczko, Jakub; Schuhmann, Wolfgang; Gebala, Magdalena

    2014-12-24

    Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.

  15. Signaling aggression.

    Science.gov (United States)

    van Staaden, Moira J; Searcy, William A; Hanlon, Roger T

    2011-01-01

    From psychological and sociological standpoints, aggression is regarded as intentional behavior aimed at inflicting pain and manifested by hostility and attacking behaviors. In contrast, biologists define aggression as behavior associated with attack or escalation toward attack, omitting any stipulation about intentions and goals. Certain animal signals are strongly associated with escalation toward attack and have the same function as physical attack in intimidating opponents and winning contests, and ethologists therefore consider them an integral part of aggressive behavior. Aggressive signals have been molded by evolution to make them ever more effective in mediating interactions between the contestants. Early theoretical analyses of aggressive signaling suggested that signals could never be honest about fighting ability or aggressive intentions because weak individuals would exaggerate such signals whenever they were effective in influencing the behavior of opponents. More recent game theory models, however, demonstrate that given the right costs and constraints, aggressive signals are both reliable about strength and intentions and effective in influencing contest outcomes. Here, we review the role of signaling in lieu of physical violence, considering threat displays from an ethological perspective as an adaptive outcome of evolutionary selection pressures. Fighting prowess is conveyed by performance signals whose production is constrained by physical ability and thus limited to just some individuals, whereas aggressive intent is encoded in strategic signals that all signalers are able to produce. We illustrate recent advances in the study of aggressive signaling with case studies of charismatic taxa that employ a range of sensory modalities, viz. visual and chemical signaling in cephalopod behavior, and indicators of aggressive intent in the territorial calls of songbirds. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Comparing transformation methods for DNA microarray data

    NARCIS (Netherlands)

    Thygesen, Helene H.; Zwinderman, Aeilko H.

    2004-01-01

    Background: When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include

  17. DNA Damage Response and Immune Defence: Links and Mechanisms

    Directory of Open Access Journals (Sweden)

    Björn Schumacher

    2016-08-01

    Full Text Available DNA damage plays a causal role in numerous human pathologies including cancer, premature aging and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signalling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signalling. We highlight evidence gained into (i which molecular and cellular pathways of DDR activate immune signalling, (ii how DNA damage drives chronic inflammation, and (iii how chronic inflammation causes DNA damage and pathology in humans.

  18. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seongman; Chul Ahn, Byung; O' Callaghan, Dennis J. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932 (United States); Kim, Seong Kee, E-mail: skim1@lsuhsc.edu [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932 (United States)

    2012-10-25

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  19. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

    International Nuclear Information System (INIS)

    Kim, Seongman; Chul Ahn, Byung; O’Callaghan, Dennis J.; Kim, Seong Kee

    2012-01-01

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)–UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  20. Molecular Mechanisms of DNA Replication Checkpoint Activation

    Directory of Open Access Journals (Sweden)

    Bénédicte Recolin

    2014-03-01

    Full Text Available The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability.

  1. Signal detection

    International Nuclear Information System (INIS)

    Tholomier, M.

    1985-01-01

    In a scanning electron microscope, whatever is the measured signal, the same set is found: incident beam, sample, signal detection, signal amplification. The resulting signal is used to control the spot luminosity with the observer cathodoscope. This is synchronized with the beam scanning on the sample; on the cathodoscope, the image in secondary electrons, backscattered electrons,... of the sample surface is reconstituted. The best compromise must be found between a register time low enough to remove eventual variations (under the incident beam) of the nature of the observed phenomenon, and a good spatial resolution of the image and a signal-to-noise ratio high enough. The noise is one of the basic limitations of the scanning electron microscope performance. The whose measurement line must be optimized to reduce it [fr

  2. DNA repair

    International Nuclear Information System (INIS)

    Setlow, R.

    1978-01-01

    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

  3. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    Science.gov (United States)

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

  4. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  5. Mechanisms of dealing with DNA damage in terminally differentiated cells

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  6. Flexibility of the genetic code with respect to DNA structure

    DEFF Research Database (Denmark)

    Baisnée, P. F.; Baldi, Pierre; Brunak, Søren

    2001-01-01

    Motivation. The primary function of DNA is to carry genetic information through the genetic code. DNA, however, contains a variety of other signals related, for instance, to reading frame, codon bias, pairwise codon bias, splice sites and transcription regulation, nucleosome positioning and DNA...... structure. Here we study the relationship between the genetic code and DNA structure and address two questions. First, to which degree does the degeneracy of the genetic code and the acceptable amino acid substitution patterns allow for the superimposition of DNA structural signals to protein coding...... sequences? Second, is the origin or evolution of the genetic code likely to have been constrained by DNA structure? Results. We develop an index for code flexibility with respect to DNA structure. Using five different di- or tri-nucleotide models of sequence-dependent DNA structure, we show...

  7. DNA repair

    International Nuclear Information System (INIS)

    Van Zeeland, A.A.

    1984-01-01

    In this chapter a series of DNA repair pathways are discussed which are available to the cell to cope with the problem of DNA damaged by chemical or physical agents. In the case of microorganisms our knowledge about the precise mechanism of each DNA repair pathway and the regulation of it has been improved considerably when mutants deficient in these repair mechanisms became available. In the case of mammalian cells in culture, until recently there were very little repair deficient mutants available, because in almost all mammalian cells in culture at least the diploid number of chromosomes is present. Therefore the frequency of repair deficient mutants in such populations is very low. Nevertheless because replica plating techniques are improving some mutants from Chinese hamsters ovary cells and L5178Y mouse lymphoma cells are now available. In the case of human cells, cultures obtained from patients with certain genetic diseases are available. A number of cells appear to be sensitive to some chemical or physical mutagens. These include cells from patients suffering from xeroderma pigmentosum, Ataxia telangiectasia, Fanconi's anemia, Cockayne's syndrome. However, only in the case of xeroderma pigmentosum cells, has the sensitivity to ultraviolet light been clearly correlated with a deficiency in excision repair of pyrimidine dimers. Furthermore the work with strains obtained from biopsies from man is difficult because these cells generally have low cloning efficiencies and also have a limited lifespan in vitro. It is therefore very important that more repair deficient mutants will become available from established cell lines from human or animal origin

  8. Small molecules, inhibitors of DNA-PK, targeting DNA repair and beyond

    Directory of Open Access Journals (Sweden)

    David eDavidson

    2013-01-01

    Full Text Available Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining (NHEJ a major mechanism for the repair of double strand breaks (DSB in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK. The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA

  9. Bystander signaling via oxidative metabolism.

    Science.gov (United States)

    Sawal, Humaira Aziz; Asghar, Kashif; Bureik, Matthias; Jalal, Nasir

    2017-01-01

    The radiation-induced bystander effect (RIBE) is the initiation of biological end points in cells (bystander cells) that are not directly traversed by an incident-radiation track, but are in close proximity to cells that are receiving the radiation. RIBE has been indicted of causing DNA damage via oxidative stress, besides causing direct damage, inducing tumorigenesis, producing micronuclei, and causing apoptosis. RIBE is regulated by signaling proteins that are either endogenous or secreted by cells as a means of communication between cells, and can activate intracellular or intercellular oxidative metabolism that can further trigger signaling pathways of inflammation. Bystander signals can pass through gap junctions in attached cell lines, while the suspended cell lines transmit these signals via hormones and soluble proteins. This review provides the background information on how reactive oxygen species (ROS) act as bystander signals. Although ROS have a very short half-life and have a nanometer-scale sphere of influence, the wide variety of ROS produced via various sources can exert a cumulative effect, not only in forming DNA adducts but also setting up signaling pathways of inflammation, apoptosis, cell-cycle arrest, aging, and even tumorigenesis. This review outlines the sources of the bystander effect linked to ROS in a cell, and provides methods of investigation for researchers who would like to pursue this field of science.

  10. Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

    Science.gov (United States)

    Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

    2016-06-01

    Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

  11. Graphene Nanopres for DNA Fingerprinting

    Science.gov (United States)

    Ahmed, Towfiq; Balatsky, Alexander V.; Haraldsen, J. T.; Schuller, Ivan K.; di Ventra, M.; Wikfeldt, K. T.

    2013-03-01

    The recent progress in nanopore experiments with transverse current is important for the development of fast, accurate and cheap finger-printing techniques for single nucleotide. Despite its enormous potential for the next generation DNA sequencing technology, the presence of large noise in the temporal spectrum of transverse current remains a big challenge for getting highly accurate interpretation of data. In this paper we present our abinitio calculations, and propose graphene based device for DNA fingerprinting. We calculate transmission current through graphene for each DNA base (A,C,G,T). As shown in our work, a proper time-series analysis of a signal provides a higher quality information in identifying single bio-molecule is translocating through the nanopores. This work is supported by LANL, Nordita, US DOE, AFOSR, and NIH.

  12. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen...

  13. Pathophysiology of Glucocorticoid Signaling.

    Science.gov (United States)

    Vitellius, Géraldine; Trabado, Séverine; Bouligand, Jérôme; Delemer, Brigitte; Lombès, Marc

    2018-06-01

    Glucocorticoids (GC), such as cortisol or dexamethasone, control various physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert most of their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene). GC signaling follows several consecutive steps leading to target gene transactivation, including ligand binding, nuclear translocation of ligand-activated GR complexes, DNA binding, coactivator interaction and recruitment of functional transcriptional machinery. Any step may be impaired and may account for altered GC signaling. Partial or generalized glucocorticoid resistance syndrome may result in a reduced level of functional GR, a decreased hormone affinity and binding, a defect in nuclear GR translocation, a decrease or lack of DNA binding and/or post-transcriptional GR modifications. To date, 26 loss-of-function NR3C1 mutations have been reported in the context of hypertension, hirsutism, adrenal hyperplasia or metabolic disorders. These clinical signs are generally associated with biological features including hypercortisolism without negative regulatory feedback loop on the hypothalamic-pituitary-adrenal axis. Patients had often low plasma aldosterone and renin levels despite hypertension. Only one GR gain-of-function mutation has been described associating Cushing's syndrome phenotype with normal urinary-free cortisol. Some GR polymorphisms (ER22/23EK, GR-9β) have been linked to glucocorticoid resistance and a healthier metabolic profile whereas some others seemed to be associated with GC hypersensitivity (N363S, BclI), increasing cardiovascular risk (diabetes type 2, visceral obesity). This review focuses on the earlier findings on the pathophysiology of GR signaling and presents criteria facilitating identification of novel NR3C1 mutations in selected patients. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  14. Signal Processing

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Signal processing techniques, extensively used nowadays to maximize the performance of audio and video equipment, have been a key part in the design of hardware and software for high energy physics detectors since pioneering applications in the UA1 experiment at CERN in 1979

  15. Sensitive Fluorescent Sensor for Recognition of HIV-1 dsDNA by Using Glucose Oxidase and Triplex DNA

    Directory of Open Access Journals (Sweden)

    Yubin Li

    2018-01-01

    Full Text Available A sensitive fluorescent sensor for sequence-specific recognition of double-stranded DNA (dsDNA was developed on the surface of silver-coated glass slide (SCGS. Oligonucleotide-1 (Oligo-1 was designed to assemble on the surface of SCGS and act as capture DNA, and oligonucleotide-2 (Oligo-2 was designed as signal DNA. Upon addition of target HIV-1 dsDNA (Oligo-3•Oligo-4, signal DNA could bind on the surface of silver-coated glass because of the formation of C•GoC in parallel triplex DNA structure. Biotin-labeled glucose oxidase (biotin-GOx could bind to signal DNA through the specific interaction of biotin-streptavidin, thereby GOx was attached to the surface of SCGS, which was dependent on the concentration of target HIV-1 dsDNA. GOx could catalyze the oxidation of glucose and yield H2O2, and the HPPA can be oxidized into a fluorescent product in the presence of HRP. Therefore, the concentration of target HIV-1 dsDNA could be estimated with fluorescence intensity. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of target HIV-1 dsDNA over the range of 10 pM to 1000 pM, the detection limit was 3 pM. Moreover, the sensor had good sequence selectivity and practicability and might be applied for the diagnosis of HIV disease in the future.

  16. DNA Repair Systems

    Indian Academy of Sciences (India)

    DNA molecule which makes it ideal for storage and propagation of genetic information. ... of these errors are broadly referred to as DNA repair. DNA can ... changes occur in the human genome per day. ..... nails, frequent physical and mental.

  17. Insulin-like growth factor-1 (IGF-1) promotes primordial follicle growth and reduces DNA fragmentation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway.

    Science.gov (United States)

    Bezerra, Maria É S; Barberino, Ricássio S; Menezes, Vanúzia G; Gouveia, Bruna B; Macedo, Taís J S; Santos, Jamile M S; Monte, Alane P O; Barros, Vanessa R P; Matos, Maria H T

    2018-05-30

    We investigated the effects of insulin-like growth factor 1 (IGF-1) on the morphology and follicular activation of ovine preantral follicles cultured in situ and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in IGF-1 action in the sheep ovary. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) analyses (fresh control) or cultured in supplemented alpha-minimum essential medium (α-MEM+; control) or α-MEM+ with IGF-1 (1, 10, 50, 100 or 200ngmL-1) for 7 days. Follicles were classified as normal or atretic, primordial or growing and the oocyte and follicle diameters were measured. DNA fragmentation was evaluated by TUNEL assay. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on the fresh control, α-MEM+ and 100ngmL-1 IGF-1 samples. Inhibition of PI3K activity was performed through pretreatment with the PI3K inhibitor LY294002 and phosphorylated AKT (pAKT) expression was analysed after culture in the absence or presence of LY294002. IGF-1 at 100ngmL-1 increased (PIGF-1. LY294002 significantly inhibited follicular activation stimulated by α-MEM+ and 100ngmL-1 IGF-1 and reduced pAKT expression in follicles. Overall, IGF-1 at 100ngmL-1 promoted primordial follicle activation, cell proliferation and reduced DNA fragmentation after in situ culture through the PI3K/AKT pathway.

  18. DNA Methylation Modulates Nociceptive Sensitization after Incision.

    Directory of Open Access Journals (Sweden)

    Yuan Sun

    Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

  19. Synthesis of DNA

    Science.gov (United States)

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  20. An information gap in DNA evidence interpretation.

    Directory of Open Access Journals (Sweden)

    Mark W Perlin

    Full Text Available Forensic DNA evidence often contains mixtures of multiple contributors, or is present in low template amounts. The resulting data signals may appear to be relatively uninformative when interpreted using qualitative inclusion-based methods. However, these same data can yield greater identification information when interpreted by computer using quantitative data-modeling methods. This study applies both qualitative and quantitative interpretation methods to a well-characterized DNA mixture and dilution data set, and compares the inferred match information. The results show that qualitative interpretation loses identification power at low culprit DNA quantities (below 100 pg, but that quantitative methods produce useful information down into the 10 pg range. Thus there is a ten-fold information gap that separates the qualitative and quantitative DNA mixture interpretation approaches. With low quantities of culprit DNA (10 pg to 100 pg, computer-based quantitative interpretation provides greater match sensitivity.

  1. Targeting DNA Replication Stress for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Jun Zhang

    2016-08-01

    Full Text Available The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit “replication stress” —a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

  2. Oxidized DNA induces an adaptive response in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kostyuk, Svetlana V., E-mail: svet.kostyuk@gmail.com [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Tabakov, Viacheslav J.; Chestkov, Valerij V.; Konkova, Marina S.; Glebova, Kristina V.; Baydakova, Galina V.; Ershova, Elizaveta S.; Izhevskaya, Vera L. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha, E-mail: abaranov@gmu.edu [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Center for the Study of Chronic Metabolic Diseases, School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2013-07-15

    Highlights: • We describe the effects of gDNAOX on human fibroblasts cultivated in serum withdrawal conditions. • gDNAOX evokes an adaptive response in human fibroblasts. • gDNAOX increases the survival rates in serum starving cell populations. • gDNAOX enhances the survival rates in cell populations irradiated at 1.2 Gy dose. • gDNAOX up-regulates NRF2 and inhibits NF-kappaB-signaling. - Abstract: Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA{sup OX}. The levels of cfDNA{sup OX} are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by H{sub 2}O{sub 2}in vitro (gDNA{sup OX}) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA{sup OX} on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA{sup OX} evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2 Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA{sup OX} leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of PSNA, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA{sup OX} and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA{sup OX} inhibits NF-κB signaling. gDNA{sup OX} is a model for oxidized cfDNA{sup OX} that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA

  3. Bystander signaling via oxidative metabolism

    Directory of Open Access Journals (Sweden)

    Sawal HA

    2017-08-01

    Full Text Available Humaira Aziz Sawal,1 Kashif Asghar,2 Matthias Bureik,3 Nasir Jalal4 1Healthcare Biotechnology Department, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, Islamabad, 2Basic Sciences Research, Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore, Pakistan; 3Health Science Platform, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China; 4Health Science Platform, Department of Molecular and Cellular Pharmacology, Tianjin University, Tianjin, China Abstract: The radiation-induced bystander effect (RIBE is the initiation of biological end points in cells (bystander cells that are not directly traversed by an incident-radiation track, but are in close proximity to cells that are receiving the radiation. RIBE has been indicted of causing DNA damage via oxidative stress, besides causing direct damage, inducing tumorigenesis, producing micronuclei, and causing apoptosis. RIBE is regulated by signaling proteins that are either endogenous or secreted by cells as a means of communication between cells, and can activate intracellular or intercellular oxidative metabolism that can further trigger signaling pathways of inflammation. Bystander signals can pass through gap junctions in attached cell lines, while the suspended cell lines transmit these signals via hormones and soluble proteins. This review provides the background information on how reactive oxygen species (ROS act as bystander signals. Although ROS have a very short half-life and have a nanometer-scale sphere of influence, the wide variety of ROS produced via various sources can exert a cumulative effect, not only in forming DNA adducts but also setting up signaling pathways of inflammation, apoptosis, cell-cycle arrest, aging, and even tumorigenesis. This review outlines the sources of the bystander effect linked to ROS in a cell, and provides methods of investigation for researchers who would like to

  4. Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

    International Nuclear Information System (INIS)

    Liu, P.; Hun, X.; Qing, H.

    2011-01-01

    We report on a highly sensitive chemiluminescent (CL) biosensor for the sequence-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H 2 O 2 and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L -1 of target DNA. (author)

  5. Raman spectroscopy for DNA quantification in cell nucleus.

    Science.gov (United States)

    Okotrub, K A; Surovtsev, N V; Semeshin, V F; Omelyanchuk, L V

    2015-01-01

    Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate. © 2014 International Society for Advancement of Cytometry.

  6. DNA sequence variants in PPARGC1A, a gene encoding a coactivator of the ω-3 LCPUFA sensing PPAR-RXR transcription complex, are associated with NV AMD and AMD-associated loci in genes of complement and VEGF signaling pathways.

    Directory of Open Access Journals (Sweden)

    John Paul SanGiovanni

    Full Text Available Increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs and use of peroxisome proliferator activator receptor (PPAR-activating drugs are associated with attenuation of pathologic retinal angiogenesis. ω-3 LCPUFAs are endogenous agonists of PPARs. We postulated that DNA sequence variation in PPAR gamma (PPARG co-activator 1 alpha (PPARGC1A, a gene encoding a co-activator of the LCPUFA-sensing PPARG-retinoid X receptor (RXR transcription complex, may influence neovascularization (NV in age-related macular degeneration (AMD.We applied exact testing methods to examine distributions of DNA sequence variants in PPARGC1A for association with NV AMD and interaction of AMD-associated loci in genes of complement, lipid metabolism, and VEGF signaling systems. Our sample contained 1858 people from 3 elderly cohorts of western European ancestry. We concurrently investigated retinal gene expression profiles in 17-day-old neonatal mice on a 2% LCPUFA feeding paradigm to identify LCPUFA-regulated genes both associated with pathologic retinal angiogenesis and known to interact with PPARs or PPARGC1A.A DNA coding variant (rs3736265 and a 3'UTR-resident regulatory variant (rs3774923 in PPARGC1A were independently associated with NV AMD (exact P = 0.003, both SNPs. SNP-SNP interactions existed for NV AMD (P<0.005 with rs3736265 and a AMD-associated variant in complement factor B (CFB, rs512559. PPARGC1A influences activation of the AMD-associated complement component 3 (C3 promoter fragment and CFB influences activation and proteolysis of C3. We observed interaction (P ≤ 0.003 of rs3736265 with a variant in vascular endothelial growth factor A (VEGFA, rs3025033, a key molecule in retinal angiogenesis. Another PPARGC1A coding variant (rs8192678 showed statistical interaction with a SNP in the VEGFA receptor fms-related tyrosine kinase 1 (FLT1, rs10507386; P ≤ 0.003. C3 expression was down-regulated 2-fold in retinas of ω-3 LCPUFA-fed mice

  7. DNA sequence variants in PPARGC1A, a gene encoding a coactivator of the ω-3 LCPUFA sensing PPAR-RXR transcription complex, are associated with NV AMD and AMD-associated loci in genes of complement and VEGF signaling pathways.

    Science.gov (United States)

    SanGiovanni, John Paul; Chen, Jing; Sapieha, Przemyslaw; Aderman, Christopher M; Stahl, Andreas; Clemons, Traci E; Chew, Emily Y; Smith, Lois E H

    2013-01-01

    Increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and use of peroxisome proliferator activator receptor (PPAR)-activating drugs are associated with attenuation of pathologic retinal angiogenesis. ω-3 LCPUFAs are endogenous agonists of PPARs. We postulated that DNA sequence variation in PPAR gamma (PPARG) co-activator 1 alpha (PPARGC1A), a gene encoding a co-activator of the LCPUFA-sensing PPARG-retinoid X receptor (RXR) transcription complex, may influence neovascularization (NV) in age-related macular degeneration (AMD). We applied exact testing methods to examine distributions of DNA sequence variants in PPARGC1A for association with NV AMD and interaction of AMD-associated loci in genes of complement, lipid metabolism, and VEGF signaling systems. Our sample contained 1858 people from 3 elderly cohorts of western European ancestry. We concurrently investigated retinal gene expression profiles in 17-day-old neonatal mice on a 2% LCPUFA feeding paradigm to identify LCPUFA-regulated genes both associated with pathologic retinal angiogenesis and known to interact with PPARs or PPARGC1A. A DNA coding variant (rs3736265) and a 3'UTR-resident regulatory variant (rs3774923) in PPARGC1A were independently associated with NV AMD (exact P = 0.003, both SNPs). SNP-SNP interactions existed for NV AMD (Pcomplement factor B (CFB, rs512559). PPARGC1A influences activation of the AMD-associated complement component 3 (C3) promoter fragment and CFB influences activation and proteolysis of C3. We observed interaction (P ≤ 0.003) of rs3736265 with a variant in vascular endothelial growth factor A (VEGFA, rs3025033), a key molecule in retinal angiogenesis. Another PPARGC1A coding variant (rs8192678) showed statistical interaction with a SNP in the VEGFA receptor fms-related tyrosine kinase 1 (FLT1, rs10507386; P ≤ 0.003). C3 expression was down-regulated 2-fold in retinas of ω-3 LCPUFA-fed mice - these animals also showed 70% reduction in retinal NV (P

  8. The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical DNA sensor: A review

    Directory of Open Access Journals (Sweden)

    Jahwarhar Izuan Abdul Rashid

    2017-11-01

    Full Text Available In recent years, electrochemical deoxyribonucleic acid (DNA sensor has recently emerged as promising alternative clinical diagnostic devices especially for infectious disease by exploiting DNA recognition events and converting them into an electrochemical signal. This is because the existing DNA diagnostic method possesses certain drawbacks such as time-consuming, expensive, laborious, low selectivity and sensitivity. DNA immobilization strategies and mechanism of electrochemical detection are two the most important aspects that should be considered before developing highly selective and sensitive electrochemical DNA sensor. Here, we focus on some recent strategies for DNA probes immobilization on the surface of electrochemical transducer such as adsorption, covalent bonding and Avidin/Streptavidin-Biotin interaction on the electrode surface for specific interaction with its complementary DNA target. A numerous approach for DNA hybridization detection based electrochemical technique that frequently used including direct DNA electrochemical detection and label based electrochemical (redox-active indicator, enzyme label and nanoparticles were also discussed in aiming to provide general guide for the design of electrochemical DNA sensor. We also discussed the challenges and suggestions to improve the application of electrochemical DNA sensor at point-care setting. Keywords: Electrochemical DNA sensor, DNA immobilization, DNA hybridization, Electrochemical mechanism

  9. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    Science.gov (United States)

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2012-01-01

    Abstract DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above...... data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated....

  11. Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2012-01-01

    DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a certain...... data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated....

  12. Integrin Signalling

    OpenAIRE

    Schelfaut, Roselien

    2005-01-01

    Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins b...

  13. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

    Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

  14. Biofunctionalization of ZnO nanowires for DNA sensory applications

    Energy Technology Data Exchange (ETDEWEB)

    Schroeder, Ulrich Christian; Gnauck, Martin; Ronning, Carsten [Institute of Solid State Physics, University of Jena, Max-Wien-Platz 1, D-07743 Jena (Germany); Moeller, Robert; Rudolph, Bettina; Fritzsche, Wolfgang [Institut fuer Photonische Technologien e.V., Albert-Einstein-Strasse 9, D-07745 Jena (Germany)

    2011-07-01

    In recent years, DNA detecting systems have received a growing interest due to promising fields of application like DNA diagnostics, gene analysis, virus detection or forensic applications. Nanowire-based DNA biosensor allows both miniaturization and easy continuous monitoring of a detection signal by electrical means. The label free detection scheme based on electrochemical changes of the surface potential during immobilization of specific DNA probes was heretofore mainly studied for silicon. In this work a surface decoration process with bifunctional molecules known as silanization was applied to VLS-grown ZnO nanowires which both feature a large sensitivity for surface modification, are biocompatible and easy to synthesize as well. Successfully bound DNA was proved by fluorescence microscopy. Dielectrophoresis (DEP) was chosen and optimized for quickly contacting the ZnO nanowires. Furthermore, electrical signal characterization was performed in preparation for DNA sensory applications.

  15. "Isogaba Maware": quality control of genome DNA by checkpoints.

    Science.gov (United States)

    Kitazono, A; Matsumoto, T

    1998-05-01

    Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

  16. Recent Development of Nano-Materials Used in DNA Biosensors

    Directory of Open Access Journals (Sweden)

    Yibin Ying

    2009-07-01

    Full Text Available As knowledge of the structure and function of nucleic acid molecules has increased, sequence-specific DNA detection has gained increased importance. DNA biosensors based on nucleic acid hybridization have been actively developed because of their specificity, speed, portability, and low cost. Recently, there has been considerable interest in using nano-materials for DNA biosensors. Because of their high surface-to-volume ratios and excellent biological compatibilities, nano-materials could be used to increase the amount of DNA immobilization; moreover, DNA bound to nano-materials can maintain its biological activity. Alternatively, signal amplification by labeling a targeted analyte with nano-materials has also been reported for DNA biosensors in many papers. This review summarizes the applications of various nano-materials for DNA biosensors during past five years. We found that nano-materials of small sizes were advantageous as substrates for DNA attachment or as labels for signal amplification; and use of two or more types of nano-materials in the biosensors could improve their overall quality and to overcome the deficiencies of the individual nano-components. Most current DNA biosensors require the use of polymerase chain reaction (PCR in their protocols. However, further development of nano-materials with smaller size and/or with improved biological and chemical properties would substantially enhance the accuracy, selectivity and sensitivity of DNA biosensors. Thus, DNA biosensors without PCR amplification may become a reality in the foreseeable future.

  17. Systematic biases in DNA copy number originate from isolation procedures

    NARCIS (Netherlands)

    van Heesch, S.; Mokry, M.; Boskova, V.; Junker, W.; Mehon, R.; Toonen, P.; de Bruijn, E.; Shull, J.D.; Aitman, T.J.; Cuppen, E.; Guryev, V.

    2013-01-01

    BACKGROUND: The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. RESULTS: While GC content has been used to correct

  18. A comparative study of the molecular evolution of signalling pathway ...

    Indian Academy of Sciences (India)

    2013-08-05

    Aug 5, 2013 ... formed using DnaSP ver. 5 (Librado and Rozas 2009) from ... a context of global divergence within each phylum we exam- ined genomewide ... suggests that the overall conserved sensory signalling cas- cade members are ...

  19. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus

  20. Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan.

    Science.gov (United States)

    Schroeder, Elizabeth A; Shadel, Gerald S

    2014-01-01

    Mitochondrial DNA (mtDNA) exists in multiple copies per cell and is essential for oxidative phosphorylation. Depleted or mutated mtDNA promotes numerous human diseases and may contribute to aging. Reduced TORC1 signaling in the budding yeast, Saccharomyces cerevisiae, extends chronological lifespan (CLS) in part by generating a mitochondrial ROS (mtROS) signal that epigenetically alters nuclear gene expression. To address the potential requirement for mtDNA maintenance in this response, we analyzed strains lacking the mitochondrial base-excision repair enzyme Ntg1p. Extension of CLS by mtROS signaling and reduced TORC1 activity, but not caloric restriction, was abrogated in ntg1Δ strains that exhibited mtDNA depletion without defects in respiration. The DNA damage response (DDR) kinase Rad53p, which transduces pro-longevity mtROS signals, is also activated in ntg1Δ strains. Restoring mtDNA copy number alleviated Rad53p activation and re-established CLS extension following mtROS signaling, indicating that Rad53p senses mtDNA depletion directly. Finally, DDR kinases regulate nucleus-mitochondria localization dynamics of Ntg1p. From these results, we conclude that the DDR pathway senses and may regulate Ntg1p-dependent mtDNA stability. Furthermore, Rad53p senses multiple mitochondrial stresses in a hierarchical manner to elicit specific physiological outcomes, exemplified by mtDNA depletion overriding the ability of Rad53p to transduce an adaptive mtROS longevity signal. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  1. Participation of ATM in cellular response to DNA damage induced by ionizing radiation

    International Nuclear Information System (INIS)

    Meng Xiangbing; Song Yi; Mao Jianping; Gong Bo; Dong Yan; Liu Bin; Sun Zhixian

    2000-01-01

    Objective: To clone ATM full length cDNA and cDNA fragments containing some functional domains and to identify proteins that interact with ATM and mediate DNA damage signal transduction in cellular response to DNA damage. Methods: ATM cDNA was amplified from MarthomTM-Ready cDNA kit of human leukocytes by LD-PCR. ATM-interacting proteins were screened by yeast two hybrid system. Results: ATM full-length cDNA and cDNA fragments containing PI3K kinase domain, leucine zipper and proline rich region were amplified from human cDNAs. Several candidate clones that interacted with ATM PI3K domain were identified. Conclusion: ATM mediates DNA damage signal transduction by interacting with many proteins

  2. Transcription and DNA Damage: Holding Hands or Crossing Swords?

    Science.gov (United States)

    D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

    2017-10-27

    Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. DNA preservation in silk.

    Science.gov (United States)

    Liu, Yawen; Zheng, Zhaozhu; Gong, He; Liu, Meng; Guo, Shaozhe; Li, Gang; Wang, Xiaoqin; Kaplan, David L

    2017-06-27

    The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50-70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

  4. Force induced DNA melting

    International Nuclear Information System (INIS)

    Santosh, Mogurampelly; Maiti, Prabal K

    2009-01-01

    When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates by roughly twice its initial contour length at a pulling force of about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying a force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA, this being a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double-strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA starts melting abruptly/smoothly above a critical force depending on the pulling direction. The critical force f m , at which DNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base pairs. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.

  5. DNA Open states and DNA hydratation

    International Nuclear Information System (INIS)

    Lema-Larre, B. de; Martin-Landrove, M

    1995-01-01

    It is a very well-known fact that an protonic exchange exists among natural DNA filaments and synthetic polynucleotides with the solvent (1--2). The existence of DNA open states, that is to say states for which the interior of the DNA molecule is exposed to the external environment, it has been demonstrated by means of proton-deuterium exchange (3). This work has carried out experiments measuring the dispersion of the traverse relaxation rate (4), as a pulsation rate function in a Carr-Purcell-Meiboom-Gill (CPMG) pulses sequence rate, to determine changes in the moist layer of the DNA molecule. The experiments were carried out under different experimental conditions in order to vary the probability that open states occurs, such as temperature or the exposure to electromagnetic fields. Some theoretical models were supposed to adjust the experimental results including those related to DNA non linear dynamic [es

  6. Immunoassay of DNA damage

    International Nuclear Information System (INIS)

    Gasparro, F.P.; Santella, R.M.

    1988-01-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

  7. Immunoassay of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Gasparro, F P; Santella, R M

    1988-09-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

  8. DNA computing models

    CERN Document Server

    Ignatova, Zoya; Zimmermann, Karl-Heinz

    2008-01-01

    In this excellent text, the reader is given a comprehensive introduction to the field of DNA computing. The book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines.

  9. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  10. Modeling DNA Replication.

    Science.gov (United States)

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  11. DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

    2010-06-01

    A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

  12. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir

    1998-01-01

    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...

  13. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  14. Fanconi anemia: a disorder defective in the DNA damage response.

    Science.gov (United States)

    Kitao, Hiroyuki; Takata, Minoru

    2011-04-01

    Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

  15. Real-time Tracking of DNA Fragment Separation by Smartphone.

    Science.gov (United States)

    Tao, Chunxian; Yang, Bo; Li, Zhenqing; Zhang, Dawei; Yamaguchi, Yoshinori

    2017-06-01

    Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

  16. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    Science.gov (United States)

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

    Science.gov (United States)

    Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

    2013-01-01

    Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

  18. ATP-dependent chromatin remodeling in the DNA-damage response

    Directory of Open Access Journals (Sweden)

    Lans Hannes

    2012-01-01

    Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

  19. Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

    Science.gov (United States)

    Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

    2015-02-25

    In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Fast phylogenetic DNA barcoding

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Willerslev, Eske

    2008-01-01

    We present a heuristic approach to the DNA assignment problem based on phylogenetic inferences using constrained neighbour joining and non-parametric bootstrapping. We show that this method performs as well as the more computationally intensive full Bayesian approach in an analysis of 500 insect...... DNA sequences obtained from GenBank. We also analyse a previously published dataset of environmental DNA sequences from soil from New Zealand and Siberia, and use these data to illustrate the fact that statistical approaches to the DNA assignment problem allow for more appropriate criteria...... for determining the taxonomic level at which a particular DNA sequence can be assigned....

  1. Radiation and DNA

    Energy Technology Data Exchange (ETDEWEB)

    Riabchenko, N I

    1979-01-01

    Consideration is given to the effects of ionizing radiation on the structure of DNA. Physical and chemical methods of determining radiation damage to the primary (polynucleotide chain and nitrogenous base) and secondary (helical) structure of DNA are discussed, and the effects of ionizing radiation on deoxyribonucleoprotein complexes are considered. The radiolysis of DNA in vitro and in bacterial and mammalian cells is examined and cellular mechanisms for the repair of radiation-damaged DNA are considered, taking into account single-strand and double-strand breaks, gamma-radiation damage and deoxyribonucleoprotein-membrane complex damage. Postradiation DNA degradation in bacteria and lymphatic cells is also discussed.

  2. DNA-Mediated Electrochemistry

    Science.gov (United States)

    Gorodetsky, Alon A.; Buzzeo, Marisa C.

    2009-01-01

    The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry. PMID:18980370

  3. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J

    2017-01-01

    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  4. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander

    2015-01-01

    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  5. Histone modifications in response to DNA damage

    International Nuclear Information System (INIS)

    Altaf, Mohammed; Saksouk, Nehme; Cote, Jacques

    2007-01-01

    The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity

  6. Detection of influenza A virus using carbon nanotubes field effect transistor based DNA sensor

    Science.gov (United States)

    Tran, Thi Luyen; Nguyen, Thi Thuy; Huyen Tran, Thi Thu; Chu, Van Tuan; Thinh Tran, Quang; Tuan Mai, Anh

    2017-09-01

    The carbon nanotubes field effect transistor (CNTFET) based DNA sensor was developed, in this paper, for detection of influenza A virus DNA. Number of factors that influence the output signal and analytical results were investigated. The initial probe DNA, decides the available DNA strands on CNTs, was 10 μM. The hybridization time for defined single helix was 120 min. The hybridization temperature was set at 30 °C to get a net change in drain current of the DNA sensor without altering properties of any biological compounds. The response time of the DNA sensor was less than one minute with a high reproducibility. In addition, the DNA sensor has a wide linear detection range from 1 pM to 10 nM, and a very low detection limit of 1 pM. Finally, after 7-month storage in 7.4 pH buffer, the output signal of DNA sensor recovered 97%.

  7. Molecular Buffers Permit Sensitivity Tuning and Inversion of Riboswitch Signals

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Genee, Hans Jasper; Jensen, Kristian

    2016-01-01

    transcription factor, while interacting DNA-binding domains mediate the transduction of signal and form an interacting molecular buffer. The molecular buffer system enables modular signal inversion through integration with repressor modules. Further, tuning of input sensitivity was achieved through perturbation...

  8. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    Science.gov (United States)

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

  9. Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

    Science.gov (United States)

    M.N. lslam-Faridi; C.D. Nelson; S.P. DiFazio; L.E. Gunter; G.A. Tuskan

    2009-01-01

    The 185-285 rDNA and 55 rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 185-285 rDNA sites and one 55 rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones...

  10. A programming language for composable DNA circuits.

    Science.gov (United States)

    Phillips, Andrew; Cardelli, Luca

    2009-08-06

    Recently, a range of information-processing circuits have been implemented in DNA by using strand displacement as their main computational mechanism. Examples include digital logic circuits and catalytic signal amplification circuits that function as efficient molecular detectors. As new paradigms for DNA computation emerge, the development of corresponding languages and tools for these paradigms will help to facilitate the design of DNA circuits and their automatic compilation to nucleotide sequences. We present a programming language for designing and simulating DNA circuits in which strand displacement is the main computational mechanism. The language includes basic elements of sequence domains, toeholds and branch migration, and assumes that strands do not possess any secondary structure. The language is used to model and simulate a variety of circuits, including an entropy-driven catalytic gate, a simple gate motif for synthesizing large-scale circuits and a scheme for implementing an arbitrary system of chemical reactions. The language is a first step towards the design of modelling and simulation tools for DNA strand displacement, which complements the emergence of novel implementation strategies for DNA computing.

  11. RevTrans: multiple alignment of coding DNA from aligned amino acid sequences

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Pedersen, Anders Gorm

    2003-01-01

    The simple fact that proteins are built from 20 amino acids while DNA only contains four different bases, means that the 'signal-to-noise ratio' in protein sequence alignments is much better than in alignments of DNA. Besides this information-theoretical advantage, protein alignments also benefit...... proteins. It is therefore preferable to align coding DNA at the amino acid level and it is for this purpose we have constructed the program RevTrans. RevTrans constructs a multiple DNA alignment by: (i) translating the DNA; (ii) aligning the resulting peptide sequences; and (iii) building a multiple DNA...

  12. Beyond DNA repair: DNA-PK function in cancer

    OpenAIRE

    Goodwin, Jonathan F.; Knudsen, Karen E.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, furthe...

  13. Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

    Science.gov (United States)

    Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

    2014-10-07

    We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-β). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-β antibody to the target protein. Polyclonal anti-NGF-β antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-β, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-β. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

  14. Activation of DNA damage repair pathways by murine polyomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Katie; Nicholas, Catherine; Garcea, Robert L., E-mail: Robert.Garcea@Colorado.edu

    2016-10-15

    Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.

  15. Eukaryotic DNA Replication Fork.

    Science.gov (United States)

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

    This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

  16. The chemical structure of DNA sequence signals for RNA transcription

    Science.gov (United States)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  17. DNA-based machines.

    Science.gov (United States)

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.

  18. DNA repair and cancer

    International Nuclear Information System (INIS)

    Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

    2012-01-01

    DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

  19. Development of a sensitive electrochemical DNA sensor by 4-aminothiophenol self-assembled on electrodeposited nanogold electrode coupled with Au nanoparticles labeled reporter ssDNA

    International Nuclear Information System (INIS)

    Li Guangjiu; Liu Lihua; Qi Xiaowei; Guo Yaqing; Sun Wei; Li Xiaolin

    2012-01-01

    Graphical abstract: - Abstract: A novel and sensitive electrochemical DNA biosensor was fabricated by using the 4-aminothiophenol (4-ATP) self-assembled on electrodeposited gold nanoparticles (NG) modified electrode to anchor capture ssDNA sequences and Au nanoparticles (AuNPs) labeled with reporter ssDNA sequences, which were further coupled with electroactive indicator of hexaammineruthenium (III) ([Ru(NH 3 ) 6 ] 3+ ) to amplify the electrochemical signal of hybridization reaction. Different modified electrodes were prepared and characterized by cyclic voltammetry, scanning electron microscope and electrochemical impedance spectroscopy. By using a sandwich model for the capture of target ssDNA sequences, which was based on the shorter probe ssDNA and AuNPs label reporter ssDNA hybridized with longer target ssDNA, the electrochemical behavior of [Ru(NH 3 ) 6 ] 3+ was monitored by differential pulse voltammetry (DPV). The fabricated electrochemical DNA sensor exhibited good distinguish capacity for the complementary ssDNA sequence and two bases mismatched ssDNA. The dynamic detection range of the target ssDNA sequences was from 1.4 × 10 −11 to 2.0 × 10 −9 mol/L with the detection limit as 9.5 × 10 −12 mol/L (3σ). So in this paper a new electrochemical DNA sensor was designed with gold nanoparticles as the immobilization platform and the signal amplifier simultaneously.

  20. Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

    Science.gov (United States)

    Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

    2016-08-16

    Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

  1. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    Science.gov (United States)

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Nonisotopic DNA probe techniques

    National Research Council Canada - National Science Library

    Kricka, Larry J

    1992-01-01

    The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labelling...

  3. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

    2016-01-01

    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  4. Forensic DNA testing.

    Science.gov (United States)

    Butler, John M

    2011-12-01

    Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

  5. Structure of DNA toroids and electrostatic attraction of DNA duplexes

    International Nuclear Information System (INIS)

    Cherstvy, A G

    2005-01-01

    DNA-DNA electrostatic attraction is considered as the driving force for the formation of DNA toroids in the presence of DNA condensing cations. This attraction comes from the DNA helical charge distribution and favours hexagonal toroidal cross-sections. The latter is in agreement with recent cryo-electron microscopy studies on DNA condensed with cobalt hexammine. We treat the DNA-DNA interactions within the modern theory of electrostatic interaction between helical macromolecules. The size and thickness of the toroids is calculated within a simple model; other models of stability of DNA toroids are discussed and compared

  6. Basic digital signal processing

    CERN Document Server

    Lockhart, Gordon B

    1985-01-01

    Basic Digital Signal Processing describes the principles of digital signal processing and experiments with BASIC programs involving the fast Fourier theorem (FFT). The book reviews the fundamentals of the BASIC program, continuous and discrete time signals including analog signals, Fourier analysis, discrete Fourier transform, signal energy, power. The text also explains digital signal processing involving digital filters, linear time-variant systems, discrete time unit impulse, discrete-time convolution, and the alternative structure for second order infinite impulse response (IIR) sections.

  7. HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

    Science.gov (United States)

    Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

    2008-01-01

    Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

  8. Development of DNA biosensor based on TiO2 nanoparticles

    Science.gov (United States)

    Nadzirah, Sh.; Hashim, U.; Rusop, M.

    2018-05-01

    A novel technique of DNA hybridization on the TiO2 nanoparticles film was developed by dropping a single droplet of target DNA onto the surface of TiO2 for the study of various concentrations of target DNA. The surface of TiO2 nanoparticle film was functionalized with APTES and covalently immobilized with 1 µM probe DNA on the silanized TiO2 nanoparticles surface. The effect of silanization, immobilization and hybridization were quantitatively measured by the output current signal obtained using a picoammeter. The 1 µM target DNA was found to be the most effective target towards the 1 µM probe DNA as the output current signal was within range; while the output current signal of the 10 µM target DNA was observed to beyond the range of the probe DNA control due to the excessive concentration as compared to the probe DNA. This approach has several advantages such as rapid, simple, low cost, and sensitive current signal during detection of different target DNA concentrations.

  9. Localized surface plasmon resonance (LSPR) study of DNA hybridization at single nanoparticle transducers

    International Nuclear Information System (INIS)

    Schneider, T.; Jahr, N.; Jatschka, J.; Csaki, A.; Stranik, O.; Fritzsche, W.

    2013-01-01

    The effect of DNA–DNA interaction on the localized surface plasmon resonance of single 80 nm gold nanoparticles is studied. Therefore, both the attachment of the capture DNA strands at the particle surface and the sequence-specific DNA binding (hybridization) of analyte DNA to the immobilized capture DNA is subject of investigations. The influence of substrate attachment chemistry, the packing density of DNA as controlled by an assisting layer of smaller molecules, and the distance as increased by a linker on the LSPR efficiency is investigated. The resulting changes in signal can be related to a higher hybridization efficiency of the analyte DNA to the immobilized capture DNA. The subsequent attachment of additional DNA strands to this system is studied, which allows for a multiple step detection of binding and an elucidation of the resulting resonance shifts. The detection limit was determined for the utilized DNA system by incubation with various concentration of analyte DNA. Although the method allows for a marker-free detection, we show that additional markers such as 20 nm gold particle labels increase the signal and thereby the sensitivity significantly. The study of resonance shift for various DNA lengths revealed that the resonance shift per base is stronger for shorter DNA molecules (20 bases) as compared to longer ones (46 bases).

  10. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

    Science.gov (United States)

    Maréchal, Alexandre; Zou, Lee

    2015-01-01

    The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

  11. KSHV strategies for host dsDNA sensing machinery.

    Science.gov (United States)

    Gao, Hang; Song, Yanyan; Liu, Chengrong; Liang, Qiming

    2016-12-01

    The innate immune system utilizes pattern recognition receptors cyclic GMP-AMP synthase (cGAS) to sense cytosolic double-stranded (ds) DNA and initiate type 1 interferon signaling and autophagy pathway, which collaborate to limit pathogen infections as well as alarm the adaptive immune response. The genomes of herpesviruses are large dsDNA, which represent a major class of pathogen signatures recognized by cellular DNA sensor cGAS. However, to successfully establish the persistent infection, herpesviruses have evolved their viral genes to modulate different aspects of host immune signaling. This review summarizes the evasion strategies of host cGAS DNA sensing pathway by Kaposi's Sarcoma-associated Herpesvirus (KSHV) and their contributions to KSHV life cycles.

  12. Microfabricated electrochemical sensor for the detection of radiation-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.; Rivas, G.; Ozsoz, M.; Grant, D.H.; Cai, X.; Parrado, C. [New Mexico State Univ., Las Cruces, NM (United States)

    1997-04-01

    An electrochemical biosensor protocol for the detection of radiation-induced DNA damage is described. The procedure employs a dsDNA-coated screen-printed electrode and relies on changes in the guanine-DNA oxidation signal upon exposure to ultraviolet radiation. The decreased signal is ascribed primarily to conformational changes in the DNA and to the photoconversion of the guanine-DNA moiety to a nonelectroactive monomeric base product. Factors influencing the response of these microfabricated DNA sensors, such as irradiation time, wavelength, and distance, are explored, and future prospects are discussed. Similar results are given for the use of bare strip electrodes in connection with irradiated DNA solutions. 8 refs., 4 figs.

  13. Extended DNA Tile Actuators

    DEFF Research Database (Denmark)

    Kristiansen, Martin; Kryger, Mille; Zhang, Zhao

    2012-01-01

    A dynamic linear DNA tile actuator is expanded to three new structures of higher complexity. The original DNA actuator was constructed from a central roller strand which hybridizes with two piston strands by forming two half-crossover junctions. A linear expansion of the actuator is obtained...

  14. Dna fingerprinting - review paper

    OpenAIRE

    Blundell, Renald

    2006-01-01

    Before the Polymerase Chain Reaction (PCR) was established, DNA fingerprinting technology has relied for years on Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandom Repeats (VNTR) analysis, a very efficient technique but quite laborious and not suitable for high throughput mapping. Since its, development, PCR has provided a new and powerful tool for DNA fingerprinting.

  15. DNA Repair Systems

    Indian Academy of Sciences (India)

    Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

  16. DNA repair genes

    International Nuclear Information System (INIS)

    Morimyo, Mitsuoki

    1995-01-01

    Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

  17. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2018-05-15

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  18. Characterization of muntjac DNA

    International Nuclear Information System (INIS)

    Davis, R.C.

    1981-01-01

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange

  19. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  20. Characterization of muntjac DNA

    Energy Technology Data Exchange (ETDEWEB)

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  1. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    Directory of Open Access Journals (Sweden)

    Andre Terzic

    2009-04-01

    Full Text Available Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7 are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.

  2. Whose DNA is this?

    DEFF Research Database (Denmark)

    Taroni, Franco; Biedermann, Alex; Vuille, Joëlle

    2013-01-01

    This communication seeks to draw the attention of researchers and practitioners dealing with forensic DNA profiling analyses to the following question: is a scientist's report, offering support to a hypothesis according to which a particular individual is the source of DNA detected during...... evoked during the international conference "The hidden side of DNA profiles. Artifacts, errors and uncertain evidence" held in Rome (April 27th to 28th, 2012). Indeed, despite the fact that this conference brought together some of the world's leading forensic DNA specialists, it appeared clearly...... talk considerably different languages. It thus is fundamental to address this issue of communication about results of forensic DNA analyses, and open a dialogue with practicing non-scientists at large who need to make meaningful use of scientific results to approach and help solve judicial cases...

  3. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  4. Racemic DNA crystallography.

    Science.gov (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

    2014-12-22

    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Retroactive signaling in short signaling pathways.

    Directory of Open Access Journals (Sweden)

    Jacques-Alexandre Sepulchre

    Full Text Available In biochemical signaling pathways without explicit feedback connections, the core signal transduction is usually described as a one-way communication, going from upstream to downstream in a feedforward chain or network of covalent modification cycles. In this paper we explore the possibility of a new type of signaling called retroactive signaling, offered by the recently demonstrated property of retroactivity in signaling cascades. The possibility of retroactive signaling is analysed in the simplest case of the stationary states of a bicyclic cascade of signaling cycles. In this case, we work out the conditions for which variables of the upstream cycle are affected by a change of the total amount of protein in the downstream cycle, or by a variation of the phosphatase deactivating the same protein. Particularly, we predict the characteristic ranges of the downstream protein, or of the downstream phosphatase, for which a retroactive effect can be observed on the upstream cycle variables. Next, we extend the possibility of retroactive signaling in short but nonlinear signaling pathways involving a few covalent modification cycles.

  6. Cascade DNA nanomachine and exponential amplification biosensing.

    Science.gov (United States)

    Xu, Jianguo; Wu, Zai-Sheng; Shen, Weiyu; Xu, Huo; Li, Hongling; Jia, Lee

    2015-11-15

    DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. [Biomarkers of radiation-induced DNA repair processes].

    Science.gov (United States)

    Vallard, Alexis; Rancoule, Chloé; Guy, Jean-Baptiste; Espenel, Sophie; Sauvaigo, Sylvie; Rodriguez-Lafrasse, Claire; Magné, Nicolas

    2017-11-01

    The identification of DNA repair biomarkers is of paramount importance. Indeed, it is the first step in the process of modulating radiosensitivity and radioresistance. Unlike tools of detection and measurement of DNA damage, DNA repair biomarkers highlight the variations of DNA damage responses, depending on the dose and the dose rate. The aim of the present review is to describe the main biomarkers of radiation-induced DNA repair. We will focus on double strand breaks (DSB), because of their major role in radiation-induced cell death. The most important DNA repair biomarkers are DNA damage signaling proteins, with ATM, DNA-PKcs, 53BP1 and γ-H2AX. They can be analyzed either using immunostaining, or using lived cell imaging. However, to date, these techniques are still time and money consuming. The development of "omics" technologies should lead the way to new (and usable in daily routine) DNA repair biomarkers. Copyright © 2017 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

  8. Nanostructures via DNA scaffold metallization

    OpenAIRE

    Ning, C.; Zinchenko, A.; Baigl, D.; Pyshkina, O.; Sergeyev, V.; Endo, Kazunaka; Yoshikawa, K.

    2005-01-01

    The critical role of polymers in process of noble metals nanostructures formation is well known, however, the use of DNA chain template in this process is yet largely unknown. In this study we demonstrate different ways of silver deposition on DNA template and report the influence of silver nanostructures formation on DNA conformational state. Metallization of DNA chain proceeds by two different scenarios depending on DNA conformation. If DNA chain is unfolded (elongated) chain, silver reduct...

  9. Stress Sensors and Signal Transducers in Cyanobacteria

    Science.gov (United States)

    Los, Dmitry A.; Zorina, Anna; Sinetova, Maria; Kryazhov, Sergey; Mironov, Kirill; Zinchenko, Vladislav V.

    2010-01-01

    In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress. PMID:22294932

  10. DNA damage and polyploidization.

    Science.gov (United States)

    Chow, Jeremy; Poon, Randy Y C

    2010-01-01

    A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

  11. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  12. Regulating DNA Self-assembly by DNA-Surface Interactions.

    Science.gov (United States)

    Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

    2017-12-14

    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Extended HSR/CARD domain mediates AIRE binding to DNA

    Energy Technology Data Exchange (ETDEWEB)

    Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-12-25

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

  14. Extended HSR/CARD domain mediates AIRE binding to DNA

    International Nuclear Information System (INIS)

    Maslovskaja, Julia; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-01-01

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

  15. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

    Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  16. Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses

    Science.gov (United States)

    Rao, Venigalla B.; Feiss, Michael

    2016-01-01

    Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead’s portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL’s N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage φ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics. PMID:26958920

  17. Dynamic and Progressive Control of DNA Origami Conformation by Modulating DNA Helicity with Chemical Adducts.

    Science.gov (United States)

    Chen, Haorong; Zhang, Hanyu; Pan, Jing; Cha, Tae-Gon; Li, Shiming; Andréasson, Joakim; Choi, Jong Hyun

    2016-05-24

    DNA origami has received enormous attention for its ability to program complex nanostructures with a few nanometer precision. Dynamic origami structures that change conformation in response to environmental cues or external signals hold great promises in sensing and actuation at the nanoscale. The reconfiguration mechanism of existing dynamic origami structures is mostly limited to single-stranded hinges and relies almost exclusively on DNA hybridization or strand displacement. Here, we show an alternative approach by demonstrating on-demand conformation changes with DNA-binding molecules, which intercalate between base pairs and unwind DNA double helices. The unwinding effect modulates the helicity mismatch in DNA origami, which significantly influences the internal stress and the global conformation of the origami structure. We demonstrate the switching of a polymerized origami nanoribbon between different twisting states and a well-constrained torsional deformation in a monomeric origami shaft. The structural transformation is shown to be reversible, and binding isotherms confirm the reconfiguration mechanism. This approach provides a rapid and reversible means to change DNA origami conformation, which can be used for dynamic and progressive control at the nanoscale.

  18. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  19. The intersection between DNA damage response and cell death pathways.

    Science.gov (United States)

    Nowsheen, S; Yang, E S

    2012-10-01

    Apoptosis is a finely regulated process that serves to determine the fate of cells in response to various stresses. One such stress is DNA damage, which not only can signal repair processes but is also intimately involved in regulating cell fate. In this review we examine the relationship between the DNA damage/repair response in cell survival and apoptosis following insults to the DNA. Elucidating these pathways and the crosstalk between them is of great importance, as they eventually contribute to the etiology of human disease such as cancer and may play key roles in determining therapeutic response. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

  20. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  1. A Novel Computational Method to Reduce Leaky Reaction in DNA Strand Displacement

    Directory of Open Access Journals (Sweden)

    Xin Li

    2015-01-01

    Full Text Available DNA strand displacement technique is widely used in DNA programming, DNA biosensors, and gene analysis. In DNA strand displacement, leaky reactions can cause DNA signals decay and detecting DNA signals fails. The mostly used method to avoid leakage is cleaning up after upstream leaky reactions, and it remains a challenge to develop reliable DNA strand displacement technique with low leakage. In this work, we address the challenge by experimentally evaluating the basic factors, including reaction time, ratio of reactants, and ion concentration to the leakage in DNA strand displacement. Specifically, fluorescent probes and a hairpin structure reporting DNA strand are designed to detect the output of DNA strand displacement, and thus can evaluate the leakage of DNA strand displacement reactions with different reaction time, ratios of reactants, and ion concentrations. From the obtained data, mathematical models for evaluating leakage are achieved by curve derivation. As a result, it is obtained that long time incubation, high concentration of fuel strand, and inappropriate amount of ion concentration can weaken leaky reactions. This contributes to a method to set proper reaction conditions to reduce leakage in DNA strand displacement.

  2. "Artifactual" arsenate DNA

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2012-01-01

    The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due...... to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present...... evidence that the identification of arsenate DNA was artifactual....

  3. Information Thermodynamics of Cytosine DNA Methylation.

    Directory of Open Access Journals (Sweden)

    Robersy Sanchez

    Full Text Available Cytosine DNA methylation (CDM is a stable epigenetic modification to the genome and a widespread regulatory process in living organisms that involves multicomponent molecular machines. Genome-wide cytosine methylation patterning participates in the epigenetic reprogramming of a cell, suggesting that the biological information contained within methylation positions may be amenable to decoding. Adaptation to a new cellular or organismal environment also implies the potential for genome-wide redistribution of CDM changes that will ensure the stability of DNA molecules. This raises the question of whether or not we would be able to sort out the regulatory methylation signals from the CDM background ("noise" induced by thermal fluctuations. Here, we propose a novel statistical and information thermodynamic description of the CDM changes to address the last question. The physical basis of our statistical mechanical model was evaluated in two respects: 1 the adherence to Landauer's principle, according to which molecular machines must dissipate a minimum energy ε = kBT ln2 at each logic operation, where kB is the Boltzmann constant, and T is the absolute temperature and 2 whether or not the binary stretch of methylation marks on the DNA molecule comprise a language of sorts, properly constrained by thermodynamic principles. The study was performed for genome-wide methylation data from 152 ecotypes and 40 trans-generational variations of Arabidopsis thaliana and 93 human tissues. The DNA persistence length, a basic mechanical property altered by CDM, was estimated with values from 39 to 66.9 nm. Classical methylome analysis can be retrieved by applying information thermodynamic modelling, which is able to discriminate signal from noise. Our finding suggests that the CDM signal comprises a language scheme properly constrained by molecular thermodynamic principles, which is part of an epigenomic communication system that obeys the same thermodynamic

  4. Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

    Science.gov (United States)

    Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

    2016-01-01

    Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample

  5. Push back to respond better: regulatory inhibition of the DNA double-strand break response.

    Science.gov (United States)

    Panier, Stephanie; Durocher, Daniel

    2013-10-01

    Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

  6. A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

    Science.gov (United States)

    Calderwood, Stuart K

    2016-05-26

    Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

  7. Sensitive electrochemical monitoring of nucleic acids coupling DNA nanostructures with hybridization chain reaction

    International Nuclear Information System (INIS)

    Zhuang, Junyang; Fu, Libing; Xu, Mingdi; Yang, Huanghao; Chen, Guonan; Tang, Dianping

    2013-01-01

    Graphical abstract: -- Highlights: •A new signal-on metallobioassay was developed for detection of nucleic acids. •Target-triggered long-range self-assembled DNA nanostructures are used for amplification of electronic signal. •Hybridization chain reaction is utilized for construction of long-range DNA nanostructures. -- Abstract: Methods based on metal nanotags have been developed for metallobioassay of nucleic acids, but most involve complicated labeling or stripping procedures and are unsuitable for routine use. Herein, we report the proof-of-concept of a novel and label-free metallobioassay for ultrasensitive electronic determination of human immunodeficiency virus (HIV)-related gene fragments at an ultralow concentration based on target-triggered long-range self-assembled DNA nanostructures and DNA-based hybridization chain reaction (HCR). The signal is amplified by silver nanotags on the DNA duplex. The assay mainly consists of capture probe, detection probe, and two different DNA hairpins. In the presence of target DNA, the capture probe immobilized on the sensor sandwiches target DNA with the 3′ end of detection probe. Another exposed part of detection probe at the 5′ end opens two alternating DNA hairpins in turn, and propagates a chain reaction of hybridization events to form a nicked double-helix. Finally, numerous silver nanotags are immobilized onto the long-range DNA nanostructures, each of which produces a strong electronic signal within the applied potentials. Under optimal conditions, the target-triggered long-range DNA nanostructures present good electrochemical behaviors for the detection of HIV DNA at a concentration as low as 0.5 fM. Importantly, the outstanding sensitivity can make this approach a promising scheme for development of next-generation DNA sensors without the need of enzyme labeling or fluorophore labeling

  8. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted

    2011-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  9. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  10. DNA Sampling Hook

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  11. Retroviral DNA Integration

    Science.gov (United States)

    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

  12. DNA damage and carcinogenesis

    International Nuclear Information System (INIS)

    Stelow, R.B.

    1980-01-01

    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10 4 fold

  13. DNA-Origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Gothelf, Kurt Vesterager

    2010-01-01

    DNA-nanostrukturer giver nye muligheder for studier af individuelle molekyler. Ved at udnytte DNAs unikke selvsamlende egenskaber kan man designe systemer, hvorpå der kan studeres kemiske reaktioner, fluoroforer og biiomolekyler på enkeltmolekyle-niveau....

  14. DNA Microarray Technology

    Science.gov (United States)

    Skip to main content DNA Microarray Technology Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research News Features Funding Divisions Funding ...

  15. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  16. Close encounters with DNA

    Science.gov (United States)

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.

    2014-01-01

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  17. Gomphid DNA sequence data

    Data.gov (United States)

    U.S. Environmental Protection Agency — DNA sequence data for several genetic loci. This dataset is not publicly accessible because: It's already publicly available on GenBank. It can be accessed through...

  18. HPV DNA test

    Science.gov (United States)

    ... test; Cancer of cervix - HPV DNA test References Hacker NF. Cervical dysplasia and cancer. In: Hacker NF, Gambone JC, Hobel CJ, eds. Hacker and Moore's Essentials of Obstetrics and Gynecology . 6th ...

  19. Close encounters with DNA.

    Science.gov (United States)

    Maffeo, C; Yoo, J; Comer, J; Wells, D B; Luan, B; Aksimentiev, A

    2014-10-15

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena. We also discuss the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field.

  20. FBI's DNA analysis program

    Science.gov (United States)

    Brown, John R.

    1994-03-01

    Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

  1. Making DNA Fingerprints.

    Science.gov (United States)

    Nunley, Kathie F.

    1996-01-01

    Presents an activity to simulate electrophoresis using everyday items. Uses adding machine paper to construct a set of DNA fingerprints that can be used to solve crime cases designed by students in any biology class. (JRH)

  2. Radiation damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, V.

    1978-01-01

    A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

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

    Science.gov (United States)

    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.

  4. DNA to DNA transcription might exist in eukaryotic cells

    OpenAIRE

    Li, Gao-De

    2016-01-01

    Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

  5. Patterning nanocrystals using DNA

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Shara Carol [Univ. of California, Berkeley, CA (United States)

    2003-01-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

  6. Das DNA-Puzzle

    Science.gov (United States)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  7. PDA: Pooled DNA analyzer

    Directory of Open Access Journals (Sweden)

    Lin Chin-Yu

    2006-04-01

    Full Text Available Abstract Background Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer, to analyze pooled DNA data. Results We develop the software, PDA, for the analysis of pooled-DNA data. PDA is originally implemented with the MATLAB® language, but it can also be executed on a Windows system without installing the MATLAB®. PDA provides estimates of the coefficient of preferential amplification and allele frequency. PDA considers an extended single-point association test, which can compare allele frequencies between two DNA pools constructed under different experimental conditions. Moreover, PDA also provides novel chromosome-wide multipoint association tests based on p-value combinations and a sliding-window concept. This new multipoint testing procedure overcomes a computational bottleneck of conventional haplotype-oriented multipoint methods in DNA pooling analyses and can handle data sets having a large pool size and/or large numbers of polymorphic markers. All of the PDA functions are illustrated in the four bona fide examples. Conclusion PDA is simple to operate and does not require that users have a strong statistical background. The software is available at http://www.ibms.sinica.edu.tw/%7Ecsjfann/first%20flow/pda.htm.

  8. Racemic DNA Crystallography

    OpenAIRE

    Mandal , Pradeep K.; Collie , Gavin W.; Kauffmann , Brice; Huc , Ivan

    2014-01-01

    International audience; Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of Land D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propens...

  9. Celebrating DNA's Repair Crew.

    Science.gov (United States)

    Kunkel, Thomas A

    2015-12-03

    This year, the Nobel Prize in Chemistry has been awarded to Tomas Lindahl, Aziz Sancar, and Paul Modrich for their seminal studies of the mechanisms by which cells from bacteria to man repair DNA damage that is generated by normal cellular metabolism and stress from the environment. These studies beautifully illustrate the remarkable power of DNA repair to influence life from evolution through disease susceptibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Agrobacterium tumefaciens responses to plant-derived signaling molecules

    Science.gov (United States)

    Subramoni, Sujatha; Nathoo, Naeem; Klimov, Eugene; Yuan, Ze-Chun

    2014-01-01

    As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium–plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its Transferred DNA (T-DNA) from its Tumor-inducing (Ti) plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA), cytokinin (CK), and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS) to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including γ-amino butyric acid and salicylic acid (SA) to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays) also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium–plant interactions. PMID:25071805

  11. Agrobacterium tumefaciens responses to plant-derived signaling molecules

    Directory of Open Access Journals (Sweden)

    Sujatha eSubramoni

    2014-07-01

    Full Text Available As a special phytopathogen, Agrobacterium tumefaciens infects a wide range of plant hosts and causes plant tumors also known as crown galls. The complexity of Agrobacterium-plant interaction has been studied for several decades. Agrobacterium pathogenicity is largely attributed to its evolved capabilities of precise recognition and response to plant-derived chemical signals. Agrobacterium perceives plant-derived signals to activate its virulence genes, which are responsible for transferring and integrating its T-DNA (Transferred DNA from its Tumour-inducing (Ti plasmid into the plant nucleus. The expression of T-DNA in plant hosts leads to the production of a large amount of indole-3-acetic acid (IAA, cytokinin (CK and opines. IAA and CK stimulate plant growth, resulting in tumor formation. Agrobacterium utilizes opines as nutrient sources as well as signals in order to activate its quorum sensing (QS to further promote virulence and opine metabolism. Intriguingly, Agrobacterium also recognizes plant-derived signals including -amino butyric acid (GABA and salicylic acid (SA to activate quorum quenching that reduces the level of QS signals, thereby avoiding the elicitation of plant defense and preserving energy. In addition, Agrobacterium hijacks plant-derived signals including SA, IAA, and ethylene (ET to down-regulate its virulence genes located on the Ti plasmid. Moreover, certain metabolites from corn (Zea mays also inhibit the expression of Agrobacterium virulence genes. Here we outline the responses of Agrobacterium to major plant-derived signals that impact Agrobacterium-plant interactions.

  12. Introduction to DNA methods

    International Nuclear Information System (INIS)

    Delincee, H.

    1991-01-01

    The purpose of this session is to discuss the various possibilities for detecting modifications in DNA after irradiation and whether these changes can be utilized as an indicator for the irradiation treatment of foods. The requirement to be fulfilled is that the method be able to distinguish irradiated food without the presence of a control sample, thus the measured response after irradiation must be large enough to supersede background levels from other treatments. Much work has been performed on the effects of radiation on DNA, particularly due to its importance in radiation biology. The main lesions of DNA as a result of irradiation are base damage, damage of the sugar moiety, single strand and double strand breaks. Crosslinking between bases also occurs, e.g. production of thymine dimers, or between DNA and protein. A valuable review on how to utilize these DNA changes for detection purposes has already appeared. Tables 1, 2 and 3 list the proposed methods of detecting changes in irradiated DNA, some identified products as examples for a possible irradiation indicator, in the case of immunoassay the substance used as antigen, and some selected literature references. In this short review, it is not intended to provide a complete literature survey

  13. Variations in brain DNA

    Directory of Open Access Journals (Sweden)

    Jesus eAvila

    2014-11-01

    Full Text Available It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain.

  14. Signal sciences workshop proceedings

    Energy Technology Data Exchange (ETDEWEB)

    Candy, J.V.

    1997-05-01

    This meeting is aimed primarily at signal processing and controls. The technical program for the 1997 Workshop includes a variety of efforts in the Signal Sciences with applications in the Microtechnology Area a new program at LLNL and a future area of application for both Signal/Image Sciences. Special sessions organized by various individuals in Seismic and Optical Signal Processing as well as Micro-Impulse Radar Processing highlight the program, while the speakers at the Signal Processing Applications session discuss various applications of signal processing/control to real world problems. For the more theoretical, a session on Signal Processing Algorithms was organized as well as for the more pragmatic, featuring a session on Real-Time Signal Processing.

  15. Signal sciences workshop. Proceedings

    International Nuclear Information System (INIS)

    Candy, J.V.

    1997-01-01

    This meeting is aimed primarily at signal processing and controls. The technical program for the 1997 Workshop includes a variety of efforts in the Signal Sciences with applications in the Microtechnology Area a new program at LLNL and a future area of application for both Signal/Image Sciences. Special sessions organized by various individuals in Seismic and Optical Signal Processing as well as Micro-Impulse Radar Processing highlight the program, while the speakers at the Signal Processing Applications session discuss various applications of signal processing/control to real world problems. For the more theoretical, a session on Signal Processing Algorithms was organized as well as for the more pragmatic, featuring a session on Real-Time Signal Processing

  16. Identifying Fishes through DNA Barcodes and Microarrays.

    Directory of Open Access Journals (Sweden)

    Marc Kochzius

    2010-09-01

    Full Text Available International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S, cytochrome b (cyt b, and cytochrome oxidase subunit I (COI for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90% renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

  17. ECG signal processing

    NARCIS (Netherlands)

    2009-01-01

    A system extracts an ECG signal from a composite signal (308) representing an electric measurement of a living subject. Identification means (304) identify a plurality of temporal segments (309) of the composite signal corresponding to a plurality of predetermined segments (202,204,206) of an ECG

  18. Blood extracellular DNA after irradiation

    International Nuclear Information System (INIS)

    Vladimirov, V.G.; Tishchenko, L.I.; Surkova, E.A.; Vasil'eva, I.N.

    1993-01-01

    It has been shown that blood extracellular DNA of irradiated rats largely consists of the low-molecular DNA and its oligomers. Molecular masses of oligomers are multiple to molecular mass of monomer fragment with nucleosome size. The low-molecular DNA has linear form. The average content of GC-pairs in low-molecular DNA is higher than in total rat's DNA (48.5% against 41.5%). The low-molecular DNA is a part of complex containing RNA, acidic proteins and lipids. It is assumed that the formation of low-molecular DNA is a result of Ca/Mg - dependent nuclear endonuclease action

  19. Quantitative phosphoproteomics to characterize signaling networks

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2012-01-01

    for analyzing protein phosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterization of signaling networks. Contemporary phosphoproteomics use highly optimized procedures for sample preparation, mass spectrometry and data analysis algorithms to identify......Reversible protein phosphorylation is involved in the regulation of most, if not all, major cellular processes via dynamic signal transduction pathways. During the last decade quantitative phosphoproteomics have evolved from a highly specialized area to a powerful and versatile platform...... and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments...

  20. ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses.

    Science.gov (United States)

    Berger, N Daniel; Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

    2017-10-05

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  1. DNAzyme-Based Logic Gate-Mediated DNA Self-Assembly.

    Science.gov (United States)

    Zhang, Cheng; Yang, Jing; Jiang, Shuoxing; Liu, Yan; Yan, Hao

    2016-01-13

    Controlling DNA self-assembly processes using rationally designed logic gates is a major goal of DNA-based nanotechnology and programming. Such controls could facilitate the hierarchical engineering of complex nanopatterns responding to various molecular triggers or inputs. Here, we demonstrate the use of a series of DNAzyme-based logic gates to control DNA tile self-assembly onto a prescribed DNA origami frame. Logic systems such as "YES," "OR," "AND," and "logic switch" are implemented based on DNAzyme-mediated tile recognition with the DNA origami frame. DNAzyme is designed to play two roles: (1) as an intermediate messenger to motivate downstream reactions and (2) as a final trigger to report fluorescent signals, enabling information relay between the DNA origami-framed tile assembly and fluorescent signaling. The results of this study demonstrate the plausibility of DNAzyme-mediated hierarchical self-assembly and provide new tools for generating dynamic and responsive self-assembly systems.

  2. Optimal Signal Quality Index for Photoplethysmogram Signals

    Directory of Open Access Journals (Sweden)

    Mohamed Elgendi

    2016-09-01

    Full Text Available A photoplethysmogram (PPG is a noninvasive circulatory signal related to the pulsatile volume of blood in tissue and is typically collected by pulse oximeters. PPG signals collected via mobile devices are prone to artifacts that negatively impact measurement accuracy, which can lead to a significant number of misleading diagnoses. Given the rapidly increased use of mobile devices to collect PPG signals, developing an optimal signal quality index (SQI is essential to classify the signal quality from these devices. Eight SQIs were developed and tested based on: perfusion, kurtosis, skewness, relative power, non-stationarity, zero crossing, entropy, and the matching of systolic wave detectors. Two independent annotators annotated all PPG data (106 recordings, 60 s each and a third expert conducted the adjudication of differences. The independent annotators labeled each PPG signal with one of the following labels: excellent, acceptable or unfit for diagnosis. All indices were compared using Mahalanobis distance, linear discriminant analysis, quadratic discriminant analysis, and support vector machine with leave-one-out cross-validation. The skewness index outperformed the other seven indices in differentiating between excellent PPG and acceptable, acceptable combined with unfit, and unfit recordings, with overall F 1 scores of 86.0%, 87.2%, and 79.1%, respectively.

  3. Optimal Signal Quality Index for Photoplethysmogram Signals.

    Science.gov (United States)

    Elgendi, Mohamed

    2016-09-22

    A photoplethysmogram (PPG) is a noninvasive circulatory signal related to the pulsatile volume of blood in tissue and is typically collected by pulse oximeters. PPG signals collected via mobile devices are prone to artifacts that negatively impact measurement accuracy, which can lead to a significant number of misleading diagnoses. Given the rapidly increased use of mobile devices to collect PPG signals, developing an optimal signal quality index (SQI) is essential to classify the signal quality from these devices. Eight SQIs were developed and tested based on: perfusion, kurtosis, skewness, relative power, non-stationarity, zero crossing, entropy, and the matching of systolic wave detectors. Two independent annotators annotated all PPG data (106 recordings, 60 s each) and a third expert conducted the adjudication of differences. The independent annotators labeled each PPG signal with one of the following labels: excellent, acceptable or unfit for diagnosis. All indices were compared using Mahalanobis distance, linear discriminant analysis, quadratic discriminant analysis, and support vector machine with leave-one-out cross-validation. The skewness index outperformed the other seven indices in differentiating between excellent PPG and acceptable, acceptable combined with unfit, and unfit recordings, with overall F 1 scores of 86.0%, 87.2%, and 79.1%, respectively.

  4. DNA Knots: Theory and Experiments

    Science.gov (United States)

    Sumners, D. W.

    Cellular DNA is a long, thread-like molecule with remarkably complex topology. Enzymes that manipulate the geometry and topology of cellular DNA perform many vital cellular processes (including segregation of daughter chromosomes, gene regulation, DNA repair, and generation of antibody diversity). Some enzymes pass DNA through itself via enzyme-bridged transient breaks in the DNA; other enzymes break the DNA apart and reconnect it to different ends. In the topological approach to enzymology, circular DNA is incubated with an enzyme, producing an enzyme signature in the form of DNA knots and links. By observing the changes in DNA geometry (supercoiling) and topology (knotting and linking) due to enzyme action, the enzyme binding and mechanism can often be characterized. This paper will discuss some personal research history, and the tangle model for the analysis of site-specific recombination experiments on circular DNA.

  5. Core nucleosomes by digestion of reconstructed histone-DNA complexes

    Energy Technology Data Exchange (ETDEWEB)

    Bryan, P N; Wright, E B; Olins, D E

    1979-04-01

    Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (..nu../sub 1/). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(dA-dT).poly(dA-dT) have been examined by circular dichroism, thermal denaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260 to 280 nm and a prominent ..cap alpha..-helix signal at 222 nm. All particles show biphasic melting except ..nu../sub 1/(dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of ..nu../sub 1/ (dA-dT) produces a ladder of DNA fragments differing in length by one base residue. ..nu../sub 1/ (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4 to 10.5 bases per turn. There appear to be two regions of different DNA pitch within ..nu../sub 1/ (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.

  6. DNA damage mediated transcription arrest: Step back to go forward.

    Science.gov (United States)

    Mullenders, Leon

    2015-12-01

    The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Cytosolic sensing of immuno-stimulatory DNA, the enemy within.

    Science.gov (United States)

    Dhanwani, Rekha; Takahashi, Mariko; Sharma, Sonia

    2018-02-01

    In the cytoplasm, DNA is sensed as a universal danger signal by the innate immune system. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor/enzyme that catalyzes formation of 2'-5'-cGAMP, an atypical cyclic di-nucleotide second messenger that binds and activates the Stimulator of Interferon Genes (STING), resulting in recruitment of Tank Binding Kinase 1 (TBK1), activation of the transcription factor Interferon Regulatory Factor 3 (IRF3), and trans-activation of innate immune response genes, including type I Interferon cytokines (IFN-I). Activation of the pro-inflammatory cGAS-STING-IRF3 response is triggered by direct recognition of the DNA genomes of bacteria and viruses, but also during RNA virus infection, neoplastic transformation, tumor immunotherapy and systemic auto-inflammatory diseases. In these circumstances, the source of immuno-stimulatory DNA has often represented a fundamental yet poorly understood aspect of the response. This review focuses on recent findings related to cGAS activation by an array of self-derived DNA substrates, including endogenous retroviral elements, mitochondrial DNA (mtDNA) and micronuclei generated as a result of genotoxic stress and DNA damage. These findings emphasize the role of the cGAS axis as a cell-intrinsic innate immune response to a wide variety of genomic insults. Copyright © 2017. Published by Elsevier Ltd.

  8. Programmable chemical controllers made from DNA

    Science.gov (United States)

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2013-10-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents.

  9. Global chromatin fibre compaction in response to DNA damage

    International Nuclear Information System (INIS)

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-01-01

    Highlights: ► Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. ► DNA repair foci are found in soluble chromatin. ► Biophysical analysis reveals global chromatin fibre compaction after DNA damage. ► DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to

  10. Protocols for dry DNA storage and shipment at room temperature.

    Science.gov (United States)

    Ivanova, Natalia V; Kuzmina, Masha L

    2013-09-01

    The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica(®) DNAstable(®) plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at -20 °C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica(®) provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica(®) at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at -20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long-term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities. © 2013 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.

  11. Second-hand signals

    DEFF Research Database (Denmark)

    Bergenholtz, Carsten

    2014-01-01

    Studies of signaling theory have traditionally focused on the dyadic link between the sender and receiver of the signal. Within a science‐based perspective this framing has led scholars to investigate how patents and publications of firms function as signals. I explore another important type...... used by various agents in their search for and assessment of products and firms. I conclude by arguing how this second‐hand nature of signals goes beyond a simple dyadic focus on senders and receivers of signals, and thus elucidates the more complex interrelations of the various types of agents...

  12. Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

    Science.gov (United States)

    Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

    2016-05-26

    RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

  13. A method for tracing exogenous DNA uptake in live spermatozoa and embryos.

    Science.gov (United States)

    Mu, Y; Jiao, M; Zhao, Y; Lv, J; Wang, J; Hao, J; Zhang, X; Kong, Q; Liu, Z

    2018-03-01

    Sperm-mediated gene transfer(SMGT) is a simple method for producing transgenic animals. Due to the lack of repeatability in spermatozoa binding and internalization of exogenous DNA, the efficiency of SMGT is still low. Considering this point, the present work aims to develop a method for evaluating the spermatozoa capacity of binding exogenous DNA after co-incubation with DNA. The main approach is using a Cy5-labelled DNA to trace the exogenous DNA and assess the ability of spermatozoa to take up exogenous DNA. Using this technique, we found that the percentage of spermatozoa that are binding and uptaking DNA is higher at concentration of 10 μg/mL and 100 μg/mL than 5 μg/mL, 1 μg/mL and 0 μg/mL after incubation with Cy5-DNA for 30min at 37oC. After fertilization, the DNA fluorescence signal was also detected in zygotes in groups where spermatozoa were incubated with 10 μg/mL and 100 μg/mL of Cy5-DNA. These results showed a simple and convenient method to trace the exogenous DNA in spermatozoa and zygote when compared to conventional methods of labeling DNA during fertilization, resulting in a real-time observation of the exogenous DNA in spermatozoa and zygote. Copyright© by the Polish Academy of Sciences.

  14. DNA-modified electrodes fabricated using copper-free click chemistry for enhanced protein detection.

    Science.gov (United States)

    Furst, Ariel L; Hill, Michael G; Barton, Jacqueline K

    2013-12-31

    A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.

  15. A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control

    International Nuclear Information System (INIS)

    Hou, Sen; Tabaka, Marcin; Sun, Lili; Trochimczyk, Piotr; Kaminski, Tomasz S; Kalwarczyk, Tomasz; Zhang, Xuzhu; Holyst, Robert

    2014-01-01

    We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5′ end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1 fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value. (letter)

  16. Proliferative signaling initiated in ACTH receptors

    Directory of Open Access Journals (Sweden)

    C.F.P. Lotfi

    2000-10-01

    Full Text Available This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK (2 to 10 min, b transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min, c induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.

  17. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.; Oke, Muse; Hamdan, Samir

    2014-01-01

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  18. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.

    2014-11-21

    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  19. Visualization of DNA double-strand break repair: From molecules to cells

    NARCIS (Netherlands)

    Krawczyk, Przemek M.; Stap, Jan; Aten, Jacob A.

    2008-01-01

    DNA double-strand break (DSB) signaling and repair processes are positioned at the crossroad of nuclear pathways that regulate DNA replication, cell division, senescence and apoptosis. Importantly, errors in DSB repair may lead to lethal or potentially tumorigenic chromosome rearrangements.

  20. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

    of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  1. Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

    DEFF Research Database (Denmark)

    Beli, Petra; Lukashchuk, Natalia; Wagner, Sebastian A

    2012-01-01

    /ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes...

  2. Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

    DEFF Research Database (Denmark)

    Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

    2016-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

  3. RNF4 is required for DNA double-strand break repair in vivo

    DEFF Research Database (Denmark)

    Vyas, R; Kumar, R; Clermont, F

    2013-01-01

    for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling...

  4. DNA replication stress restricts ribosomal DNA copy number

    Science.gov (United States)

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  5. DNA replication stress restricts ribosomal DNA copy number.

    Science.gov (United States)

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  6. DNA replication stress restricts ribosomal DNA copy number.

    Directory of Open Access Journals (Sweden)

    Devika Salim

    2017-09-01

    Full Text Available Ribosomal RNAs (rRNAs in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  7. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.

    1988-01-01

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  8. Histone H2AX in DNA repair

    International Nuclear Information System (INIS)

    Lewandowska, H.; Szumiel, I.

    2002-01-01

    The paper reviews the recent reports on the role of the phosphorylated histone H2AX (γ-H2AX). The modification of this histone is an important part of the cellular response to the induction of DNA double strand brakes (DSB) by ionising radiation and other DSB-generating factors. In irradiated cells the modification is carried out mainly by ATM (ataxia-telangiectasia mutated) kinase, the enzyme that starts the alarm signalling upon induction of DSB.γ-H2AX molecules are formed within 1-3 min after irradiation and form foci at the sites of DSB. This seems to be necessary for the recruitment of repair factors that are later present in foci of damaged nuclei. Modification of a constant percentage of H2AX molecules per DSB takes place, corresponding to chromatin domains of megabase of DNA. (author)

  9. DNA alterations photosensitized by tetracycline and some of its derivatives

    International Nuclear Information System (INIS)

    Piette, J.; Decuyper, J.; Van de Vorst, A.

    1986-01-01

    Bacteriophage M13 mp10 DNA were irradiated with near-UV light in the presence of tetracycline derivatives and primed with synthetic oligonucleotide to be used for DNA synthesis using Escherichia coli DNA polymerase. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis and mapped precisely. All the synthesis stops occurred before or at the level of guanine residues, showing that the photoreaction mediated by tetracycline derivatives led to a preferential alteration of guanine residues. These lesions were demonstrated to be induced in DNA through a pathway involving singlet oxygen. Tetracycline derivatives also photoinduced the breakage of the DNA sugar-phosphate backbone monitored by the conversion of supercoiled phi X174 DNA to a relaxed form. This lesion was shown to be initiated by hydroxyl radicals. The production of this free radical has been confirmed by electron paramagnetic resonance (EPR) spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap. In addition to the EPR signal due to OH radicals trapping another unassigned signal has been detected

  10. The interferon response to intracellular DNA: why so many receptors?

    Science.gov (United States)

    Unterholzner, Leonie

    2013-11-01

    The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA. Copyright © 2013 Elsevier GmbH. All rights reserved.

  11. Complete cDNA sequence coding for human docking protein

    Energy Technology Data Exchange (ETDEWEB)

    Hortsch, M; Labeit, S; Meyer, D I

    1988-01-11

    Docking protein (DP, or SRP receptor) is a rough endoplasmic reticulum (ER)-associated protein essential for the targeting and translocation of nascent polypeptides across this membrane. It specifically interacts with a cytoplasmic ribonucleoprotein complex, the signal recognition particle (SRP). The nucleotide sequence of cDNA encoding the entire human DP and its deduced amino acid sequence are given.

  12. The DNA-damage response in human biology and disease

    DEFF Research Database (Denmark)

    Jackson, Stephen P; Bartek, Jiri

    2009-01-01

    , signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management....

  13. Comparing transformation methods for DNA microarray data

    Directory of Open Access Journals (Sweden)

    Zwinderman Aeilko H

    2004-06-01

    Full Text Available Abstract Background When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include subtraction of an estimated background signal, subtracting the reference signal, smoothing (to account for nonlinear measurement effects, and more. Different authors use different approaches, and it is generally not clear to users which method they should prefer. Results We used the ratio between biological variance and measurement variance (which is an F-like statistic as a quality measure for transformation methods, and we demonstrate a method for maximizing that variance ratio on real data. We explore a number of transformations issues, including Box-Cox transformation, baseline shift, partial subtraction of the log-reference signal and smoothing. It appears that the optimal choice of parameters for the transformation methods depends on the data. Further, the behavior of the variance ratio, under the null hypothesis of zero biological variance, appears to depend on the choice of parameters. Conclusions The use of replicates in microarray experiments is important. Adjustment for the null-hypothesis behavior of the variance ratio is critical to the selection of transformation method.

  14. Principles of DNA architectonics: design of DNA-based nanoobjects

    International Nuclear Information System (INIS)

    Vinogradova, O A; Pyshnyi, D V

    2012-01-01

    The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

  15. DNA Topoisomerases in Transcription

    DEFF Research Database (Denmark)

    Rødgaard, Morten Terpager

    2015-01-01

    This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most of the ex......This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most...... topoisomerase-DNA cleavage complex. The second study is an investigation of how topoisomerases influence gene regulation by keeping the genome in an optimal topological state....

  16. Duplication in DNA Sequences

    Science.gov (United States)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  17. DNA methylation in obesity

    Directory of Open Access Journals (Sweden)

    Małgorzata Pokrywka

    2014-11-01

    Full Text Available The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes, have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

  18. Proximity hybridization-regulated catalytic DNA hairpin assembly for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles

    International Nuclear Information System (INIS)

    Zhou, Fuyi; Yao, Yao; Luo, Jianjun; Zhang, Xing; Zhang, Yu; Yin, Dengyang; Gao, Fenglei; Wang, Po

    2017-01-01

    Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH 4 oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10 −15 to 10 −11  g mL −1 and a detection limit of 0.43 × 10 −15  g mL −1 . Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10 −16  g mL −1 . And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. - Graphical abstract: A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA. - Highlights: • A novel enzyme-free electrochemical immunosensor was developed for detection of CEA. • The signal amplification was based on catalytic DNA hairpin assembly and DNA-template-synthesized Pd nanoparticles. • The biosensor could detect CEA down to 0.52 × 10 −16  g mL −1 level with a dynamic range spanning 5 orders of magnitude.

  19. Repeated DNA sequences in fungi

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, S K

    1974-11-01

    Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10 to 20 percent repeated DNA sequences. There are approximately 100 to 110 copies of repeated DNA sequences of approximately 4 x 10/sup 7/ daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5/sup 0/C difference of T/sub e/50 (temperature at which 50 percent duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA. (auth)

  20. Method of signal analysis

    International Nuclear Information System (INIS)

    Berthomier, Charles

    1975-01-01

    A method capable of handling the amplitude and the frequency time laws of a certain kind of geophysical signals is described here. This method is based upon the analytical signal idea of Gabor and Ville, which is constructed either in the time domain by adding an imaginary part to the real signal (in-quadrature signal), or in the frequency domain by suppressing negative frequency components. The instantaneous frequency of the initial signal is then defined as the time derivative of the phase of the analytical signal, and his amplitude, or envelope, as the modulus of this complex signal. The method is applied to three types of magnetospheric signals: chorus, whistlers and pearls. The results obtained by analog and numerical calculations are compared to results obtained by classical systems using filters, i.e. based upon a different definition of the concept of frequency. The precision with which the frequency-time laws are determined leads then to the examination of the principle of the method and to a definition of instantaneous power density spectrum attached to the signal, and to the first consequences of this definition. In this way, a two-dimensional representation of the signal is introduced which is less deformed by the analysis system properties than the usual representation, and which moreover has the advantage of being obtainable practically in real time [fr

  1. Measurand transient signal suppressor

    Science.gov (United States)

    Bozeman, Richard J., Jr. (Inventor)

    1994-01-01

    A transient signal suppressor for use in a controls system which is adapted to respond to a change in a physical parameter whenever it crosses a predetermined threshold value in a selected direction of increasing or decreasing values with respect to the threshold value and is sustained for a selected discrete time interval is presented. The suppressor includes a sensor transducer for sensing the physical parameter and generating an electrical input signal whenever the sensed physical parameter crosses the threshold level in the selected direction. A manually operated switch is provided for adapting the suppressor to produce an output drive signal whenever the physical parameter crosses the threshold value in the selected direction of increasing or decreasing values. A time delay circuit is selectively adjustable for suppressing the transducer input signal for a preselected one of a plurality of available discrete suppression time and producing an output signal only if the input signal is sustained for a time greater than the selected suppression time. An electronic gate is coupled to receive the transducer input signal and the timer output signal and produce an output drive signal for energizing a control relay whenever the transducer input is a non-transient signal which is sustained beyond the selected time interval.

  2. Virulent poxviruses inhibit DNA sensing by preventing STING activation.

    Science.gov (United States)

    Georgana, Iliana; Sumner, Rebecca P; Towers, Greg J; Maluquer de Motes, Carlos

    2018-02-28

    Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerises and translocates from the ER to a perinuclear region to mediate IRF-3 activation. Poxviruses are dsDNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here we investigated the activation of innate immune signalling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerised and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerisation and phosphorylation during infection and in response to transfected DNA and cGAMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence. IMPORTANCE Poxviruses are dsDNA viruses infecting a wide range of vertebrates and include the causative agent of smallpox (variola virus) and its vaccine vaccinia virus (VACV). Despite smallpox eradication VACV remains of interest as a therapeutic. Attenuated strains are popular vaccine candidates, whereas replication-competent strains are emerging as

  3. Pharmacodynamic Assay Panel for Monitoring Phospho-Signaling Networks | Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The DNA damage response (DDR) is a highly regulated signal transduction network that orchestrates the temporal and spatial organization of protein complexes required to repair (or tolerate) DNA damage (e.g., nucleotide excision repair, base excision repair, homologous recombination, non-homologous end joining, post-replication repair).

  4. Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, S.; Alavaren, M.; Varlaro, J. [Roche Molecular Systems, Alameda, CA (United States)] [and others

    1994-09-01

    The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.

  5. The ATM signaling network in development and disease

    Science.gov (United States)

    Stracker, Travis H.; Roig, Ignasi; Knobel, Philip A.; Marjanović, Marko

    2013-01-01

    The DNA damage response (DDR) rapidly recognizes DNA lesions and initiates the appropriate cellular programs to maintain genome integrity. This includes the coordination of cell cycle checkpoints, transcription, translation, DNA repair, metabolism, and cell fate decisions, such as apoptosis or senescence (Jackson and Bartek, 2009). DNA double-strand breaks (DSBs) represent one of the most cytotoxic DNA lesions and defects in their metabolism underlie many human hereditary diseases characterized by genomic instability (Stracker and Petrini, 2011; McKinnon, 2012). Patients with hereditary defects in the DDR display defects in development, particularly affecting the central nervous system, the immune system and the germline, as well as aberrant metabolic regulation and cancer predisposition. Central to the DDR to DSBs is the ataxia-telangiectasia mutated (ATM) kinase, a master controller of signal transduction. Understanding how ATM signaling regulates various aspects of the DDR and its roles in vivo is critical for our understanding of human disease, its diagnosis and its treatment. This review will describe the general roles of ATM signaling and highlight some recent advances that have shed light on the diverse roles of ATM and related proteins in human disease. PMID:23532176

  6. Yeast signaling pathways in the oxidative stress response

    Energy Technology Data Exchange (ETDEWEB)

    Ikner, Aminah [Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616 (United States); Shiozaki, Kazuhiro [Section of Microbiology, Division of Biological Sciences, University of California, Davis, CA 95616 (United States)]. E-mail: kshiozaki@ucdavis.edu

    2005-01-06

    Oxidative stress that generates the reactive oxygen species (ROS) is one of the major causes of DNA damage and mutations. The 'DNA damage checkpoint' that arrests cell cycle and repairs damaged DNA has been a focus of recent studies, and the genetically amenable model systems provided by yeasts have been playing a leading role in the eukaryotic checkpoint research. However, means to eliminate ROS are likely to be as important as the DNA repair mechanisms in order to suppress mutations in the chromosomal DNA, and yeasts also serve as excellent models to understand how eukaryotes combat oxidative stress. In this article, we present an overview of the signaling pathways that sense oxidative stress and induce expression of various anti-oxidant genes in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the pathogenic yeast Candida albicans. Three conserved signaling modules have been identified in the oxidative stress response of these diverse yeast species: the stress-responsive MAP kinase cascade, the multistep phosphorelay and the AP-1-like transcription factor. The structure and function of these signaling modules are discussed.

  7. Yeast signaling pathways in the oxidative stress response

    International Nuclear Information System (INIS)

    Ikner, Aminah; Shiozaki, Kazuhiro

    2005-01-01

    Oxidative stress that generates the reactive oxygen species (ROS) is one of the major causes of DNA damage and mutations. The 'DNA damage checkpoint' that arrests cell cycle and repairs damaged DNA has been a focus of recent studies, and the genetically amenable model systems provided by yeasts have been playing a leading role in the eukaryotic checkpoint research. However, means to eliminate ROS are likely to be as important as the DNA repair mechanisms in order to suppress mutations in the chromosomal DNA, and yeasts also serve as excellent models to understand how eukaryotes combat oxidative stress. In this article, we present an overview of the signaling pathways that sense oxidative stress and induce expression of various anti-oxidant genes in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the pathogenic yeast Candida albicans. Three conserved signaling modules have been identified in the oxidative stress response of these diverse yeast species: the stress-responsive MAP kinase cascade, the multistep phosphorelay and the AP-1-like transcription factor. The structure and function of these signaling modules are discussed

  8. The ATM signaling network in development and disease

    Directory of Open Access Journals (Sweden)

    Travis H. Stracker

    2013-03-01

    Full Text Available The DNA damage response (DDR rapidly recognizes DNA lesions and initiates the appropriate cellular programs to maintain genome integrity. This includes the coordination of cell cycle checkpoints, transcription, translation, DNA repair, metabolism and cell fate decisions, such as apoptosis or senescence(Jackson and Bartek, 2009. DNA double-strand breaks (DSBs represent one of the most cytotoxic DNA lesions and defects in their metabolism underlie many human hereditary diseases characterized by genomic instability(Stracker and Petrini, 2011;McKinnon, 2012. Patients with hereditary defects in the DDR display defects in development, particularly affecting the central nervous system (CNS, the immune system and the germline, as well as aberrant metabolic regulation and cancer predisposition. Central to the DDR to DSBs is the ATM kinase, a master controller of signal transduction. Understanding how ATM signaling regulates various aspects of the DDR and its roles in vivo is critical for our understanding of human disease, its diagnosis and its treatment. This review will describe the general roles of ATM signaling and highlight some recent advances that have shed light on the diverse roles of ATM and related proteins in human disease.

  9. DNA Nanotechnology for Cancer Therapy

    Science.gov (United States)

    Kumar, Vinit; Palazzolo, Stefano; Bayda, Samer; Corona, Giuseppe; Toffoli, Giuseppe; Rizzolio, Flavio

    2016-01-01

    DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as stability, loading capability, release and immunocompatibility, which mainly limit in vivo applications. Special attention was dedicated to highlighting the boundaries to be overcome to bring DNA nanostructures closer to the bedside of patients. PMID:27022418

  10. Viral evasion of DNA-stimulated innate immune responses

    Science.gov (United States)

    Christensen, Maria H; Paludan, Søren R

    2017-01-01

    Cellular sensing of virus-derived nucleic acids is essential for early defenses against virus infections. In recent years, the discovery of DNA sensing proteins, including cyclic GMP–AMP synthase (cGAS) and gamma-interferon-inducible protein (IFI16), has led to understanding of how cells evoke strong innate immune responses against incoming pathogens carrying DNA genomes. The signaling stimulated by DNA sensors depends on the adaptor protein STING (stimulator of interferon genes), to enable expression of antiviral proteins, including type I interferon. To facilitate efficient infections, viruses have evolved a wide range of evasion strategies, targeting host DNA sensors, adaptor proteins and transcription factors. In this review, the current literature on virus-induced activation of the STING pathway is presented and we discuss recently identified viral evasion mechanisms targeting different steps in this antiviral pathway. PMID:26972769

  11. Initiation of DNA replication requires actin dynamics and formin activity.

    Science.gov (United States)

    Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

    2017-11-02

    Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

  12. Chromosomal instability drives metastasis through a cytosolic DNA response.

    Science.gov (United States)

    Bakhoum, Samuel F; Ngo, Bryan; Laughney, Ashley M; Cavallo, Julie-Ann; Murphy, Charles J; Ly, Peter; Shah, Pragya; Sriram, Roshan K; Watkins, Thomas B K; Taunk, Neil K; Duran, Mercedes; Pauli, Chantal; Shaw, Christine; Chadalavada, Kalyani; Rajasekhar, Vinagolu K; Genovese, Giulio; Venkatesan, Subramanian; Birkbak, Nicolai J; McGranahan, Nicholas; Lundquist, Mark; LaPlant, Quincey; Healey, John H; Elemento, Olivier; Chung, Christine H; Lee, Nancy Y; Imielenski, Marcin; Nanjangud, Gouri; Pe'er, Dana; Cleveland, Don W; Powell, Simon N; Lammerding, Jan; Swanton, Charles; Cantley, Lewis C

    2018-01-25

    Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

  13. Viral evasion of DNA-stimulated innate immune responses.

    Science.gov (United States)

    Christensen, Maria H; Paludan, Søren R

    2017-01-01

    Cellular sensing of virus-derived nucleic acids is essential for early defenses against virus infections. In recent years, the discovery of DNA sensing proteins, including cyclic GMP-AMP synthase (cGAS) and gamma-interferon-inducible protein (IFI16), has led to understanding of how cells evoke strong innate immune responses against incoming pathogens carrying DNA genomes. The signaling stimulated by DNA sensors depends on the adaptor protein STING (stimulator of interferon genes), to enable expression of antiviral proteins, including type I interferon. To facilitate efficient infections, viruses have evolved a wide range of evasion strategies, targeting host DNA sensors, adaptor proteins and transcription factors. In this review, the current literature on virus-induced activation of the STING pathway is presented and we discuss recently identified viral evasion mechanisms targeting different steps in this antiviral pathway.

  14. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    Science.gov (United States)

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  15. DNA damage response in nephrotoxic and ischemic kidney injury

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Mingjuan; Tang, Chengyuan [Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 (China); Ma, Zhengwei [Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA 30912 (United States); Huang, Shuang [Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL (United States); Dong, Zheng, E-mail: zdong@augusta.edu [Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 (China); Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA 30912 (United States)

    2016-12-15

    DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore relies on a thorough elucidation of the DDR pathways in various forms of AKI.

  16. Wnt signaling in cancer

    Science.gov (United States)

    Zhan, T; Rindtorff, N; Boutros, M

    2017-01-01

    Wnt signaling is one of the key cascades regulating development and stemness, and has also been tightly associated with cancer. The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer, but aberrant Wnt signaling is observed in many more cancer entities. Here, we review current insights into novel components of Wnt pathways and describe their impact on cancer development. Furthermore, we highlight expanding functions of Wnt signaling for both solid and liquid tumors. We also describe current findings how Wnt signaling affects maintenance of cancer stem cells, metastasis and immune control. Finally, we provide an overview of current strategies to antagonize Wnt signaling in cancer and challenges that are associated with such approaches. PMID:27617575

  17. DNA profiling of trace DNA recovered from bedding.

    Science.gov (United States)

    Petricevic, Susan F; Bright, Jo-Anne; Cockerton, Sarah L

    2006-05-25

    Trace DNA is often detected on handled items and worn clothing examined in forensic laboratories. In this study, the potential transfer of trace DNA to bedding by normal contact, when an individual sleeps in a bed, is examined. Volunteers slept one night on a new, lower bed sheet in their own bed and one night in a bed foreign to them. Samples from the sheets were collected and analysed by DNA profiling. The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples. This transfer may have important repercussions in sexual assault investigations.

  18. Cooperative ethylene receptor signaling

    OpenAIRE

    Liu, Qian; Wen, Chi-Kuang

    2012-01-01

    The gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members in the dicotyledonous model plant Arabidopsis. Genetic and biochemical studies suggest that the ethylene response is suppressed by ethylene receptor complexes, but the biochemical nature of the receptor signal is unknown. Without appropriate biochemical measures to trace the ethylene receptor signal and quantify the signal strength, the biological significance of the modulation of ethylene responses ...

  19. Traffic signal synchronization.

    Science.gov (United States)

    Huang, Ding-wei; Huang, Wei-neng

    2003-05-01

    The benefits of traffic signal synchronization are examined within the cellular automata approach. The microsimulations of traffic flow are obtained with different settings of signal period T and time delay delta. Both numerical results and analytical approximations are presented. For undersaturated traffic, the green-light wave solutions can be realized. For saturated traffic, the correlation among the traffic signals has no effect on the throughput. For oversaturated traffic, the benefits of synchronization are manifest only when stochastic noise is suppressed.

  20. Danger-signaler og inflammasomer ved autoinflammatoriske og autoimmune sygdomme

    DEFF Research Database (Denmark)

    Bendtzen, Klaus

    2011-01-01

    Cytoplasmic inflammasomes are formed through activation of pattern recognition receptors (PRR) of the innate immune system. Endogenous and exogenous danger signals, e.g. DNA- and RNA-fragments, urate- and cholesterol crystals, silica and asbestos, ß-amyloid, UV-light and skin irritants, may induce...

  1. Nanochannel Device with Embedded Nanopore: a New Approach for Single-Molecule DNA Analysis and Manipulation

    Science.gov (United States)

    Zhang, Yuning; Reisner, Walter

    2013-03-01

    Nanopore and nanochannel based devices are robust methods for biomolecular sensing and single DNA manipulation. Nanopore-based DNA sensing has attractive features that make it a leading candidate as a single-molecule DNA sequencing technology. Nanochannel based extension of DNA, combined with enzymatic or denaturation-based barcoding schemes, is already a powerful approach for genome analysis. We believe that there is revolutionary potential in devices that combine nanochannels with embedded pore detectors. In particular, due to the fast translocation of a DNA molecule through a standard nanopore configuration, there is an unfavorable trade-off between signal and sequence resolution. With a combined nanochannel-nanopore device, based on embedding a pore inside a nanochannel, we can in principle gain independent control over both DNA translocation speed and sensing signal, solving the key draw-back of the standard nanopore configuration. We demonstrate that we can optically detect successful translocation of DNA from the nanochannel out through the nanopore, a possible method to 'select' a given barcode for further analysis. In particular, we show that in equilibrium DNA will not escape through an embedded sub-persistence length nanopore, suggesting that the pore could be used as a nanoscale window through which to interrogate a nanochannel extended DNA molecule. Furthermore, electrical measurements through the nanopore are performed, indicating that DNA sensing is feasible using the nanochannel-nanopore device.

  2. Managing Single-Stranded DNA during Replication Stress in Fission Yeast

    Directory of Open Access Journals (Sweden)

    Sarah A. Sabatinos

    2015-09-01

    Full Text Available Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

  3. DNA tagged microparticles

    Science.gov (United States)

    Farquar, George R.; Leif, Roald N.; Wheeler, Elizabeth

    2016-03-22

    In one embodiment, a product includes a plurality of particles, each particle including: a carrier that includes a non-toxic material; and at least one DNA barcode coupled to the carrier, where the particles each have a diameter in a range from about 1 nanometer to about 100 microns.

  4. Digital signal processing the Tevatron BPM signals

    International Nuclear Information System (INIS)

    Cancelo, G.; James, E.; Wolbers, S.

    2005-01-01

    The Beam Position Monitor (TeV BPM) readout system at Fermilab's Tevatron has been updated and is currently being commissioned. The new BPMs use new analog and digital hardware to achieve better beam position measurement resolution. The new system reads signals from both ends of the existing directional stripline pickups to provide simultaneous proton and antiproton measurements. The signals provided by the two ends of the BPM pickups are processed by analog band-pass filters and sampled by 14-bit ADCs at 74.3MHz. A crucial part of this work has been the design of digital filters that process the signal. This paper describes the digital processing and estimation techniques used to optimize the beam position measurement. The BPM electronics must operate in narrow-band and wide-band modes to enable measurements of closed-orbit and turn-by-turn positions. The filtering and timing conditions of the signals are tuned accordingly for the operational modes. The analysis and the optimized result for each mode are presented

  5. Nichtkontinuierliche (zeitdiskrete) Signale

    Science.gov (United States)

    Plaßmann, Wilfried

    Zeitdiskrete Signale werden häufig aus zeitkontinuierlichen Signalen durch Abtastung erzeugt. Dass beide Signale gleichwertig sind, zeigt das Abtasttheorem (Kap. 116) von Shannon, sofern die Bedingung nach (116.2), f_{ab}≈(2{,}2 {\\ldots} 4)\\cdot fg) eingehalten wird. Digitale Signale haben Vorteile: Einfache Speicherung, Weiterverarbeitung in Rechnern, wenig störanfällige Übertragung. Für die Bearbeitung dieser Signale dienen die im Kapitel dargestellten Hilfsmittel: Diskrete Fouriertransformation; Schnelle Fouriertransformation; z-Transformation: Darstellung, Sätze zur z-Transformation, Korrespondenzen zu Zeitfunktionen, Beispiele.

  6. Biomedical signals and systems

    CERN Document Server

    Tranquillo, Joseph V

    2013-01-01

    Biomedical Signals and Systems is meant to accompany a one-semester undergraduate signals and systems course. It may also serve as a quick-start for graduate students or faculty interested in how signals and systems techniques can be applied to living systems. The biological nature of the examples allows for systems thinking to be applied to electrical, mechanical, fluid, chemical, thermal and even optical systems. Each chapter focuses on a topic from classic signals and systems theory: System block diagrams, mathematical models, transforms, stability, feedback, system response, control, time

  7. Radiation signal processing system

    International Nuclear Information System (INIS)

    Bennett, M.; Knoll, G.; Strange, D.

    1980-01-01

    An improved signal processing system for radiation imaging apparatus comprises: a radiation transducer producing transducer signals proportional to apparent spatial coordinates of detected radiation events; means for storing true spatial coordinates corresponding to a plurality of predetermined apparent spatial coordinates relative to selected detected radiation events said means for storing responsive to said transducer signal and producing an output signal representative of said true spatial coordinates; and means for interpolating the true spatial coordinates of the detected radiation events located intermediate the stored true spatial coordinates, said means for interpolating communicating with said means for storing

  8. Digital signal processing

    CERN Document Server

    O'Shea, Peter; Hussain, Zahir M

    2011-01-01

    In three parts, this book contributes to the advancement of engineering education and that serves as a general reference on digital signal processing. Part I presents the basics of analog and digital signals and systems in the time and frequency domain. It covers the core topics: convolution, transforms, filters, and random signal analysis. It also treats important applications including signal detection in noise, radar range estimation for airborne targets, binary communication systems, channel estimation, banking and financial applications, and audio effects production. Part II considers sel

  9. The dynamic interplay between DNA topoisomerases and DNA topology.

    Science.gov (United States)

    Seol, Yeonee; Neuman, Keir C

    2016-11-01

    Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

  10. Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

    Science.gov (United States)

    Stejskalová, Eva; Horáková, Petra; Vacek, Jan; Bowater, Richard P; Fojta, Miroslav

    2014-07-01

    DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

  11. Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome.

    Science.gov (United States)

    Robicheau, Brent M; Susko, Edward; Harrigan, Amye M; Snyder, Marlene

    2017-02-01

    Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. Conformation-dependent DNA attraction

    Science.gov (United States)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-05-01

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by

  13. Melting curves of gammairradiated DNA

    International Nuclear Information System (INIS)

    Hofer, H.; Altmann, H.; Kehrer, M.

    1978-08-01

    Melting curves of gammairradiated DNA and data derived of them, are reported. The diminished stability is explained by basedestruction. DNA denatures completely at room temperature, if at least every fifth basepair is broken or weakened by irradiation. (author)

  14. An Introduction to DNA Fingerprinting.

    Science.gov (United States)

    Hepfer, Carol Ely; And Others

    1993-01-01

    Provides background information on DNA fingerprinting, and describes exercises for introducing general biology students at the high school or college level to the methodology and applications of DNA fingerprinting. (PR)

  15. Esitleti kakskeelset luulekogu "Luule DNA"

    Index Scriptorium Estoniae

    2007-01-01

    Magrelli, Valerio. Luule DNA = Il DNA della poesia / tõlkinud [ja saatesõna:] Maarja Kangro ja Kalju Kruusa. Tallinn : Koma, 2006. Sisaldab autori teksti. Esitlus 24. jaan. Kirjanike majas Tallinnas

  16. DNA damage repair and radiosensitivity

    International Nuclear Information System (INIS)

    Suzuki, Norio

    2003-01-01

    Tailored treatment is not new in radiotherapy; it has been the major subject for the last 20-30 years. Radiation responses and RBE (relative biological effectiveness) depend on assay systems, endpoints, type of tissues and tumors, radiation quality, dose rate, dose fractionation, physiological and environmental factors etc, Latent times to develop damages also differ among tissues and endpoints depending on doses and radiation quality. Recent progress in clarification of radiation induced cell death, especially of apoptotic cell death, is quite important for understanding radiosensitivity of tumor cure process as well as of tumorigenesis. Apoptotic cell death as well as dormant cells had been unaccounted and missed into a part of reproductive cell death. Another area of major progress has been made in clarifying repair mechanisms of radiation damage, i.e., non-homologous end joining (NHEJ) and homologous recombinational repair (HRR). New approaches and developments such as cDNA or protein micro arrays and so called informatics in addition to basic molecular biological analysis are expected to aid identifying molecules and their roles in signal transduction pathways, which are multi-factorial and interactive each other being involved in radiation responses. (authors)

  17. Conformation-dependent DNA attraction.

    Science.gov (United States)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-06-21

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg(2+) ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg(2+) or Na(+), benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg(2+) bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.

  18. Alterations of ultraviolet irradiated DNA

    International Nuclear Information System (INIS)

    Davila, C.; Garces, F.

    1980-01-01

    Thymine dimers production has been studied in several DNA- 3 H irradiated at various wave lenght of U.V. Light. The influence of dimers on the hydrodynamic and optic properties, thermal structural stability and transformant capacity of DNA have been studied too. At last the recognition and excision of dimers by the DNA-UV-Endonuclease and DNA-Polimerase-I was also studied. (author)

  19. Amplified Detection of the Aptamer-Vanillin Complex with the Use of Bsm DNA Polymerase.

    Science.gov (United States)

    Andrianova, Mariia; Komarova, Natalia; Grudtsov, Vitaliy; Kuznetsov, Evgeniy; Kuznetsov, Alexander

    2017-12-26

    The electrochemical detection of interactions between aptamers and low-molecular-weight targets often lacks sensitivity. Signal amplification improves the detection of the aptamer-analyte complex; Bsm DNA polymerase was used to amplify the signal from the interaction of vanillin and its aptamer named Van_74 on an ion-sensitive field-effect transistor (ISFET)-based biosensor. The aptamer was immobilized on the ISFET sensitive surface. A short DNA probe was hybridized with the aptamer and dissociated from it upon vanillin addition. A free probe interacted with a special DNA molecular beacon initiated the Bsm DNA polymerase reaction that was detected by ISFET. A buffer solution suitable for both aptamer action and Bsm DNA polymerase activity was determined. The ISFET was shown to detect the Bsm DNA polymerase reaction under the selected conditions. Vanillin at different concentrations (1 × 10 -6 -1 × 10 -8 M) was detected using the biosensor with signal amplification. The developed detection system allowed for the determination of vanillin, starting at a 10 -8 M concentration. Application of the Bsm DNA polymerase resulted in a 15.5 times lower LoD when compared to the biosensor without signal amplification (10.1007/s00604-017-2586-4).

  20. Amplified Detection of the Aptamer–Vanillin Complex with the Use of Bsm DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Mariia Andrianova

    2017-12-01

    Full Text Available The electrochemical detection of interactions between aptamers and low-molecular-weight targets often lacks sensitivity. Signal amplification improves the detection of the aptamer-analyte complex; Bsm DNA polymerase was used to amplify the signal from the interaction of vanillin and its aptamer named Van_74 on an ion-sensitive field-effect transistor (ISFET-based biosensor. The aptamer was immobilized on the ISFET sensitive surface. A short DNA probe was hybridized with the aptamer and dissociated from it upon vanillin addition. A free probe interacted with a special DNA molecular beacon initiated the Bsm DNA polymerase reaction that was detected by ISFET. A buffer solution suitable for both aptamer action and Bsm DNA polymerase activity was determined. The ISFET was shown to detect the Bsm DNA polymerase reaction under the selected conditions. Vanillin at different concentrations (1 × 10−6–1 × 10−8 M was detected using the biosensor with signal amplification. The developed detection system allowed for the determination of vanillin, starting at a 10−8 M concentration. Application of the Bsm DNA polymerase resulted in a 15.5 times lower LoD when compared to the biosensor without signal amplification (10.1007/s00604-017-2586-4.

  1. Drought Signaling in Plants

    Indian Academy of Sciences (India)

    depending upon the source and nature of signaling: (i) hormone signal, (ii) .... plants to regulate the rate of transpiration through minor structural .... cell has to keep spending energy (in the form of A TP) to maintain a .... enzymes and proteins in the regulation of cellular metabolism can be determined by either inactivating.

  2. Ubiquitination in apoptosis signaling

    NARCIS (Netherlands)

    van de Kooij, L.W.

    2014-01-01

    The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

  3. Multiresolution signal decomposition schemes

    NARCIS (Netherlands)

    J. Goutsias (John); H.J.A.M. Heijmans (Henk)

    1998-01-01

    textabstract[PNA-R9810] Interest in multiresolution techniques for signal processing and analysis is increasing steadily. An important instance of such a technique is the so-called pyramid decomposition scheme. This report proposes a general axiomatic pyramid decomposition scheme for signal analysis

  4. SignalR blueprints

    CERN Document Server

    Ingebrigtsen, Einar

    2015-01-01

    This book is designed for software developers, primarily those with knowledge of C#, .NET, and JavaScript. Good knowledge and understanding of SignalR is assumed to allow efficient programming of core elements and applications in SignalR.

  5. Optimal fault signal estimation

    NARCIS (Netherlands)

    Stoorvogel, Antonie Arij; Niemann, H.H.; Saberi, A.; Sannuti, P.

    2002-01-01

    We consider here both fault identification and fault signal estimation. Regarding fault identification, we seek either exact or almost fault identification. On the other hand, regarding fault signal estimation, we seek either $H_2$ optimal, $H_2$ suboptimal or Hinfinity suboptimal estimation. By

  6. Signal sampling circuit

    NARCIS (Netherlands)

    Louwsma, S.M.; Vertregt, Maarten

    2011-01-01

    A sampling circuit for sampling a signal is disclosed. The sampling circuit comprises a plurality of sampling channels adapted to sample the signal in time-multiplexed fashion, each sampling channel comprising a respective track-and-hold circuit connected to a respective analogue to digital

  7. Signal sampling circuit

    NARCIS (Netherlands)

    Louwsma, S.M.; Vertregt, Maarten

    2010-01-01

    A sampling circuit for sampling a signal is disclosed. The sampling circuit comprises a plurality of sampling channels adapted to sample the signal in time-multiplexed fashion, each sampling channel comprising a respective track-and-hold circuit connected to a respective analogue to digital

  8. Interfacing DNA nanodevices with biology

    DEFF Research Database (Denmark)

    Vinther, Mathias; Kjems, Jørgen

    2016-01-01

    in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular...... environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology....

  9. Human Genome Research: Decoding DNA

    Science.gov (United States)

    dropdown arrow Site Map A-Z Index Menu Synopsis Human Genome Research: Decoding DNA Resources with of the DNA double helix during April 2003. James D. Watson, Francis Crick, and Maurice Wilkins were company Celera announced the completion of a "working draft" reference DNA sequence of the human

  10. My journey to DNA repair.

    Science.gov (United States)

    Lindahl, Tomas

    2013-02-01

    I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mammalian exonucleases DNase III (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O(6)-methylguanine (O(6)mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation. Copyright © 2013. Production and hosting by Elsevier Ltd.

  11. Bioelectric Signal Measuring System

    Science.gov (United States)

    Guadarrama-Santana, A.; Pólo-Parada, L.; García-Valenzuela, A.

    2015-01-01

    We describe a low noise measuring system based on interdigitated electrodes for sensing bioelectrical signals. The system registers differential voltage measurements in order of microvolts. The base noise during measurements was in nanovolts and thus, the sensing signals presented a very good signal to noise ratio. An excitation voltage of 1Vrms with 10 KHz frequency was applied to an interdigitated capacitive sensor without a material under test and to a mirror device simultaneously. The output signals of both devices was then subtracted in order to obtain an initial reference value near cero volts and reduce parasitic capacitances due to the electronics, wiring and system hardware as well. The response of the measuring system was characterized by monitoring temporal bioelectrical signals in real time of biological materials such as embryo chicken heart cells and bovine suprarenal gland cells.

  12. Acoustic Signals and Systems

    DEFF Research Database (Denmark)

    2008-01-01

    The Handbook of Signal Processing in Acoustics will compile the techniques and applications of signal processing as they are used in the many varied areas of Acoustics. The Handbook will emphasize the interdisciplinary nature of signal processing in acoustics. Each Section of the Handbook...... will present topics on signal processing which are important in a specific area of acoustics. These will be of interest to specialists in these areas because they will be presented from their technical perspective, rather than a generic engineering approach to signal processing. Non-specialists, or specialists...... from different areas, will find the self-contained chapters accessible and will be interested in the similarities and differences between the approaches and techniques used in different areas of acoustics....

  13. Molecular and Cellular Signaling

    CERN Document Server

    Beckerman, Martin

    2005-01-01

    A small number of signaling pathways, no more than a dozen or so, form a control layer that is responsible for all signaling in and between cells of the human body. The signaling proteins belonging to the control layer determine what kinds of cells are made during development and how they function during adult life. Malfunctions in the proteins belonging to the control layer are responsible for a host of human diseases ranging from neurological disorders to cancers. Most drugs target components in the control layer, and difficulties in drug design are intimately related to the architecture of the control layer. Molecular and Cellular Signaling provides an introduction to molecular and cellular signaling in biological systems with an emphasis on the underlying physical principles. The text is aimed at upper-level undergraduates, graduate students and individuals in medicine and pharmacology interested in broadening their understanding of how cells regulate and coordinate their core activities and how diseases ...

  14. Adaptive signal processor

    Energy Technology Data Exchange (ETDEWEB)

    Walz, H.V.

    1980-07-01

    An experimental, general purpose adaptive signal processor system has been developed, utilizing a quantized (clipped) version of the Widrow-Hoff least-mean-square adaptive algorithm developed by Moschner. The system accommodates 64 adaptive weight channels with 8-bit resolution for each weight. Internal weight update arithmetic is performed with 16-bit resolution, and the system error signal is measured with 12-bit resolution. An adapt cycle of adjusting all 64 weight channels is accomplished in 8 ..mu..sec. Hardware of the signal processor utilizes primarily Schottky-TTL type integrated circuits. A prototype system with 24 weight channels has been constructed and tested. This report presents details of the system design and describes basic experiments performed with the prototype signal processor. Finally some system configurations and applications for this adaptive signal processor are discussed.

  15. Adaptive signal processor

    International Nuclear Information System (INIS)

    Walz, H.V.

    1980-07-01

    An experimental, general purpose adaptive signal processor system has been developed, utilizing a quantized (clipped) version of the Widrow-Hoff least-mean-square adaptive algorithm developed by Moschner. The system accommodates 64 adaptive weight channels with 8-bit resolution for each weight. Internal weight update arithmetic is performed with 16-bit resolution, and the system error signal is measured with 12-bit resolution. An adapt cycle of adjusting all 64 weight channels is accomplished in 8 μsec. Hardware of the signal processor utilizes primarily Schottky-TTL type integrated circuits. A prototype system with 24 weight channels has been constructed and tested. This report presents details of the system design and describes basic experiments performed with the prototype signal processor. Finally some system configurations and applications for this adaptive signal processor are discussed

  16. Zinc Signals and Immunity.

    Science.gov (United States)

    Maywald, Martina; Wessels, Inga; Rink, Lothar

    2017-10-24

    Zinc homeostasis is crucial for an adequate function of the immune system. Zinc deficiency as well as zinc excess result in severe disturbances in immune cell numbers and activities, which can result in increased susceptibility to infections and development of especially inflammatory diseases. This review focuses on the role of zinc in regulating intracellular signaling pathways in innate as well as adaptive immune cells. Main underlying molecular mechanisms and targets affected by altered zinc homeostasis, including kinases, caspases, phosphatases, and phosphodiesterases, will be highlighted in this article. In addition, the interplay of zinc homeostasis and the redox metabolism in affecting intracellular signaling will be emphasized. Key signaling pathways will be described in detail for the different cell types of the immune system. In this, effects of fast zinc flux, taking place within a few seconds to minutes will be distinguish from slower types of zinc signals, also designated as "zinc waves", and late homeostatic zinc signals regarding prolonged changes in intracellular zinc.

  17. Response to DNA damage: why do we need to focus on protein phosphatases?

    Directory of Open Access Journals (Sweden)

    Midori eShimada

    2013-01-01

    Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

  18. DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

    Science.gov (United States)

    Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R

    2017-01-11

    DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

  19. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    Directory of Open Access Journals (Sweden)

    Nynne Sharma

    2013-01-01

    Full Text Available DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

  20. Construction of a fuzzy and all Boolean logic gates based on DNA

    DEFF Research Database (Denmark)

    M. Zadegan, Reza; Jepsen, Mette D E; Hildebrandt, Lasse

    2015-01-01

    to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive...... DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics....