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Sample records for egfr-initiated signalling dna

  1. Invariants of DNA genomic signals

    Science.gov (United States)

    Cristea, Paul Dan A.

    2005-02-01

    For large scale analysis purposes, the conversion of genomic sequences into digital signals opens the possibility to use powerful signal processing methods for handling genomic information. The study of complex genomic signals reveals large scale features, maintained over the scale of whole chromosomes, that would be difficult to find by using only the symbolic representation. Based on genomic signal methods and on statistical techniques, the paper defines parameters of DNA sequences which are invariant to transformations induced by SNPs, splicing or crossover. Re-orienting concatenated coding regions in the same direction, regularities shared by the genomic material in all exons are revealed, pointing towards the hypothesis of a regular ancestral structure from which the current chromosome structures have evolved. This property is not found in non-nuclear genomic material, e.g., plasmids.

  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

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    Gerardo Mendizabal-Ruiz

    2018-01-01

    Full Text Available 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. 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.

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

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    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.

  6. Oxidized Extracellular DNA as a Stress Signal in Human Cells

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    Aleksei V. Ermakov

    2013-01-01

    Full Text Available The term “cell-free DNA” (cfDNA was recently coined for DNA fragments from plasma/serum, while DNA present in in vitro cell culture media is known as extracellular DNA (ecDNA. Under oxidative stress conditions, the levels of oxidative modification of cellular DNA and the rate of cell death increase. Dying cells release their damaged DNA, thus, contributing oxidized DNA fragments to the pool of cfDNA/ecDNA. Oxidized cell-free DNA could serve as a stress signal that promotes irradiation-induced bystander effect. Evidence points to TLR9 as a possible candidate for oxidized DNA sensor. An exposure to oxidized ecDNA stimulates a synthesis of reactive oxygen species (ROS that evokes an adaptive response that includes transposition of the homologous loci within the nucleus, polymerization and the formation of the stress fibers of the actin, as well as activation of the ribosomal gene expression, and nuclear translocation of NF-E2 related factor-2 (NRF2 that, in turn, mediates induction of phase II detoxifying and antioxidant enzymes. In conclusion, the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells and, possibly, in the human body; in particular, it might contribute to systemic abscopal effects of localized irradiation treatments.

  7. 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.

  8. 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.

  9. PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors.

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    Chen, Dongshi; Tong, Jingshan; Yang, Liheng; Wei, Liang; Stolz, Donna B; Yu, Jian; Zhang, Jianke; Zhang, Lin

    2018-04-10

    Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA , a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD -deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo. Copyright © 2018 the Author(s). Published by PNAS.

  10. Functionalization of DNA Nanostructures for Cell Signaling Applications

    Science.gov (United States)

    Pedersen, Ronnie O.

    Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling. The predictable Watson Crick base pairing allows for designing self-assembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures. To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization. The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity. The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by

  11. Nanomaterial-Assisted Signal Enhancement of Hybridization for DNA Biosensors: A Review

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    Minqiang Li

    2009-09-01

    Full Text Available Detection of DNA sequences has received broad attention due to its potential applications in a variety of fields. As sensitivity of DNA biosensors is determined by signal variation of hybridization events, the signal enhancement is of great significance for improving the sensitivity in DNA detection, which still remains a great challenge. Nanomaterials, which possess some unique chemical and physical properties caused by nanoscale effects, provide a new opportunity for developing novel nanomaterial-based signal-enhancers for DNA biosensors. In this review, recent progress concerning this field, including some newly-developed signal enhancement approaches using quantum-dots, carbon nanotubes and their composites reported by our group and other researchers are comprehensively summarized. Reports on signal enhancement of DNA biosensors by non-nanomaterials, such as enzymes and polymer reagents, are also reviewed for comparison. Furthermore, the prospects for developing DNA biosensors using nanomaterials as signal-enhancers in future are also indicated.

  12. 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.

  13. 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.

  14. 1Restoration of ATM expression in DNA-PKcs deficient cells inhibits signal end joining

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    Neal, Jessica A.; Xu, Yao; Abe, Masumi; Hendrickson, Eric; Meek, Katheryn

    2016-01-01

    Unlike most DNA-PKcs deficient mouse cell strains, we show here that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those studied here) and in DNA-PKcs deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the “classical” non-homologous end-joining pathway. PMID:26921311

  15. 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.

  16. Effect of structure on sensing performance of a target induced signaling probe shifting DNA-based (TISPS-DNA) sensor.

    Science.gov (United States)

    Yu, Xiang; Yu, Zhigang; Li, Fengqin; Xu, Yanmei; He, Xunjun; Xu, Lan; Shi, Wenbing; Zhang, Guiling; Yan, Hong

    2017-05-15

    A type of "signal on" displacement-based sensors named target induced signaling probe shifting DNA-based (TISPS-DNA) sensor were developed for a designated DNA detection. The signaling mechanism of the signaling probe (SP) shifting different from the classical conformation/flexibility change mode endows the sensor with high sensitivity. Through using thiolated or no thiolated capturing probe (CP), two 3-probe sensing structures, sensor-1 and sensor-2, were designed and constructed. The systematical comparing research results show that both sensors exhibit some similarities or big differences in sensing performance. On the one hand, the similarity in structures determines the similarity in some aspects of signaling mechanism, background signal, signal changing form, anti-fouling ability and versatility; on the other hand, the slight difference in structures also results in two opposite hybridization modes of gradual increasing resistance and gradual decreasing resistance which can affect the hybridization efficiency between the assistant probe (AP) and the SP, further producing some big differences in sensing performance, for example, apparently different signal enhancement (SE) change, point mutation discrimination ability and response speed. Under the optimized fabrication and detection conditions, both sensors feature high sensitivity for target DNAs with the detection limits of ∼10 fM for sensor-1 and ∼7 fM for sensor-2, respectively. Among many acquired sensing virtues, the sensor-1 shows a peculiar specificity adjustability which is also a highlight in this work. Copyright © 2017 Elsevier B.V. All rights reserved.

  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. 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.

  19. Bidirectional coupling of splicing and ATM signaling in response to transcription-blocking DNA damage

    NARCIS (Netherlands)

    M. Tresini (Maria); J.A. Marteijn (Jurgen); W. Vermeulen (Wim)

    2016-01-01

    textabstractIn response to DNA damage cells activate intricate protein networks to ensure genomic fidelity and tissue homeostasis. DNA damage response signaling pathways coordinate these networks and determine cellular fates, in part, by modulating RNA metabolism. Here we discuss a

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

    Science.gov (United States)

    Velic, Denis; Couturier, Anthony M.; Ferreira, Maria Tedim; Rodrigue, Amélie; Poirier, Guy G.; Fleury, Fabrice; Masson, Jean-Yves

    2015-01-01

    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. PMID:26610585

  1. 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

  2. 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

  3. Human papillomavirus E1 and E2 mediated DNA replication is not arrested by DNA damage signalling.

    Science.gov (United States)

    King, Lauren E; Fisk, John C; Dornan, Edward S; Donaldson, Mary M; Melendy, Thomas; Morgan, Iain M

    2010-10-10

    Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer. Copyright © 2010 Elsevier Inc. 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

    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......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...... barriers by making chromatin permissive for DNA damage signaling, whereas the ensuing exclusion of SAFB1 may help prevent excessive signaling....

  5. Initiation signals for complementary strand DNA synthesis on single-stranded plasmid DNA

    NARCIS (Netherlands)

    van der Ende, A.; Teertstra, R.; van der Avoort, H. G.; Weisbeek, P. J.

    1983-01-01

    The bacteriophage 0X174 origin for (+) strand DNA synthesis, when inserted in a plasmid, is in vivo a substrate for the initiator A protein, that is produced by infecting phages. The result of this interaction is the packaging of single-stranded plasmid DNA into preformed phage coats. These plasmid

  6. DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas

    DEFF Research Database (Denmark)

    Herrtwich, Laura; Nanda, Indrajit; Evangelou, Konstantinos

    2016-01-01

    granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent...

  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. 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.

  9. Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition.

    Science.gov (United States)

    Huang, Yong; Shi, Ming; Zhao, Limin; Zhao, Shulin; Hu, Kun; Chen, Zheng-Feng; Chen, Jia; Liang, Hong

    2014-04-15

    A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dye-labeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π-π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs. © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  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. 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

    Signal transfer is central to the controlled exchange of information in biology and advanced technologies. Therefore, the development of reliable, long-ranging signal transfer systems for artificial nanoscale assemblies is of great scientific interest. We have designed such a system for signal...... 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......, and therefore has the potential to be applied to larger multi-component DNA assemblies...

  12. 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

    -dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition...... and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR......-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage....

  13. 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...

  14. 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

  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. DNA stabilization by the upregulation of estrogen signaling in BRCA gene mutation carriers

    Directory of Open Access Journals (Sweden)

    Suba Z

    2015-05-01

    Full Text Available Zsuzsanna Suba Surgical and Molecular Tumor Pathology Centre, National Institute of Oncology, Budapest, Hungary Abstract: Currently available scientific evidence erroneously suggests that mutagenic weakness or loss of the BRCA1/2 genes may liberate the proliferative effects of estrogen signaling, which provokes DNA damage and genomic instability. Conversely, BRCA mutation seems to be an imbalanced defect, crudely inhibiting the upregulation of estrogen receptor expression and liganded transcriptional activity, whereas estrogen receptor-repressor functions become predominant. In BRCA-proficient cases, estrogen signaling orchestrates the activity of cell proliferation and differentiation with high safety, while upregulating the expression and DNA-stabilizing impact of BRCA genes. In turn, BRCA proteins promote estrogen signaling by proper estrogen synthesis via CYP19 gene regulation and by induction of the appropriate expression and transcriptional activity of estrogen receptors. In this exquisitely organized regulatory system, the dysfunction of each player may jeopardize genome stability and lead to severe chronic diseases, such as cancer development. Female organs, such as breast, endometrium, and ovary, exhibiting regular cyclic proliferative activity are particularly vulnerable in case of disturbances in either estrogen signaling or BRCA-mediated DNA repair. BRCA mutation carrier women may apparently be healthy or exhibit clinical signs of deficient estrogen signaling in spite of hyperestrogenism. Even women who enjoy sufficient compensatory DNA-defending activities are at risk of tumor development because many endogenous and environmental factors may jeopardize the mechanisms of extreme compensatory processes. Natural estrogens have numerous benefits in tumor prevention and therapy even in BRCA mutation carriers. There are no toxic effects even in sky-high doses and all physiologic cellular functions are strongly upregulated, while malignant

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

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    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.

  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.

    Science.gov (United States)

    Kellenberger, Roman T; Schlüter, Philipp M; Schiestl, Florian P

    2016-01-01

    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. 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.

  3. Spatial Representativeness of Environmental DNA Metabarcoding Signal for Fish Biodiversity Assessment in a Natural Freshwater System.

    Science.gov (United States)

    Civade, Raphaël; Dejean, Tony; Valentini, Alice; Roset, Nicolas; Raymond, Jean-Claude; Bonin, Aurélie; Taberlet, Pierre; Pont, Didier

    2016-01-01

    In the last few years, the study of environmental DNA (eDNA) has drawn attention for many reasons, including its advantages for monitoring and conservation purposes. So far, in aquatic environments, most of eDNA research has focused on the detection of single species using species-specific markers. Recently, species inventories based on the analysis of a single generalist marker targeting a larger taxonomic group (eDNA metabarcoding) have proven useful for bony fish and amphibian biodiversity surveys. This approach involves in situ filtering of large volumes of water followed by amplification and sequencing of a short discriminative fragment from the 12S rDNA mitochondrial gene. In this study, we went one step further by investigating the spatial representativeness (i.e. ecological reliability and signal variability in space) of eDNA metabarcoding for large-scale fish biodiversity assessment in a freshwater system including lentic and lotic environments. We tested the ability of this approach to characterize large-scale organization of fish communities along a longitudinal gradient, from a lake to the outflowing river. First, our results confirm that eDNA metabarcoding is more efficient than a single traditional sampling campaign to detect species presence, especially in rivers. Second, the species list obtained using this approach is comparable to the one obtained when cumulating all traditional sampling sessions since 1995 and 1988 for the lake and the river, respectively. In conclusion, eDNA metabarcoding gives a faithful description of local fish biodiversity in the study system, more specifically within a range of a few kilometers along the river in our study conditions, i.e. longer than a traditional fish sampling site.

  4. On nanopore DNA sequencing by signal and noise analysis of ionic current.

    Science.gov (United States)

    Wen, Chenyu; Zeng, Shuangshuang; Zhang, Zhen; Hjort, Klas; Scheicher, Ralph; Zhang, Shi-Li

    2016-05-27

    DNA sequencing, i.e., the process of determining the succession of nucleotides on a DNA strand, has become a standard aid in biomedical research and is expected to revolutionize medicine. With the capability of handling single DNA molecules, nanopore technology holds high promises to become speedier in sequencing at lower cost than what are achievable with the commercially available optics- or semiconductor-based massively parallelized technologies. Despite tremendous progress made with biological and solid-state nanopores, high error rates and large uncertainties persist with the sequencing results. Here, we employ a nano-disk model to quantitatively analyze the sequencing process by examining the variations of ionic current when a DNA strand translocates a nanopore. Our focus is placed on signal-boosting and noise-suppressing strategies in order to attain the single-nucleotide resolution. Apart from decreasing pore diameter and thickness, it is crucial to also reduce the translocation speed and facilitate a stepwise translocation. Our best-case scenario analysis points to severe challenges with employing plain nanopore technology, i.e., without recourse to any signal amplification strategy, in achieving sequencing with the desired single-nucleotide resolution. A conceptual approach based on strand synthesis in the nanopore of the translocating DNA from single-stranded to double-stranded is shown to yield a 10-fold signal amplification. Although it involves no advanced physics and is very simple in mathematics, this simple model captures the essence of nanopore sequencing and is useful in guiding the design and operation of nanopore sequencing.

  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. A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround.

    Directory of Open Access Journals (Sweden)

    Mark A Baumeister

    Full Text Available Branched DNA (bDNA is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA ("Versant Assay" currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16-18 h to 2.5 h, composition of only the "Lysis Diluent" solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135 showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.

  7. 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.

  8. 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)

  9. 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.

  10. 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...

  11. Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

    Science.gov (United States)

    Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

    2012-11-09

    To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

  12. Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons*

    Science.gov (United States)

    Huelsenbeck, Stefanie C.; Schorr, Anne; Roos, Wynand P.; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

    2012-01-01

    To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons. PMID:23012366

  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. 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.

  15. Drosophila proteins involved in metabolism of uracil-DNA possess different types of nuclear localization signals.

    Science.gov (United States)

    Merényi, Gábor; Kónya, Emese; Vértessy, Beáta G

    2010-05-01

    Adequate transport of large proteins that function in the nucleus is indispensable for cognate molecular events within this organelle. Selective protein import into the nucleus requires nuclear localization signals (NLS) that are recognized by importin receptors in the cytoplasm. Here we investigated the sequence requirements for nuclear targeting of Drosophila proteins involved in the metabolism of uracil-substituted DNA: the recently identified uracil-DNA degrading factor, dUTPase, and the two uracil-DNA glycosylases present in Drosophila. For the uracil-DNA degrading factor, NLS prediction identified two putative NLS sequences [PEKRKQE(320-326) and PKRKKKR(347-353)]. Truncation and site-directed mutagenesis using YFP reporter constructs showed that only one of these basic stretches is critically required for efficient nuclear localization in insect cells. This segment corresponds to the well-known prototypic NLS of SV40 T-antigen. An almost identical NLS segment is also present in the Drosophila thymine-DNA glycosylase, but no NLS elements were predicted in the single-strand-specific monofunctional uracil-DNA glycosylase homolog protein. This latter protein has a molecular mass of 31 kDa, which may allow NLS-independent transport. For Drosophila dUTPase, two isoforms with distinct features regarding molecular mass and subcellular distribution were recently described. In this study, we characterized the basic PAAKKMKID(10-18) segment of dUTPase, which has been predicted to be a putative NLS by in silico analysis. Deletion studies, using YFP reporter constructs expressed in insect cells, revealed the importance of the PAA(10-12) tripeptide and the ID(17-18) dipeptide, as well as the role of the PAAK(10-13) segment in nuclear localization of dUTPase. We constructed a structural model that shows the molecular basis of such recognition in three dimensions.

  16. Gene delivery: A single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus

    OpenAIRE

    Zanta, Maria Antonietta; Belguise-Valladier, Pascale; Behr, Jean-Paul

    1999-01-01

    Translocation of exogenous DNA through the nuclear membrane is a major concern of gene delivery technologies. To take advantage of the cellular import machinery, we have synthesized a capped 3.3-kbp CMVLuciferase-NLS gene containing a single nuclear localization signal peptide (PKKKRKVEDPYC). Transfection of cells with the tagged gene remained effective down to nanogram amounts of DNA. Transfection enhancement (10- to 1,000-fold) as a result of the signal peptide was observed irrespective of ...

  17. 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.)

  18. Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

    International Nuclear Information System (INIS)

    Park, Iha; Avraham, Hava Karsenty

    2006-01-01

    Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving γ-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation

  19. A novel method using edge detection for signal extraction from cDNA microarray image analysis.

    Science.gov (United States)

    Kim, J H; Kim, H Y; Lee, Y S

    2001-06-30

    Gene expression analyses by probes of hybridization from mRNA to cDNA targets arrayed on membranes or activated glass surfaces have revolutionized the way of profiling mega level gene expression. The main remaining problems however are sensitivity of detection, reproducibility and data processing. During processing of microarray images, especially irregularities of spot position and shape could generate significant errors: small regions of signal spots can be mis-included into background area and vice versa. Here we report a novel method to eliminate such obstacles by sensing their edges. Application of edge detection technology on separating spots from the background decreases the probability of the errors and gives more accurate information about the states of spots such as the pixel number, degree of fragmentation, width and height of spot, and circumference of spot. Such information can be used for the quality control of cDNA microarray experiments and filtering of low quality spots. We analyzed the cDNA microarray image that contains 10,368 genes using edge detection and compared the result with that of conventional method which draws circle around the spot.

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

    Directory of Open Access Journals (Sweden)

    Sune Agersnap

    Full Text Available For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from 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 and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1, including separate assays for two clades of A. leptodactylus. The limit of detection (LOD was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1 comparing traditional and eDNA based estimates of abundance, and 2 representing a broader geographical range for the involved crayfish species.

  1. [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.

  2. Slow conformational changes in MutS and DNA direct ordered transitions between mismatch search, recognition and signaling of DNA repair

    Science.gov (United States)

    Sharma, Anushi; Doucette, Christopher; Biro, F. Noah; Hingorani, Manju M.

    2013-01-01

    MutS functions in mismatch repair (MMR) to scan DNA for errors, identify a target site and trigger subsequent events in the pathway leading to error removal and DNA re-synthesis. These actions, enabled by the ATPase activity of MutS, are now beginning to be analyzed from the perspective of the protein itself. This study provides the first ensemble transient kinetic data on MutS conformational dynamics as it works with DNA and ATP in MMR. Using a combination of fluorescence probes (on T. aquaticus MutS and DNA) and signals (intensity, anisotropy and resonance energy transfer), we have monitored the timing of key conformational changes in MutS that are coupled to mismatch binding and recognition, ATP binding and hydrolysis, as well as sliding clamp formation and signaling of repair. Significant findings include: (a) a slow step that follows weak initial interaction between MutS and DNA, in which concerted conformational changes in both macromolecules control mismatch recognition, (b) rapid, binary switching of MutS conformations that is concerted with ATP binding and hydrolysis, and (c) is stalled after mismatch recognition to control formation of the ATP-bound MutS sliding clamp. These rate-limiting pre- and post-mismatch recognition events outline the mechanism of action of MutS on DNA during initiation of MMR. PMID:23973435

  3. 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...DNA elements thatrespond to T cell activation signals. PubmedID 1492121 Title Activation of lymphokine genes... in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. Authors Arai N, Naito

  4. 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

  5. The first successful observation of in-cell NMR signals of DNA and RNA in living human cells.

    Science.gov (United States)

    Yamaoki, Yudai; Kiyoishi, Ayaka; Miyake, Masayuki; Kano, Fumi; Murata, Masayuki; Nagata, Takashi; Katahira, Masato

    2018-01-31

    In order to understand intracellular biological events, information on the structure, dynamics and interaction of proteins and nucleic acids in living cells is of crucial importance. In-cell NMR is a promising method to obtain this information. Although NMR signals of proteins in human cells have been reported, those of nucleic acids were reported only in Xenopus laevis oocytes, i.e., not in human cells. Here, DNA and RNA were introduced into human cells by means of pore formation by bacterial toxin streptolysin O and subsequent resealing. Then, NMR signals of DNA and RNA were successfully observed for the first time in living human cells. The observed signals directly suggested the formation of DNA and RNA hairpin structures in living human cells.

  6. 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

    Medulloblastomas are the most common, and often fatal, paediatric brain tumours that feature high genomic instability, frequent infection by human cytomegalovirus (HCMV) and resistance to radiation and chemotherapy. The causes of the pronounced chromosomal instability and its potential links...... with HCMV infection and/or resistance to genotoxic therapies remain largely unknown. To address these issues, here we have combined immunohistochemical analysis of a series of 25 paediatric medulloblastomas, complemented by medulloblastoma cell culture models including experimental HCMV infection. Using...... 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...

  7. Bacterial Intoxication Evokes Cellular Senescence with Persistent DNA Damage and Cytokine Signaling

    DEFF Research Database (Denmark)

    Blazkova, Hana; Krejcikova, Katerina; Moudry, Pavel

    2009-01-01

    features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may...... to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic...... phenotype included persistently activated DNA damage signaling (detected as 53BP1/gammaH2AX-positive foci), enhanced senescence-associated beta-galactosidase activity, expansion of PML nuclear compartments, and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall...

  8. Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

    Science.gov (United States)

    Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

    2014-11-26

    Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

  9. Direct visualization of the reaction transformation and signal amplification in a DNA molecular machine with total internal reflection fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Rui eRen

    2013-10-01

    Full Text Available In this study, as a proof of concept, the signal amplification in an artificial DNA molecular machine was directly visualized via total internal reflection fluorescence microscopy (TIRFM. The molecular machine brought about obvious morphology change in DNA nanostructures as well as signal amplifications. On one hand, through a triggered and autonomically repeated RCA, a DNA nano-complex featuring a locked circular DNA template (serving as raw feed was converted into a long periodically repeated strand, i.e. the RCA products. Furthermore, this RCA was repeated in three controllable reaction phases, bring about progressive signal amplification. It was testified that the RCA products (presented as long thread-like fluorescent objects can be easily distinguished from the inputted DNA probes (presented as fluorescent dots, thus the transformation in reaction can be visualized. Also, by quantitive counting of the aforementioned fluorescence objects, the progress of the reaction through the phases, along with time, and over the lysozyme concentration can be demonstrated through TIRFM visualization. Overall, it was demonstrated that TIRFM is an efficient approach to quantitively visualize the biochemical processes at single-molecule level.

  10. 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.

  11. 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.

  12. 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.

  13. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    Science.gov (United States)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  14. 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

  15. Physalin H from Solanum nigrum as an Hh signaling inhibitor blocks GLI1–DNA-complex formation

    Directory of Open Access Journals (Sweden)

    Midori A. Arai

    2014-01-01

    Full Text Available Hedgehog (Hh signaling plays an important role in embryonic development, cell maintenance and cell proliferation. Moreover, Hh signaling contributes to the growth of cancer cells. Physalins are highly oxidized natural products with a complex structure. Physalins (1–7 were isolated from Solanum nigrum (Solanaceae collected in Bangladesh by using our cell-based assay. The isolated physalins included the previously reported Hh inhibitors 5 and 6. Compounds 1 and 4 showed strong inhibition of GLI1 transcriptional activity, and exhibited cytotoxicity against cancer cell lines with an aberrant activation of Hh signaling. Compound 1 inhibited the production of the Hh-related proteins patched (PTCH and BCL2. Analysis of the structures of different physalins showed that the left part of the physalins was important for Hh inhibitory activity. Interestingly, physalin H (1 disrupted GLI1 binding to its DNA binding domain, while the weak inhibitor physalin G (2 did not show inhibition of GLI1-DNA complex formation.

  16. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence

    Energy Technology Data Exchange (ETDEWEB)

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. (Univ. of Washington, Seattle (United States)); Chapline, C.; Crabb, J.W. (W.Alton Jones Cell Science Center, Lake Placid, NY (United States))

    1991-10-22

    Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

  17. Tryptophan-Dependent Control of Colony Formation After DNA Damage via Sea3-Regulated TORC1 Signaling in Saccharomyces cerevisiae.

    Science.gov (United States)

    Polleys, Erica J; Bertuch, Alison A

    2015-05-04

    The Saccharomyces cerevisiae Iml1 complex inhibits TORC1 signaling and SEACAT antagonizes the Iml1 complex. Conditions in which SEACAT functions to inhibit Iml1 and, hence, TORC1 signaling, remain largely unknown. The SEACAT member Sea3 was linked previously to telomere maintenance and DNA repair via genome-wide genetic and physical interaction studies. Therefore, we questioned whether Sea3 functioned through TORC1 to influence these pathways. Deletion of SEA3 delayed the emergence of telomerase-independent survivors that use break-induced replication (BIR) to maintain their telomeres. Similarly, sea3∆ mutants exhibited a delay in colony formation in a BIR assay strain after double-strand break (DSB) induction as well as on the DNA-damaging agent bleomycin. Deletion of IML1 rescued the impaired growth of sea3∆ mutants after DNA damage, consistent with Sea3 functioning as a regulator of TORC1 signaling. The delay was not attributable to slowed DSB repair or termination of the DNA damage checkpoint but to tryptophan auxotrophy. High levels of tryptophan in yeast peptone dextrose media did not rescue the delay in colony formation, suggesting a defect in tryptophan import, although levels of the high-affinity tryptophan permease Tat2 were not perturbed in the sea3Δ mutant. Addition of quinolinic acid, an intermediate of the de novo NAD+ biosynthetic pathway, however, rescued the delay in colony formation in the sea3Δ mutant. Together, these findings highlight the importance of enforcement of TORC1 signaling and suggest that internal tryptophan levels influence growth recovery post DNA damage through the role of tryptophan in NAD+ synthesis. Copyright © 2015 Polleys and Bertuch.

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

    International Nuclear Information System (INIS)

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala; Glas, Rickard

    2010-01-01

    Research highlights: → Nuclear translocation of TPPII occurs in response to different DNA damage inducers. → Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. → MAPKs control nuclear accumulation of TPPII. → Inhibited nuclear accumulation of TPPII decreases DNA damage-induced γ-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 γ-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 γ-hexa-chloro-cyclohexane (γ-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon γ-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 γ-H2AX in γ-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  19. 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.

  20. 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.

  1. 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.

  2. Fluorescence Regulation of Copper Nanoclusters via DNA Template Manipulation toward Design of a High Signal-to-Noise Ratio Biosensor.

    Science.gov (United States)

    Li, Junyao; Fu, Wenxin; Bao, Jianchun; Wang, Zhaoyin; Dai, Zhihui

    2018-02-28

    Because of bioaccumulation of food chain and disability of biodegradation, concentration of toxic mercury ions (Hg 2+ ) in the environment dramatically varies from picomolar to micromolar, indicating the importance of well-performed Hg 2+ analytical methods. Herein, reticular DNA is constructed by introducing thymine (T)-Hg 2+ -T nodes in poly(T) DNA, and copper nanoclusters (CuNCs) with aggregate morphology are prepared using this reticular DNA as a template. Intriguingly, the prepared CuNCs exhibit enhanced fluorescence. Meanwhile, the reticular DNA reveals evident resistance to enzyme digestion, further clarifying the fluorescence enhancement of CuNCs. Relying on the dual function of DNA manipulation, a high signal-to-noise ratio biosensor is designed. This analytical approach can quantify Hg 2+ in a very wide range (50 pM to 500 μM) with an ultralow detection limit (16 pM). Besides, depending on the specific interaction between Hg 2+ and reduced l-glutathione (GSH), this biosensor is able to evaluate the inhibition of GSH toward Hg 2+ . In addition, pollution of Hg 2+ in three lakes is tested using this method, and the obtained results are in accord with those from inductively coupled plasma mass spectrometry. In general, this work provides an alternative way to regulate the properties of DNA-templated nanomaterials and indicates the applicability of this way by fabricating an advanced biosensor.

  3. 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

  4. 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...

  5. Hypersensitivity of primordial germ cells to compromised replication-associated DNA repair involves ATM-p53-p21 signaling.

    Directory of Open Access Journals (Sweden)

    Yunhai Luo

    2014-07-01

    Full Text Available Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs, which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA, a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.

  6. 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.

  7. 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...

  8. Exposure to welding fumes activates DNA damage response and redox-sensitive transcription factor signalling in Sprague-Dawley rats.

    Science.gov (United States)

    Krishnaraj, Jayaraman; Kowshik, Jaganathan; Sebastian, Robin; Raghavan, Sathees C; Nagini, Siddavaram

    2017-05-15

    Occupational exposure to welding fumes containing a complex mixture of genotoxic heavy metals, radiation, gases and nanoparticles poses a serious health hazard to welders. Since their categorization as possible carcinogens, welding fumes have gained increasing attention as high priority agents for risk assessment. The present study was undertaken to investigate the effects of welding fume inhalation on oxidative stress, DNA damage response (DDR), and nuclear factor erythroid 2-related factor-2 (Nrf2) and nuclear factor kappa B (NFκB) signalling in the lung tissues of male Sprague-Dawley rats . METHODS: Animals were divided into five groups. Group 1 animals served as control. Rats in groups 2-5 were exposed to 50mg/m 3 stainless steel (SS) welding fumes for 1h for 1day, 1 week, 2 weeks, and 4 weeks respectively. Reactive oxygen species (ROS) generation, 8-oxo-2'-deoxyguanosine (8-oxodG), xenobiotic-metabolizing enzymes (XMEs) and antioxidants were analysed. DNA damage sensors, DNA repair enzymes, inflammatory mediators, cell cycle progression, apoptosis and key players in Nrf2 and NFκB signalling were assessed by flow cytometry, quantitative real-time reverse transcriptase PCR, immunoblotting, immunohistochemistry and immunofluorescence. Rats exposed to welding fumes showed increased levels of chromium and ROS in lung tissues associated with accumulation of 8-oxodG and enhanced expression of XMEs and antioxidants. This was accompanied by upregulation of DNA damage sensors, cell cycle arrest in G1/S phase, overexpression of a multitude of DNA repair enzymes and caspase-mediated apoptosis. In addition, exposure to welding fumes induced activation of Nrf2 and NFκB signalling with enhanced expression of inflammatory mediators. The results of the present study unequivocally demonstrate that exposure of rats to SS welding fumes alters the expression of 37 genes involved in oxidative stress, detoxification, inflammation, DNA repair, cell cycle progression, and apoptosis

  9. 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.

  10. 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.

  11. The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage

    DEFF Research Database (Denmark)

    Stewart, Grant S.; Panier, Stephanie; Townsend, Kelly

    2009-01-01

    The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such di...

  12. 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.

  13. 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)

  14. Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: A review

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shaohong; Yuan, Liang; Hua, Xin; Xu, Lingling; Liu, Songqin, E-mail: liusq@seu.edu.cn

    2015-06-02

    Highlights: • We review the innovative advances in polymer-based signal amplification. • Conceptual connectivity between different amplified methodologies is illustrated. • Examples explain the mechanisms of polymers/polymerizations-based amplification. • Several elegant applications are summarized that illustrate underlying concept. - Abstract: Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted.

  15. 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...

  16. The role of the epigenetic signal, DNA methylation, in gene regulation during erythroid development.

    Science.gov (United States)

    Ginder, Gordon D; Gnanapragasam, Merlin N; Mian, Omar Y

    2008-01-01

    The sequence complexity of the known vertebrate genomes alone is insufficient to account for the diversity between individuals of a species. Although our knowledge of vertebrate biology has evolved substantially with the growing compilation of sequenced genomes, understanding the temporal and spatial regulation of genes remains fundamental to fully exploiting this information. The importance of epigenetic factors in gene regulation was first hypothesized decades ago when biologists posited that methylation of DNA could heritably alter gene expression [Holliday and Pugh, 1975. Science 187(4173), 226-232; Riggs, 1975. Cytogenet. and Cell Genet.14(1), 9-25; Scarano et al., 1967. Proc. Natl. Acad. Sci. USA 57(5), 1394-1400)]. It was subsequently shown that vertebrate DNA methylation, almost exclusively at the 5' position of cytosine in the dinucleotide CpG, played a role in a number of processes including embryonic development, genetic imprinting, cell differentiation, and tumorigenesis. At the time of this writing, a large and growing list of genes is known to exhibit DNA methylation-dependent regulation, and we understand in some detail the mechanisms employed by cells in using methylation as a regulatory modality. In this context, we revisit one of the original systems in which the role of DNA methylation in vertebrate gene regulation during development was described and studied: erythroid cells. We briefly review the recent advances in our understanding of DNA methylation and, in particular, its regulatory role in red blood cells during differentiation and development. We also address DNA methylation as a component of erythroid chromatin architecture, and the interdependence of CpG methylation and histone modification.

  17. 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...

  18. 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...

  19. Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation.

    Science.gov (United States)

    Naumann, Marcel; Pal, Arun; Goswami, Anand; Lojewski, Xenia; Japtok, Julia; Vehlow, Anne; Naujock, Maximilian; Günther, René; Jin, Mengmeng; Stanslowsky, Nancy; Reinhardt, Peter; Sterneckert, Jared; Frickenhaus, Marie; Pan-Montojo, Francisco; Storkebaum, Erik; Poser, Ina; Freischmidt, Axel; Weishaupt, Jochen H; Holzmann, Karlheinz; Troost, Dirk; Ludolph, Albert C; Boeckers, Tobias M; Liebau, Stefan; Petri, Susanne; Cordes, Nils; Hyman, Anthony A; Wegner, Florian; Grill, Stephan W; Weis, Joachim; Storch, Alexander; Hermann, Andreas

    2018-01-23

    Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.

  20. Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair.

    Directory of Open Access Journals (Sweden)

    Qi Wu

    2008-09-01

    Full Text Available DNA interstrand crosslinks (ICLs are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSalpha and MutSbeta mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents.

  1. 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

  2. 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.

  3. The DNA damage response and immune signaling alliance: Is it good or bad? Nature decides when and where.

    Science.gov (United States)

    Pateras, Ioannis S; Havaki, Sophia; Nikitopoulou, Xenia; Vougas, Konstantinos; Townsend, Paul A; Panayiotidis, Michalis I; Georgakilas, Alexandros G; Gorgoulis, Vassilis G

    2015-10-01

    The characteristic feature of healthy living organisms is the preservation of homeostasis. Compelling evidence highlight that the DNA damage response and repair (DDR/R) and immune response (ImmR) signaling networks work together favoring the harmonized function of (multi)cellular organisms. DNA and RNA viruses activate the DDR/R machinery in the host cells both directly and indirectly. Activation of DDR/R in turn favors the immunogenicity of the incipient cell. Hence, stimulation of DDR/R by exogenous or endogenous insults triggers innate and adaptive ImmR. The immunogenic properties of ionizing radiation, a prototypic DDR/R inducer, serve as suitable examples of how DDR/R stimulation alerts host immunity. Thus, critical cellular danger signals stimulate defense at the systemic level and vice versa. Disruption of DDR/R-ImmR cross talk compromises (multi)cellular integrity, leading to cell-cycle-related and immune defects. The emerging DDR/R-ImmR concept opens up a new avenue of therapeutic options, recalling the Hippocrates quote "everything in excess is opposed by nature." Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Phosphates in the Z-DNA dodecamer are flexible, but their P-SAD signal is sufficient for structure solution.

    Science.gov (United States)

    Luo, Zhipu; Dauter, Miroslawa; Dauter, Zbigniew

    2014-07-01

    A large number of Z-DNA hexamer duplex structures and a few oligomers of different lengths are available, but here the first crystal structure of the d(CGCGCGCGCGCG)2 dodecameric duplex is presented. Two synchrotron data sets were collected; one was used to solve the structure by the single-wavelength anomalous dispersion (SAD) approach based on the anomalous signal of P atoms, the other set, extending to an ultrahigh resolution of 0.75 Å, served to refine the atomic model to an R factor of 12.2% and an R(free) of 13.4%. The structure consists of parallel duplexes arranged into practically infinitely long helices packed in a hexagonal fashion, analogous to all other known structures of Z-DNA oligomers. However, the dodecamer molecule shows a high level of flexibility, especially of the backbone phosphate groups, with six out of 11 phosphates modeled in double orientations corresponding to the two previously observed Z-DNA conformations: Z(I), with the phosphate groups inclined towards the inside of the helix, and Z(II), with the phosphate groups rotated towards the outside of the helix.

  5. 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/.

  6. 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.

  7. 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...

  8. 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.

  9. Proximity hybridization triggered hemin/G-quadruplex formation for construction a label-free and signal-on electrochemical DNA sensor.

    Science.gov (United States)

    Gao, Fenglei; Fan, Taotao; Wu, Jing; Liu, Sha; Du, Yan; Yao, Yao; Zhou, Fuyi; Zhang, Yu; Liao, Xianjiu; Geng, Deqin

    2017-10-15

    We describe a novel label-free and signal-on electrochemical DNA sensing platform via proximity hybridization triggered hemin/G-quadruplex formation based on the direct electron transfer of hemin. The thiolated modified G-DNA1 was first immobilized onto the Au electrode surface. In the presence of target DNA, Y-junction-structure ternary complex can be formed to trigger the proximity assembly of G-DNA1, hemin, and G-DNA2, which leads to the formation of hemin/G-quadruplex for generation an amplified electrochemical signal by differential pulse voltammetry. The observed signal gain was sufficient to achieve a demonstrated detection limit of 54 fM, with a wide linear dynamic range from 10 -13 to 10 -9 M and discriminated mismatched DNA from perfect matched target DNA with a high selectivity. Benefiting from the one step proximity dependent hemin/G-quadruplex formation, the assay method is extremely simple and can be carried out within 40min. The advantages of free of any label conjugation step, and versatility make it a promising candidate for point-of-care testing and commercial application. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. 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

  11. 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.

  12. Recovering phylogenetic signal from DNA sequences: relationships within the corvine assemblage (class aves) as inferred from complete sequences of the mitochondrial DNA cytochrome-b gene.

    Science.gov (United States)

    Helm-Bychowski, K; Cracraft, J

    1993-11-01

    Phylogenetic analysis of cytochrome-b sequences and cranial osteological characters for nine genera of corvine passerine birds supports the hypothesis that the two major groups of birds of paradise, the manucodines and paradisaeinines, constitute a monophyletic group and that their postulated sister group is the Corvidae (crows, jays, and allies). The data are also consistent with the hypothesis that the bowerbirds are not closely related to the birds of paradise but instead lie near the base of the corvine assemblage. The corvine radiation exemplifies a case of multiple star phylogenies embedded within a major clade, with the branching pattern characterized by very short internodal divergence times. Such histories are difficult to resolve no matter what type of data is employed, because little change accumulates between branching events. With respect to sequence data, reconstructed tree topologies are sensitive to the choice of outgroup and to the method of analysis (e.g., transversion vs. global parsimony). In such cases, assessing the "reliability" of a best-fit or most-parsimonious tree inferred from any particular data set becomes problematic. Statistical tests of tree topologies that depend on random sampling of characters will generally be inconclusive in that all cladistic components will tend to be poorly supported because relatively few character-state changes will be recorded between branching events. It is suggested, on the other hand, that congruence in cladistic signal across different data sets may be a potentially more useful method for evaluating the reliability of the signal of any one data set. Resolution of star phylogenies will probably be possible only if DNA sequence and morphological characters are combined in a single analysis.

  13. 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.

  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. 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.

  16. New Insights into p53 Signaling and Cancer Cell Response to DNA Damage: Implications for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Razmik Mirzayans

    2012-01-01

    Full Text Available Activation of the p53 signaling pathway by DNA-damaging agents was originally proposed to result either in cell cycle checkpoint activation to promote survival or in apoptotic cell death. This model provided the impetus for numerous studies focusing on the development of p53-based cancer therapies. According to recent evidence, however, most p53 wild-type human cell types respond to ionizing radiation by undergoing stress-induced premature senescence (SIPS and not apoptosis. SIPS is a sustained growth-arrested state in which cells remain viable and secrete factors that may promote cancer growth and progression. The p21WAF1 (hereafter p21 protein has emerged as a key player in the p53 pathway. In addition to its well-studied role in cell cycle checkpoints, p21 regulates p53 and its upstream kinase (ATM, controls gene expression, suppresses apoptosis, and induces SIPS. Herein, we review these and related findings with human solid tumor-derived cell lines, report new data demonstrating dynamic behaviors of p53 and p21 in the DNA damage response, and examine the gain-of-function properties of cancer-associated p53 mutations. We point out obstacles in cancer-therapeutic strategies that are aimed at reactivating the wild-type p53 function and highlight some alternative approaches that target the apoptotic threshold in cancer cells with differing p53 status.

  17. 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.

  18. Disruption of GRM1-mediated signalling using riluzole results in DNA damage in melanoma cells.

    Science.gov (United States)

    Wall, Brian A; Wangari-Talbot, Janet; Shin, Seung S; Schiff, Devora; Sierra, Jairo; Yu, Lumeng J; Khan, Atif; Haffty, Bruce; Goydos, James S; Chen, Suzie

    2014-03-01

    Gain of function of the neuronal receptor, metabotropic glutamate receptor 1 (Grm1), was sufficient to induce melanocytic transformation in vitro and spontaneous melanoma development in vivo when ectopically expressed in melanocytes. The human form of this receptor, GRM1, has been shown to be ectopically expressed in a subset of human melanomas but not benign nevi or normal melanocytes, suggesting that misregulation of GRM1 is involved in the pathogenesis of certain human melanomas. Sustained stimulation of Grm1 by the ligand, glutamate, is required for the maintenance of transformed phenotypes in vitro and tumorigenicity in vivo. In this study, we investigate the mechanism of an inhibitor of glutamate release, riluzole, on human melanoma cells that express metabotropic glutamate receptor 1 (GRM1). Various in vitro assays conducted show that inhibition of glutamate release in several human melanoma cell lines resulted in an increase of oxidative stress and DNA damage response markers. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. 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.

  20. 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.

  1. Expression of Caveolin 1 is enhanced by DNA demethylation during adipocyte differentiation. status of insulin signaling.

    Science.gov (United States)

    Palacios-Ortega, Sara; Varela-Guruceaga, Maider; Milagro, Fermín Ignacio; Martínez, José Alfredo; de Miguel, Carlos

    2014-01-01

    Caveolin 1 (Cav-1) is an essential constituent of adipocyte caveolae which binds the beta subunit of the insulin receptor (IR) and is implicated in the regulation of insulin signaling. We have found that, during adipocyte differentiation of 3T3-L1 cells the promoter, exon 1 and first intron of the Cav-1 gene undergo a demethylation process that is accompanied by a strong induction of Cav-1 expression, indicating that epigenetic mechanisms must have a pivotal role in this differentiation process. Furthermore, IR, PKB-Akt and Glut-4 expression are also increased during the differentiation process suggesting a coordinated regulation with Cav-1. Activation of Cav-1 protein by phosphorylation arises during the differentiation process, yet in fully mature adipocytes insulin is no longer able to significantly increase Cav-1 phosphorylation. However, these long-term differentiated cells are still able to respond adequately to insulin, increasing IR and PKB-Akt phosphorylation and glucose uptake. The activation of Cav-1 during the adipocyte differentiation process could facilitate the maintenance of insulin sensitivity by these fully mature adipocytes isolated from additional external stimuli. However, under the influence of physiological conditions associated to obesity, such as chronic inflammation and hypoxia, insulin sensitivity would finally be compromised.

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

    Science.gov (United States)

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Genome-wide analysis of the nucleus accumbens identifies DNA methylation signals differentiating low/binge from heavy alcohol drinking.

    Science.gov (United States)

    Cervera-Juanes, Rita; Wilhelm, Larry J; Park, Byung; Grant, Kathleen A; Ferguson, Betsy

    2017-05-01

    Alcohol-use disorders encompass a range of drinking levels and behaviors, including low, binge, and heavy drinking. In this regard, investigating the neural state of individuals who chronically self-administer lower doses of alcohol may provide insight into mechanisms that prevent the escalation of alcohol use. DNA methylation is one of the epigenetic mechanisms that stabilizes adaptations in gene expression and has been associated with alcohol use. Thus, we investigated DNA methylation, gene expression, and the predicted neural effects in the nucleus accumbens core (NAcc) of male rhesus macaques categorized as "low" or "binge" drinkers, compared to "alcohol-naïve" and "heavy" drinkers based on drinking patterns during a 12-month alcohol self-administration protocol. Using genome-wide CpG-rich region enrichment and bisulfite sequencing, the methylation levels of 2.6 million CpGs were compared between alcohol-naïve (AN), low/binge (L/BD), and heavy/very heavy (H/VHD) drinking subjects (n = 24). Through regional clustering analysis, we identified nine significant differential methylation regions (DMRs) that specifically distinguished ANs and L/BDs, and then compared those DMRs among H/VHDs. The DMRs mapped to genes encoding ion channels, receptors, cell adhesion molecules, and cAMP, NF-κβ and Wnt signaling pathway proteins. Two of the DMRs, linked to PDE10A and PKD2L2, were also differentially methylated in H/VHDs, suggesting an alcohol-dose independent effect. However, two other DMRs, linked to the CCBE1 and FZD5 genes, had L/BD methylation levels that significantly differed from both ANs and H/VHDs. The remaining five DMRs also differentiated L/BDs and ANs. However, H/VHDs methylation levels were not distinguishable from either of the two groups. Functional validation of two DMRs, linked to FZD5 and PDE10A, support their role in regulating gene expression and exon usage, respectively. In summary, the findings demonstrate that L/BD is associated with unique

  4. Global DNA cytosine methylation as an evolving trait: phylogenetic signal and correlated evolution with genome size in Angiosperms

    Directory of Open Access Journals (Sweden)

    Conchita eAlonso

    2015-01-01

    Full Text Available DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value. Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis and 39.2% (Narcissus. Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages.

  5. 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.

  6. The ER-associated protein ZDHHC1 is a positive regulator of DNA virus-triggered, MITA/STING-dependent innate immune signaling.

    Science.gov (United States)

    Zhou, Qian; Lin, Heng; Wang, Suyun; Wang, Shuai; Ran, Yong; Liu, Ying; Ye, Wen; Xiong, Xiaozhe; Zhong, Bo; Shu, Hong-Bing; Wang, Yan-Yi

    2014-10-08

    Viral DNA sensing within the cytosol of infected cells activates type I interferon (IFN) expression. MITA/STING plays an essential role in this pathway by acting as both a sensor for the second messenger cGAMP and as an adaptor for downstream signaling components. In an expression screen for proteins that can activate the IFNB1 promoter, we identified the ER-associated protein ZDHHC1 as a positive regulator of virus-triggered, MITA/STING-dependent immune signaling. Zdhhc1(-/-) cells failed to effectively produce IFNs and other cytokines in response to infection with DNA but not RNA viruses. Zdhhc1(-/-) mice infected with the neurotropic DNA virus HSV-1 exhibited lower cytokine levels and higher virus titers in the brain, resulting in higher lethality. ZDHHC1 constitutively associated with MITA/STING and mediates dimerization/aggregation of MITA/STING and recruitment of the downstream signaling components TBK1 and IRF3. These findings support a role for ZDHHC1 in mediating MITA/STING-dependent innate immune response against DNA viruses. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. 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.

  8. RAG-mediated DNA double-strand breaks activate a cell type–specific checkpoint to inhibit pre–B cell receptor signals

    Science.gov (United States)

    Bednarski, Jeffrey J.; Pandey, Ruchi; Schulte, Emily; White, Lynn S.; Chen, Bo-Ruei; Sandoval, Gabriel J.; Kohyama, Masako; Haldar, Malay; Nickless, Andrew; Trott, Amanda; Cheng, Genhong; Murphy, Kenneth M.; Bassing, Craig H.; Payton, Jacqueline E.

    2016-01-01

    DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes. PMID:26834154

  9. 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.

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

    Science.gov (United States)

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

    2017-10-01

    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.

  11. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

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    Renfeng Li

    2015-12-01

    Full Text Available Epstein-Barr virus (EBV is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1. Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR, mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been

  12. Effect of tetrahedral DNA nanostructures on proliferation and osteo/odontogenic differentiation of dental pulp stem cells via activation of the notch signaling pathway.

    Science.gov (United States)

    Zhou, Mi; Liu, Nan-Xin; Shi, Si-Rong; Li, Yong; Zhang, Qi; Ma, Quan-Quan; Tian, Tao-Ran; Ma, Wen-Juan; Cai, Xiao-Xiao; Lin, Yun-Feng

    2018-02-17

    Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration. Copyright © 2018. Published by Elsevier Inc.

  13. 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

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

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    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.

  15. 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.

  16. 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.

  17. 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

  18. Irreparable telomeric DNA damage and persistent DDR signalling as a shared causative mechanism of cellular senescence and ageing.

    Science.gov (United States)

    Rossiello, Francesca; Herbig, Utz; Longhese, Maria Pia; Fumagalli, Marzia; d'Adda di Fagagna, Fabrizio

    2014-06-01

    The DNA damage response (DDR) orchestrates DNA repair and halts cell cycle. If damage is not resolved, cells can enter into an irreversible state of proliferative arrest called cellular senescence. Organismal ageing in mammals is associated with accumulation of markers of cellular senescence and DDR persistence at telomeres. Since the vast majority of the cells in mammals are non-proliferating, how do they age? Are telomeres involved? Also oncogene activation causes cellular senescence due to altered DNA replication and DDR activation in particular at the telomeres. Is there a common mechanism shared among apparently distinct types of cellular senescence? And what is the role of telomeric DNA damage? Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. 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.

  20. 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.

  1. Accumulation of amplified target DNAs using thiol/biotin labeling, S1 nuclease, and ferrocene–streptavidin–magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

    Directory of Open Access Journals (Sweden)

    Piyasak Chaumpluk et al

    2007-01-01

    Full Text Available Combinations of PCR-based amplification platform using 5' thiolated and biotinylated specific primers, S1 nuclease–PCR products treatment, ferrocene–streptavidin (Fc–Stv–magnetic binding for DNA accumulation, and screen printed gold electrode for the DNA allocation, were applied to Hoechst 33258-induced DNA aggregation and signals induction system for direct signals detection and DNA quantification in food samples. Thiolated and biotinylated at each 5' terminus enabled DNA purification through S1 nuclease treatment for primers and non-specific DNA elimination and enabled DNA trapping with a ferrocene–streptavidin–magnetic system. This facilitated the accumulation of target DNAs at higher concentration, resulting in enhanced signals. After allocation of DNA on the surface of gold electrode via thiol binding, intensity of DNA signals through these treatments could be measured directly after being induced by Hoechst 33258. Wider amplitude changes in anodic current peaks between negative and positive samples (increasing from 3.70 to 10.10 μA compared with those applied with no treatment combinations (decreasing from 3.92 to 1.23 μA were observed. This enhancement of the signals allowed a greater efficiency of DNA quantification. When this combination was used for GMOs content estimation in reference samples, results revealed an improved accuracy from 66% to 96%. The combined biosensor system, although more costly than the standard Hoechst 33258/carbon electrode system, provided an alternative choice for DNA quantification, offering labor-free immobilization of probe onto electrode surface, easy test administration, and efficient semi-quantitative test without expensive instruments.

  2. 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.

  3. IL-1alpha is a DNA damage sensor linking genotoxic stress signaling to sterile inflammation and innate immunity

    NARCIS (Netherlands)

    Idan, C.; Peleg, R.; Elena, V.; Martin, T.; Cicerone, T.; Mareike, W.; Lydia, B.; Marina, F.; Gerhard, M.; Elisa, F.M.; Dinarello, C.A.; Ron, A.N.; Robert, S.

    2015-01-01

    Environmental signals can be translated into chromatin changes, which alter gene expression. Here we report a novel concept that cells can signal chromatin damage from the nucleus back to the surrounding tissue through the cytokine interleukin-1alpha (IL-1alpha). Thus, in addition to its role as a

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

    Science.gov (United States)

    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 O6meG: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. PMID:23391514

  5. 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...

  6. 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

  7. 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

  8. 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

  9. 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)

  10. 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

  11. 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.

  12. 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

  13. Why Human Papillomaviruses Activate the DNA Damage Response (DDR) and How Cellular and Viral Replication Persists in the Presence of DDR Signaling.

    Science.gov (United States)

    Bristol, Molly L; Das, Dipon; Morgan, Iain M

    2017-09-21

    Human papillomaviruses (HPV) require the activation of the DNA damage response (DDR) in order to undergo a successful life cycle. This activation presents a challenge for the virus and the infected cell: how does viral and host replication proceed in the presence of a DDR that ordinarily arrests replication; and how do HPV16 infected cells retain the ability to proliferate in the presence of a DDR that ordinarily arrests the cell cycle? This raises a further question: why do HPV activate the DDR? The answers to these questions are only partially understood; a full understanding could identify novel therapeutic strategies to target HPV cancers. Here, we propose that the rapid replication of an 8 kb double stranded circular genome during infection creates aberrant DNA structures that attract and activate DDR proteins. Therefore, HPV replication in the presence of an active DDR is a necessity for a successful viral life cycle in order to resolve these DNA structures on viral genomes; without an active DDR, successful replication of the viral genome would not proceed. We discuss the essential role of TopBP1 in this process and also how viral and cellular replication proceeds in HPV infected cells in the presence of DDR signals.

  14. 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.

  15. Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

    Science.gov (United States)

    Nones, Katia; Waddell, Nic; Song, Sarah; Patch, Ann-Marie; Miller, David; Johns, Amber; Wu, Jianmin; Kassahn, Karin S; Wood, David; Bailey, Peter; Fink, Lynn; Manning, Suzanne; Christ, Angelika N; Nourse, Craig; Kazakoff, Stephen; Taylor, Darrin; Leonard, Conrad; Chang, David K; Jones, Marc D; Thomas, Michelle; Watson, Clare; Pinese, Mark; Cowley, Mark; Rooman, Ilse; Pajic, Marina; Butturini, Giovanni; Malpaga, Anna; Corbo, Vincenzo; Crippa, Stefano; Falconi, Massimo; Zamboni, Giuseppe; Castelli, Paola; Lawlor, Rita T; Gill, Anthony J; Scarpa, Aldo; Pearson, John V; Biankin, Andrew V; Grimmond, Sean M

    2014-09-01

    The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy. The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

  16. 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.

  17. Large-scale genomic analyses link reproductive aging to hypothalamic signaling, breast cancer susceptibility and BRCA1-mediated DNA repair

    DEFF Research Database (Denmark)

    Day, Felix R; Ruth, Katherine S; Thompson, Deborah J

    2015-01-01

    Menopause timing has a substantial impact on infertility and risk of disease, including breast cancer, but the underlying mechanisms are poorly understood. We report a dual strategy in ∼70,000 women to identify common and low-frequency protein-coding variation associated with age at natural...... lifespan. Pathway analyses identified major association with DNA damage response (DDR) genes, including the first common coding variant in BRCA1 associated with any complex trait. Mendelian randomization analyses supported a causal effect of later ANM on breast cancer risk (∼6% increase in risk per year; P...

  18. 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.

  19. A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents.

    Science.gov (United States)

    Low, Kim-Fatt; Zain, Zainiharyati Mohd; Yean, Chan Yean

    2017-01-15

    A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R 2 =0.992) and log 10 (1-10 4 CFU/ml) (R 2 =0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. 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.

  1. Protection by taurine against INOS-dependent DNA damage in heavily exercised skeletal muscle by inhibition of the NF-κB signaling pathway.

    Science.gov (United States)

    Sugiura, Hiromichi; Okita, Shinya; Kato, Toshihiro; Naka, Toru; Kawanishi, Shosuke; Ohnishi, Shiho; Oshida, Yoshiharu; Ma, Ning

    2013-01-01

    Taurine protects against tissue damage in a variety of models involving inflammation, especially the muscle. We set up a heavy exercise bout protocol for rats consisting of climbing ran on a treadmill to examine the effect of an intraabdominal dose of taurine (300 mg/kg/day) administered 1 h before heavy exercise for ten consecutive days. Each group ran on the treadmill at 20 m/min, 25% grade, for 20 min or until exhaustion within 20 min once each 10 days. Exhaustion was the point when an animal was unable to right itself when placed on its side. The muscle damage was associated with an increased accumulation of 8-nitroguanine and 8-OHdG in the nuclei of skeletal muscle cells. The immunoreactivities for NF-κB and iNOS were also increased in the exercise group. Taurine ameliorated heavy exercise-induced muscle DNA damage to a significant extent since it reduced the accumulation of 8-nitroguanine and 8-OHdG, possibly by down-regulating the expression of iNOS through a modulatory action on NF-κB signaling pathway. This study demonstrates for the first time that taurine can protect against intense exercise-induced nitrosative inflammation and ensuing DNA damage in the skeletal muscle of rats by preventing iNOS expression and the nitrosative stress generated by heavy exercise.

  2. 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.

  3. Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses.

    Science.gov (United States)

    Biacchesi, Stéphane; LeBerre, Monique; Lamoureux, Annie; Louise, Yoann; Lauret, Emilie; Boudinot, Pierre; Brémont, Michel

    2009-08-01

    Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.

  4. 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)

  5. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  6. 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)

  7. Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling.

    Science.gov (United States)

    Kobayashi, Alisa; Tengku Ahmad, Tengku Ahbrizal Farizal; Autsavapromporn, Narongchai; Oikawa, Masakazu; Homma-Takeda, Shino; Furusawa, Yoshiya; Wang, Jun; Konishi, Teruaki

    2017-10-01

    Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) "targeted"/irradiated A549-GFP cells; (2) "neighboring"/non-irradiated cells directly contacting the "targeted" cells; and (3) "distant"/non-irradiated cells, which were not in direct contact with the "targeted" cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi

  8. 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

  9. When mtDNA COI is misleading: congruent signal of ITS2 molecular marker and morphology for North European Melanostoma Schiner, 1860 (Diptera, Syrphidae

    Directory of Open Access Journals (Sweden)

    Antti Haarto

    2014-08-01

    Full Text Available The northern European taxa of genus Melanostoma Schiner, 1860 (Syrphidae, Diptera are revised. A longstanding question concerning the number of Melanostoma taxa occurring in northern Europe prompted us to contrast and compare their morphological and molecular variability. Particular uncertainty concerned the putative existence of a sibling species of Melanostoma mellinum, and the identity of the taxon Melanostoma dubium in northern Europe due to existence of morphologically similar dark forms of M. mellinum in the northern parts of its distributional range. Partial sequences of two DNA markers, the mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI-3' and the nuclear second internal transcribed spacer (ITS2 were analysed separately under parsimony. The obtained COI-3'gene fragment showed taxon-specific haplotypes and haplotypes that were shared among the taxa. The ITS2 sequences presented genotypes unique to each species, and congruence with our independently established taxonomic entities. Based on congruent signal of the ITS2 sequences and study of morphological characters we establish the presence of four taxa in northern Europe: Melanostoma mellium (= M. dubium nec auctt., syn. n., M. certum sp. n. (= M. dubium auctt., M. mellarium stat. n. (= M. mellinum auctt. partim and M. scalare. Lectotype designations were made for Musca mellina, Syrphus mellarius and Melanostoma mellinum var. melanatus.The following synonymies were established: Melanostoma mellarium = Melanostoma melanatum syn. n.; Melanostoma mellinum = Scaeva dubia syn. n., Melanostoma tschernovi syn. n., and Melanostoma clausseni syn. n. Morphological circumscriptions of the taxa and an identification key are presented.

  10. 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

  11. 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...

  12. Critical Role of TGF-β and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation.

    Science.gov (United States)

    Freudenberg, Kristin; Lindner, Nadja; Dohnke, Sebastian; Garbe, Annette I; Schallenberg, Sonja; Kretschmer, Karsten

    2018-01-01

    Under physiological conditions, CD4 + regulatory T (Treg) cells expressing the transcription factor Foxp3 are generated in the thymus [thymus-derived Foxp3 + Treg (tTregs) cells] and extrathymically at peripheral sites [peripherally induced Foxp3 + Treg (pTreg) cell], and both developmental subsets play non-redundant roles in maintaining self-tolerance throughout life. In addition, a variety of experimental in vitro and in vivo modalities can extrathymically elicit a Foxp3 + Treg cell phenotype in peripheral CD4 + Foxp3 - T cells, which has attracted much interest as an approach toward cell-based therapy in clinical settings of undesired immune responses. A particularly notable example is the in vitro induction of Foxp3 expression and Treg cell activity (iTreg cells) in initially naive CD4 + Foxp3 - T cells through T cell receptor (TCR) and IL-2R ligation, in the presence of exogenous TGF-β. Clinical application of Foxp3 + iTreg cells has been hampered by the fact that TGF-β-driven Foxp3 induction is not sufficient to fully recapitulate the epigenetic and transcriptional signature of in vivo induced Foxp3 + tTreg and pTreg cells, which includes the failure to imprint iTreg cells with stable Foxp3 expression. This hurdle can be potentially overcome by pharmacological interference with DNA methyltransferase activity and CpG methylation [e.g., by the cytosine nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC)] to stabilize TGF-β-induced Foxp3 expression and to promote a Foxp3 + iTreg cell phenotype even in the absence of added TGF-β. However, the molecular mechanisms of 5-aza-dC-mediated Foxp3 + iTreg cell generation have remained incompletely understood. Here, we show that in the absence of exogenously added TGF-β and IL-2, efficient 5-aza-dC-mediated Foxp3 + iTreg cell generation from TCR-stimulated CD4 + Foxp3 - T cells is critically dependent on TGF-βR and IL-2R signaling and that this process is driven by TGF-β and IL-2, which could either be FCS derived

  13. 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

  14. 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

  15. 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.

  16. 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

  17. Low folate metabolic stress reprograms DNA methylation-activated sonic hedgehog signaling to mediate cancer stem cell-like signatures and invasive tumour stage-specific malignancy of human colorectal cancers.

    Science.gov (United States)

    Feng, Hsin-Chun; Lin, Jhuan-Yu; Hsu, Shu-Han; Lan, Wen-Yu; Kuo, Chang-Sheng; Tian, Yu-Feng; Sun, Ding-Ping; Huang, Rwei-Fen Syu

    2017-12-15

    The mechanistic role of colonic low folate metabolic stress (LFMS) in colorectal cancer (CRC) malignancy development remains unknown. Folate analysis on the 99 paired human CRC tissues localized LFMS to the deep invasive T3/T4 staged tumours with hypo-methylated sonic hedgehog (Shh) promoter region and amplified expressions of Shh ligand and Gli1 effector, which coincided with deregulated expressions of the epithelial-mesenchymal transition (EMT) mediators. Colonic folate levels of CRC were inversely correlated with pluripotent expressions of the SOX2, NANOG and OCT4 markers (p cells to LFMS microenvironment significantly hypomethylated Shh promoter region, activated Shh signaling, induced transcript and protein expressions of the pluripotent markers, promoted trans-differentiation as EMT by deregulation of Snail mediator and epithelial marker E-cadherin, increased MMP2/MMP9 enzymatic digestion on matrix protein for invasion, and promoted self-renewal capability of anchorage-independent tumor-spheroid formation. LFMS-induced cancer stem cell (CSC) signature and CRC invasion is synergized with inhibition of DNA methylation by 5-Aza-2-deoxycytidine (5AZA) in rewiring EMT genotypes, which can be blockade by the Shh inhibitor (cyclopamine). The in vivo and in vitro data corroboratively identify CSC-like molecular targets specific to the LFMS-predisposed invasive CRC through reprogramming DNA methylation-activated Shh signaling. The study highlights CSC targets specific to LFMS-predisposed invasive CRC in optimizing folate co-chemotherapy to minimize tumour metastasis potential of CRC patients. © 2017 UICC.

  18. 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.

  19. 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.

    Science.gov (United States)

    Spanou, Evagelia; Kalisperati, Polyxeni; Pateras, Ioannis S; Papalampros, Alexandros; Barbouti, Alexandra; Tzioufas, Athanasios G; Kotsinas, Athanassios; Sougioultzis, Stavros

    2017-01-01

    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.

  20. 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

    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...... damage. In support of this, we show that SMARCA5 and RNF168 interact in a DNA damage- and PARP-dependent manner. RNF168 became poly(ADP-ribosyl)ated after DNA damage, while RNF168 and poly(ADP-ribose) chains were required for SMARCA5 binding in vivo, explaining how SMARCA5 is linked to the RNF168...

  1. 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

  2. [Enrichment of extracellular DNA from the cultivation medium of human peripheral blood mononuclears with genomic CpG rich fragments results in increased cell production of IL-6 and TNF-a via activation of the NF-kB signaling pathway].

    Science.gov (United States)

    Speranskii, A I; Kostyuk, S V; Kalashnikova, E A; Veiko, N N

    2016-03-01

    Previously, it was found that blood plasma extracellular DNA (ecDNA) of patients with rheumatoid arthritis (RA) is enriched with CpG-rich genomic DNA fragments, which contain TLR9 ligands (Veiko et al., 2006). In this study we have demonstrated that ecDNA of a RA patient and model fragments added to a cultivation medium of peripheral blood mononuclear cells (PBMC) of healthy donors stimulate expression of genes for the TLR9-MyD88-NF-kB signaling pathway; this leads to a significant increase in concentrations of the proinflammatory cytokines IL-6 and TNF-a in the cultivation medium. Human genomic DNA non-enriched with the CpG sequences did not stimulate IL-6 and TNF-a synthesis in PBMC. A scheme explaining the potential role ecDNA in the induction and maintenance of increased levels of the proinflammatory cytokines under conditions damaging the human cells has been proposed.

  3. 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

  4. 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.)

  5. Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

    Directory of Open Access Journals (Sweden)

    Naimuddin Mohammed

    2011-01-01

    Full Text Available Abstract Background Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F. 3F proteins are small (~7 kDa, structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution. Results We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R. Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function. Conclusions This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.

  6. Hyperglycemia-induced oxidative stress induces apoptosis by inhibiting PI3-kinase/Akt and ERK1/2 MAPK mediated signaling pathway causing downregulation of 8-oxoG-DNA glycosylase levels in glial cells.

    Science.gov (United States)

    Kumar, Premranjan; Rao, G Nageswar; Pal, Bibhuti Bhusan; Pal, Arttatrana

    2014-08-01

    Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8

  7. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.

    Science.gov (United States)

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui

    2016-08-31

    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. D-Serine/N-methyl-D-aspartate receptor signaling decreases DNA-binding activity of the transcriptional repressor DREAM in Müller glia from the retina.

    Science.gov (United States)

    Chavira-Suárez, Erika; Ramírez, Mónica; Lamas, Mónica

    2008-02-20

    In the adult retina, N-methyl-D-aspartate (NMDA) neurotoxicity induces Müller cell reactive gliosis which is characterized by changes in gene expression that lead to proliferation and affect retinal physiology. The amino acid D-serine is synthesized in Müller cells and modulates these processes acting as a coagonist of NMDA receptors. We have found that the transcription factor DREAM (downstream regulatory element antagonist modulator), which acts as a transcriptional repressor by binding as a tetramer to regulatory elements located in the promoter region of target genes, is expressed in these cells and that its DNA-binding activity is modulated by NMDA receptor activation. Consistently, immunocytochemical analysis demonstrates that NMDA receptor activation induces changes in the nuclear localization of this transcription factor. DREAM is a pleiotropic transcription factor capable to repress and activate genes involved in several physiological events in different tissues. These results link, for the first time, this transcription factor with NMDA-receptor activation. Given the relevance of glutamatergic transmission in the retina and the remarkable functional plasticity of Müller cells, these findings support the notion that the NMDA receptor-dependent modulation of DREAM activity could play a role in relevant physiological processes ranging from retinal response to injury to differentiation capacity of retinal progenitor cells.

  9. 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.

  10. 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...

  11. 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.

  12. Genomic Signals of Reoriented ORFs

    Directory of Open Access Journals (Sweden)

    Paul Dan Cristea

    2004-01-01

    Full Text Available Complex representation of nucleotides is used to convert DNA sequences into complex digital genomic signals. The analysis of the cumulated phase and unwrapped phase of DNA genomic signals reveals large-scale features of eukaryote and prokaryote chromosomes that result from statistical regularities of base and base-pair distributions along DNA strands. By reorienting the chromosome coding regions, a “hidden” linear variation of the cumulated phase has been revealed, along with the conspicuous almost linear variation of the unwrapped phase. A model of chromosome longitudinal structure is inferred on these bases.

  13. A Serpentine Way to Signaling

    Indian Academy of Sciences (India)

    IAS Admin

    tude inside the cells, leading to activation of effectors molecules. Effectors are protein molecules that bind to DNA and cause. Figure 1. Simplified diagram of a signal transduction cas- cade. A signal transduction cascade represents the route of transfer of information from outside to inside the cell. There are many molecules.

  14. A general method of analysis of ligand binding to competing macromolecules using the spectroscopic signal originating from a reference macromolecule. Application to Escherichia coli replicative helicase DnaB protein nucleic acid interactions.

    Science.gov (United States)

    Jezewska, M J; Bujalowski, W

    1996-02-20

    MCT method by applying it to the binding of the Escherichia coli DnaB helicase to unmodified, nonfluorescent single-stranded nucleic acids where the interactions are not accompanied by any adequate spectroscopic signal changes. In order to analyze simultaneous binding of a ligand to different competing nucleic acid lattices, we introduced the combined application of the McGhee-von Hippel theory and the Epstein combinatorial approach for the binding of a large ligand to a linear, homogeneous nucleic acid lattice. Our approach allows one to perform a direct fit of the entire experimental isotherm for the protein binding to two competing nucleic acid lattices without resorting to complex numerical calculations.

  15. Mitochondrial DNA.

    Science.gov (United States)

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  16. 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…

  17. Retrograde signaling

    DEFF Research Database (Denmark)

    Kleine, Tatjana; Leister, Dario Michael

    2016-01-01

    The term retrograde signaling refers to the fact that chloroplasts and mitochondria utilize specific signaling molecules to convey information on their developmental and physiological states to the nucleus and modulate the expression of nuclear genes accordingly. Signals emanating from plastids...... of retrograde signaling has since been extended and revised. Elements of several 'operational' signaling circuits have come to light, including metabolites, signaling cascades in the cytosol and transcription factors. Here, we review recent advances in the identification and characterization of retrograde...

  18. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  19. Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

    Science.gov (United States)

    Nair, Nidhi; Shoaib, Muhammad

    2017-01-01

    Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage. PMID:28698521

  20. Instructing cells with programmable peptide DNA hybrids

    Science.gov (United States)

    Freeman, Ronit; Stephanopoulos, Nicholas; Álvarez, Zaida; Lewis, Jacob A; Sur, Shantanu; Serrano, Chris M; Boekhoven, Job; Lee, Sungsoo S.; Stupp, Samuel I.

    2017-01-01

    The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Here we describe a molecular system that can be programmed to control these three characteristics. In this approach we immobilize peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers directing cells to adhere and spread reversibly over multiple cycles. The DNA can also serve as a molecular ruler to control the distance-dependent synergy between two peptides. Finally, we use two orthogonal DNA handles to regulate two different bioactive signals, with the ability to independently up- or downregulate each over time. This enabled us to discover that neural stem cells, derived from the murine spinal cord and organized as neurospheres, can be triggered to migrate out in response to an exogenous signal but then regroup into a neurosphere as the signal is removed. PMID:28691701

  1. Development of an electrochemical DNA biosensor for detection of ...

    Indian Academy of Sciences (India)

    long oligonucleotides related to DNA sequence of Mycobacterium tuberculosis in optimal condition. Keywords. Poly(L-glutamic acid); DNA .... (TBS) (pH 7.0) containing 20 mM MDB.12,13 Then, the biosensors were immersed in washing ... signal when interacting with single strand DNA since this kind of DNA does not have ...

  2. The intraflagellar transport machinery in ciliary signaling

    DEFF Research Database (Denmark)

    Mourão, André; Christensen, Søren Tvorup; Lorentzen, Esben

    2016-01-01

    environmental cues necessary for organ development and maintenance of human health. Pathways reported to rely on the cilium organelle include Hedgehog, TGF-β, Wnt, PDGFRα, integrin and DNA damage repair signaling. An emerging theme in ciliary signaling is the requirement for active transport of signaling...

  3. Chromatin dynamics coupled to DNA repair.

    Science.gov (United States)

    Huertas, Dori; Sendra, Ramon; Muñoz, Purificación

    2009-01-01

    In order to protect and preserve the integrity of the genome, eukaryotic cells have developed accurate DNA repair pathways involving a coordinated network of DNA repair and epigenetic factors. The DNA damage response has to proceed in the context of chromatin, a packaged and compact structure that is flexible enough to regulate the accession of the DNA repair machinery to DNA-damaged sites. Chromatin modifications and ATP-remodeling activities are both necessary to ensure efficient DNA repair. Here we review the current progress of research into the importance of chromatin modifications and the ATP-remodeling complex to the DNA damage response, with respect to the sensing and signaling of DNA lesions, DNA repair and the processes that restore chromatin structure.

  4. 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...

  5. DNA Vaccines

    Indian Academy of Sciences (India)

    DNA vaccine, immune response, antibodies, infectious diseases. GENERAL I ARTICLE. DNA Vaccines. P N Rangarajan. History of Vaccine Development. The year 1996 marked the 200th anniversary of the first vaccine developed against smallpox by Edward Jenner. In the now- famous 1796 experiment, Jenner scratched ...

  6. 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

  7. Signalization and repair of the DNA double-strand breaks of in the cerebral tumors: modulation of the radiation response with the chemotherapy treatments; Signalization et reparation des cassures double-brin de l'ADN dans les gliomes: modulation de la reponse aux traitements chimio-radiotherapeutiques

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkova-Bencokova, Z

    2007-07-15

    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

  8. The core spliceosome as target and effector of non-canonical ATM signalling

    NARCIS (Netherlands)

    Tresini, Maria; Warmerdam, Daniël O; Kolovos, Petros; Snijder, Loes; Vrouwe, Mischa G; Demmers, Jeroen A A; van IJcken, Wilfred F J; Grosveld, Frank G; Medema, René H|info:eu-repo/dai/nl/107536129; Hoeijmakers, Jan H J; Mullenders, Leon H F; Vermeulen, Wim; Marteijn, Jurgen A

    2015-01-01

    In response to DNA damage, tissue homoeostasis is ensured by protein networks promoting DNA repair, cell cycle arrest or apoptosis. DNA damage response signalling pathways coordinate these processes, partly by propagating gene-expression-modulating signals. DNA damage influences not only the

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.,...

  15. 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...

  16. 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.

  17. 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

  18. 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

  19. 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 ...

  20. 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.

  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 binding domains and nuclear localization signal of LEDGF: contribution of two helix-turn-helix (HTH)-like domains and a stretch of 58 amino acids of the N-terminal to the trans-activation potential of LEDGF.

    Science.gov (United States)

    Singh, Dhirendra P; Kubo, E; Takamura, Y; Shinohara, T; Kumar, A; Chylack, Leo T; Fatma, N

    2006-01-20

    Lens epithelium derived growth factor (LEDGF), a nuclear protein, plays a role in regulating the transcription of stress-associated genes such as heat shock proteins by binding to consensus core DNA sequences nAGGn or nGAAn or their repeats, and in doing so helps to provide cyto-protection. However, additional information is required to identify the specific structural features of LEDGF involved in gene transcription. Here we have investigated the functional domains activating and repressing DNA-binding modules, by using a DNA binding assay and trans-activation experiments performed by analyzing proteins prepared from deletion constructs. The results disclosed the DNA-binding domain of N-terminal LEDGF mapped between amino acid residues 5 and 62, a 58 amino acid residue stretch PWWP domain which binds to stress response elements (STRE; A/TGGGGA/T). C-terminal LEDGF contains activation domains, an extensive loop-region (aa 418-530) with two helix-turn-helix (HTH)-like domains, and binds to a heat shock element (HSE; nGAAn). A trans-activation assay using Hsp27 promoter revealed that both HTH domains contribute in a cooperative manner to the trans-activation potential of LEDGF. Interestingly, removal of N-terminal LEDGF (aa 1-187) significantly enhances the gene activation potential of C-terminal LEDGF (aa 199-530); thus the N-terminal domain (aa 5-62), exhibits auto-transcriptional repression activity. It appears that this domain is involved in stabilizing the LEDGF-DNA binding complex. Collectively, our results demonstrate that LEDGF contains three DNA-binding domains, which regulate gene expression depending on cellular microenvironment and thus modify the physiology of cells to maintain cellular homeostasis.

  3. DNA nanotechnology

    OpenAIRE

    Nadrian C Seeman

    2003-01-01

    Since Watson and Crick’s determination of its structure nearly 50 years ago, DNA has come to fill our lives in many areas, from genetic counseling to forensics, from genomics to gene therapy. These, and other ways in which DNA affects human activities, are related to its function as genetic material, not just our genetic material, but the genetic material of all living organisms. Here, we will ignore DNA’s biological role; rather, we will discuss how the properties that make it so successful ...

  4. Phosphoinositide signaling.

    Science.gov (United States)

    Boss, Wendy F; Im, Yang Ju

    2012-01-01

    "All things flow and change…even in the stillest matter there is unseen flux and movement." Attributed to Heraclitus (530-470 BC), from The Story of Philosophy by Will Durant. Heraclitus, a Greek philosopher, was thinking on a much larger scale than molecular signaling; however, his visionary comments are an important reminder for those studying signaling today. Even in unstimulated cells, signaling pathways are in constant metabolic flux and provide basal signals that travel throughout the organism. In addition, negatively charged phospholipids, such as the polyphosphorylated inositol phospholipids, provide a circuit board of on/off switches for attracting or repelling proteins that define the membranes of the cell. This template of charged phospholipids is sensitive to discrete changes and metabolic fluxes-e.g., in pH and cations-which contribute to the oscillating signals in the cell. The inherent complexities of a constantly fluctuating system make understanding how plants integrate and process signals challenging. In this review we discuss one aspect of lipid signaling: the inositol family of negatively charged phospholipids and their functions as molecular sensors and regulators of metabolic flux in plants.

  5. 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)

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Chromosomal localization of rDNA genes and genomic organization ...

    Indian Academy of Sciences (India)

    Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples.

  11. Yap1: a DNA damage responder in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rowe, Lori A; Degtyareva, Natalya; Doetsch, Paul W

    2012-04-01

    Activation of signaling pathways in response to genotoxic stress is crucial for cells to properly repair DNA damage. In response to DNA damage, intracellular levels of reactive oxygen species increase. One important function of such a response could be to initiate signal transduction processes. We have employed the model eukaryote Saccharomyces cerevisiae to delineate DNA damage sensing mechanisms. We report a novel, unanticipated role for the transcription factor Yap1 as a DNA damage responder, providing direct evidence that reactive oxygen species are an important component of the DNA damage signaling process. Our findings reveal an epistatic link between Yap1 and the DNA base excision repair pathway. Corruption of the Yap1-mediated DNA damage response influences cell survival and genomic stability in response to exposure to genotoxic agents. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  12. 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.

  13. 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.

  14. Compressive Sensing DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Sheikh Mona A

    2009-01-01

    Full Text Available Compressive sensing microarrays (CSMs are DNA-based sensors that operate using group testing and compressive sensing (CS principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed.

  15. 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.

  16. DNA Chip

    Indian Academy of Sciences (India)

    Imagine a world without identity cards; no I-cards for the college or office or bank account or anything! All you are carrying is a small (say, 2cm x 2cm) 'DNA-chip', which has the whole of your genetic profile on it. Your identity cannot get more authentic than that. Imagine a world where marriages are not decided by matching ...

  17. 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

  18. Stool DNA Test

    Science.gov (United States)

    ... The stool DNA test is a noninvasive laboratory test that identifies DNA changes in the cells of a stool sample. ... the presence of cancer. If a stool DNA test detects abnormal DNA, additional testing may be used to investigate the ...

  19. 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.

  20. 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.

  1. Single molecule Studies of DNA Mismatch Repair

    Science.gov (United States)

    Erie, Dorothy A.; Weninger, Keith R.

    2015-01-01

    DNA mismatch repair involves is a widely conserved set of proteins that is essential to limit genetic drift in all organisms. The same system of proteins plays key roles in many cancer related cellular transactions in humans. Although the basic process has been reconstituted in vitro using purified components, many fundamental aspects of DNA mismatch repair remain hidden due in part to the complexity and transient nature of the interactions between the mismatch repair proteins and DNA substrates. Single molecule methods offer the capability to uncover these transient but complex interactions and allow novel insights into mechanisms that underlie DNA mismatch repair. In this review, we discuss applications of single molecule methodology including electron microscopy, atomic force microscopy, particle tracking, FRET, and optical trapping to studies of DNA mismatch repair. These studies have led to formulation of mechanistic models of how proteins identify single base mismatches in the vast background of matched DNA and signal for their repair. PMID:24746644

  2. 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...

  3. 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

  4. 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

  5. DNA Microarrays

    Science.gov (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  6. 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.

  7. Bromodomain proteins: repairing DNA damage within chromatin.

    Science.gov (United States)

    Chiu, Li-Ya; Gong, Fade; Miller, Kyle M

    2017-10-05

    Genome surveillance and repair, termed the DNA damage response (DDR), functions within chromatin. Chromatin-based DDR mechanisms sustain genome and epigenome integrity, defects that can disrupt cellular homeostasis and contribute to human diseases. An important chromatin DDR pathway is acetylation signalling which is controlled by histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes, which regulate acetylated lysines within proteins. Acetylated proteins, including histones, can modulate chromatin structure and provide molecular signals that are bound by acetyl-lysine binders, including bromodomain (BRD) proteins. Acetylation signalling regulates several DDR pathways, as exemplified by the preponderance of HATs, HDACs and BRD proteins that localize at DNA breaks to modify chromatin for lesion repair. Here, we explore the involvement of acetylation signalling in the DDR, focusing on the involvement of BRD proteins in promoting chromatin remodelling to repair DNA double-strand breaks. BRD proteins have widespread DDR functions including chromatin remodelling, chromatin modification and transcriptional regulation. We discuss mechanistically how BRD proteins read acetylation signals within chromatin to trigger DDR and chromatin activities to facilitate genome-epigenome maintenance. Thus, DDR pathways involving BRD proteins represent key participants in pathways that preserve genome-epigenome integrity to safeguard normal genome and cellular functions.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  8. Image analysis for DNA sequencing

    International Nuclear Information System (INIS)

    Palaniappan, K.; Huang, T.S.

    1991-01-01

    This paper reports that there is a great deal of interest in automating the process of DNA (deoxyribonucleic acid) sequencing to support the analysis of genomic DNA such as the Human and Mouse Genome projects. In one class of gel-based sequencing protocols autoradiograph images are generated in the final step and usually require manual interpretation to reconstruct the DNA sequence represented by the image. The need to handle a large volume of sequence information necessitates automation of the manual autoradiograph reading step through image analysis in order to reduce the length of time required to obtain sequence data and reduce transcription errors. Various adaptive image enhancement, segmentation and alignment methods were applied to autoradiograph images. The methods are adaptive to the local characteristics of the image such as noise, background signal, or presence of edges. Once the two-dimensional data is converted to a set of aligned one-dimensional profiles waveform analysis is used to determine the location of each band which represents one nucleotide in the sequence. Different classification strategies including a rule-based approach are investigated to map the profile signals, augmented with the original two-dimensional image data as necessary, to textual DNA sequence information

  9. Genetic and environmental influence on DNA strand break repair

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander

    2013-01-01

    factors are likely to influence DNA repair capacity. In order to gain more insight into the genetic and environmental contribution to the molecular basis of DNA repair, we have performed a human twin study, where we focused on the consequences of some of the most abundant types of DNA damage (single-strand...... breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104...... dizygotic). We did not detect genetic effects on the DNA-strand break variables in our study. Environ. Mol. Mutagen., 2013. © 2013 Wiley Periodicals, Inc....

  10. 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

  11. Prosocial Signalling

    DEFF Research Database (Denmark)

    Kahsay, Goytom Abraha

    signalling can cause reverse price reactions resembling the crowding-out of pre-existing motives for prosocial behavior seen in situations of volunteering and charitable giving. Using a unique combination of questionnaire and purchase panel data, it presents evidence of such reputation-driven reverse price...... reactions in the Danish market for organic milk. The second paper proposes a self-image model to account consumers’ behaviour under PWYW. It finds that when a good’s fixed price is lower than an exogenously given threshold fair value, PWYW can lead to a lower utility, which may lead to lower purchase rate...

  12. 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.

  13. Plasmonic SERS biosensing nanochips for DNA detection.

    Science.gov (United States)

    Ngo, Hoan T; Wang, Hsin-Neng; Fales, Andrew M; Vo-Dinh, Tuan

    2016-03-01

    The development of rapid, cost-effective DNA detection methods for molecular diagnostics at the point-of-care (POC) has been receiving increasing interest. This article reviews several DNA detection techniques based on plasmonic-active nanochip platforms developed in our laboratory over the last 5 years, including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). DNA probes were used as the recognition elements, and surface-enhanced Raman scattering (SERS) was used as the signal detection method. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the nanochip's plasmonic-active surface. As the field intensity of the surface plasmon decays exponentially as a function of distance, the distance change in turn affects SERS signal intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized nanochips and measuring the SERS signal after appropriate incubation times. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost-effective. The usefulness of the nanochip platform-based techniques for medical diagnostics was illustrated by the detection of host genetic biomarkers for respiratory viral infection and of the dengue virus gene.

  14. HPV DNA test

    Science.gov (United States)

    ... HPV testing in women; Cervical cancer - HPV DNA test; Cancer of cervix - HPV DNA test ... The HPV DNA test may be done during a Pap smear . You lie on a table and place your feet in stirrups. The ...

  15. 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.

  16. Terahertz molecular resonance of cancer DNA.

    Science.gov (United States)

    Cheon, Hwayeong; Yang, Hee-Jin; Lee, Sang-Hun; Kim, Young A; Son, Joo-Hiuk

    2016-11-15

    Carcinogenesis involves the chemical and structural alteration of biomolecules in cells. Aberrant methylation of DNA is a well-known carcinogenic mechanism and a common chemical modification of DNA. Terahertz waves can directly observe changes in DNA because the characteristic energies lie in the same frequency region. In addition, terahertz energy levels are not high enough to damage DNA by ionization. Here, we present terahertz molecular resonance fingerprints of DNA methylation in cancer DNA. Methylated cytidine, a nucleoside, has terahertz characteristic energies that give rise to the molecular resonance of methylation in DNA. Molecular resonance is monitored in aqueous solutions of genomic DNA from cancer cell lines using a terahertz time-domain spectroscopic technique. Resonance signals can be quantified to identify the types of cancer cells with a certain degree of DNA methylation. These measurements reveal the existence of molecular resonance fingerprints of cancer DNAs in the terahertz region, which can be utilized for the early diagnosis of cancer cells at the molecular level.

  17. Programmable DNA-Mediated Multitasking Processor.

    Science.gov (United States)

    Shu, Jian-Jun; Wang, Qi-Wen; Yong, Kian-Yan; Shao, Fangwei; Lee, Kee Jin

    2015-04-30

    Because of DNA appealing features as perfect material, including minuscule size, defined structural repeat and rigidity, programmable DNA-mediated processing is a promising computing paradigm, which employs DNAs as information storing and processing substrates to tackle the computational problems. The massive parallelism of DNA hybridization exhibits transcendent potential to improve multitasking capabilities and yield a tremendous speed-up over the conventional electronic processors with stepwise signal cascade. As an example of multitasking capability, we present an in vitro programmable DNA-mediated optimal route planning processor as a functional unit embedded in contemporary navigation systems. The novel programmable DNA-mediated processor has several advantages over the existing silicon-mediated methods, such as conducting massive data storage and simultaneous processing via much fewer materials than conventional silicon devices.

  18. 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.

  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. 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

  1. 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

  2. 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.

  3. 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.

  4. Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System*

    Science.gov (United States)

    Lindsey-Boltz, Laura A.; Kemp, Michael G.; Reardon, Joyce T.; DeRocco, Vanessa; Iyer, Ravi R.; Modrich, Paul; Sancar, Aziz

    2014-01-01

    DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response. PMID:24403078

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™.

    Science.gov (United States)

    Ip, Stephen C Y; Lin, Sze-Wah; Lai, Kam-Ming

    2015-05-01

    DNA left at a crime scene was often limited in amount and far from pristine. To maximize the chance of recovering as much information as possible from such compromised samples, an appropriate extraction method using the available technologies needs to be devised. In this study, we used human blood, buffy coat and a total of 76 simulated touch DNA samples to test the effectiveness of the following five common DNA extraction methods, namely, Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™ system, in the recovery of such DNA. We demonstrated that the QIAamp® and QIAsymphony® DNA Investigator® Kits, and the DNA IQ™ system, exhibited a better effectiveness in DNA recovery amongst these methods and yielded extracts with higher success rate in subsequent DNA profiling. These extracts also generated profiles with better intra-colour signal balance. The findings in this work allowed us to propose an extraction approach as follows: 1) casework samples shall be extracted with the QIAamp®/QIAsymphony® DNA Investigator® Kits or the DNA IQ™ system, viz., QIAsymphony® DNA Investigator® Kit and DNA IQ™, due to their higher throughput, are for the touched DNA evidence from the volume crime, while QIAamp® DNA Investigator Kit is preferable for challenging bloodstain samples; and 2) control samples, such as buccal swab, with known identity can be extracted with the Chelex, due to their cheaper cost per sample. Copyright © 2015 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

  10. DNA damage response: multilevel proteomics gains momentum.

    Science.gov (United States)

    Daub, Henrik

    2012-04-27

    In this issue of Molecular Cell, Beli et al. (2012) introduce a multilevel proteomics approach for parallel quantification of protein phosphorylation, acetylation, and abundance and apply this to the complex signaling network of the DNA damage response. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. 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

  12. DNA nanotechnology for nucleic acid analysis: multifunctional molecular DNA machine for RNA detection.

    Science.gov (United States)

    Cox, A J; Bengtson, H N; Rohde, K H; Kolpashchikov, D M

    2016-12-06

    The Nobel prize in chemistry in 2016 was awarded for 'the design and synthesis of molecular machines'. Here we designed and assembled a molecular machine for the detection of specific RNA molecules. An association of several DNA strands, named multifunctional DNA machine for RNA analysis (MDMR1), was designed to (i) unwind RNA with the help of RNA-binding arms, (ii) selectively recognize a targeted RNA fragment, (iii) attract a signal-producing substrate and (iv) amplify the fluorescent signal by catalysis. MDMR1 enabled detection of 16S rRNA at concentrations ∼24 times lower than that by a traditional deoxyribozyme probe.

  13. Classification of genomic signals using dynamic time warping.

    Science.gov (United States)

    Skutkova, Helena; Vitek, Martin; Babula, Petr; Kizek, Rene; Provaznik, Ivo

    2013-01-01

    Classification methods of DNA most commonly use comparison of the differences in DNA symbolic records, which requires the global multiple sequence alignment. This solution is often inappropriate, causing a number of imprecisions and requires additional user intervention for exact alignment of the similar segments. The similar segments in DNA represented as a signal are characterized by a similar shape of the curve. The DNA alignment in genomic signals may adjust whole sections not only individual symbols. The dynamic time warping (DTW) is suitable for this purpose and can replace the multiple alignment of symbolic sequences in applications, such as phylogenetic analysis. The proposed method is composed of three main parts. The first part represent conversion of symbolic representation of DNA sequences in the form of a string of A,C,G,T symbols to signal representation in the form of cumulated phase of complex components defined for each symbol. Next part represents signals size adjustment realized by standard signal preprocessing methods: median filtration, detrendization and resampling. The final part necessary for genomic signals comparison is position and length alignment of genomic signals by dynamic time warping (DTW). The application of the DTW on set of genomic signals was evaluated in dendrogram construction using cluster analysis. The resulting tree was compared with a classical phylogenetic tree reconstructed using multiple alignment. The classification of genomic signals using the DTW is evolutionary closer to phylogeny of organisms. This method is more resistant to errors in the sequences and less dependent on the number of input sequences. Classification of genomic signals using dynamic time warping is an adequate variant to phylogenetic analysis using the symbolic DNA sequences alignment; in addition, it is robust, quick and more precise technique.

  14. DNA Damage, DNA Repair, Aging, and Neurodegeneration

    Science.gov (United States)

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.

    2015-01-01

    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

  15. 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

  16. Carbon nanostructures as immobilization platform for DNA: A review on current progress in electrochemical DNA sensors.

    Science.gov (United States)

    Rasheed, P Abdul; Sandhyarani, N

    2017-11-15

    Development of a sensitive, specific and cost-effective DNA detection method is motivated by increasing demand for the early stage diagnosis of genetic diseases. Recent developments in the design and fabrication of efficient sensor platforms based on nanostructures make the highly sensitive sensors which could indicate very low detection limit to the level of few molecules, a realistic possibility. Electrochemical detection methods are widely used in DNA diagnostics as it provide simple, accurate and inexpensive platform for DNA detection. In addition, the electrochemical DNA sensors provide direct electronic signal without the use of expensive signal transduction equipment and facilitates the immobilization of single stranded DNA (ssDNA) probe sequences on a wide variety of electrode substrates. It has been found that a range of nanomaterials such as metal nanoparticles (MNPs), carbon based nanomaterials, quantum dots (QDs), magnetic nanoparticles and polymeric NPs have been introduced in the sensor design to enhance the sensing performance of electrochemical DNA sensor. In this review, we discuss recent progress in the design and fabrication of efficient electrochemical genosensors based on carbon nanostructures such as carbon nanotubes, graphene, graphene oxide and nanodiamonds. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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.

  18. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  19. Detection of Transgenes on DNA Fibers.

    Science.gov (United States)

    Shibata, Fukashi

    2016-01-01

    Fluorescence in situ hybridization (FISH) was developed for detecting specific DNA sequences directly on mitotic or meiotic chromosomes. However, the resolution of FISH on chromosomes is limited by condensed structure of chromatin, and it is difficult to differentiate two target sites close to each other. To overcome this issue, the objects was changed to stretched DNA fibers, and this fiber FISH technique has now been used for revealing genome structure at molecular level. Hybridization and detection procedures of fiber FISH are common with FISH on chromosomes. Therefore, application of fiber FISH is not difficult for the researchers of some experience in ordinary FISH. DNA fibers can be released from nuclei fixed on glass slides using a detergent. The DNA fibers were shred in FISH procedure, and the resultant fragments became small bead-like shape. This makes FISH signals on DNA fibers a series of dots. The size of DNA in the dot is estimated to be approximately 1 kb, it corresponding to the resolution of fiber FISH. This makes it possible to analyze structures of transgenes on DNA fibers in detail.

  20. Chromatin remodelers in the DNA double strand break response

    NARCIS (Netherlands)

    Smeenk, Godelieve

    2012-01-01

    During my PhD project, I studied the role of several chromatin remodelers in the DNA double strand break (DSB) response. We discovered that both CHD4 and SMARCA5 are required for ubiquitin signaling through the E3 ubiquitin ligases RNF8 and RNF168, which is a central signaling event in the response

  1. 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...

  2. DNA Computing Systems Activated by Electrochemically-triggered DNA Release from a Polymer-brush-modified Electrode Array

    Science.gov (United States)

    Gamella, Maria; Zakharchenko, Andrey; Guz, Nataliia; Masi, Madeline; Minko, Sergiy; Kolpashchikov, Dmitry M.; Iken, Heiko; Poghossian, Arshak; Schöning, Michael J.; Katz, Evgeny

    2017-01-01

    An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA-based Identity, AND and XOR logic gates. Single-stranded DNA molecules were loaded on the mixed poly(N,N-di-methylaminoethyl methacrylate) (PDMAEMA)/poly-(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAE-MA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals. PMID:29379265

  3. 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...

  4. 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.

  5. Subcloning of DNA fragments.

    Science.gov (United States)

    Struhl, K

    2001-05-01

    The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNA synthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.

  6. 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.

  7. 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.

  8. DNA Repair Systems

    Indian Academy of Sciences (India)

    exogenous damage). Endogenous damage ... of spontaneous DNA-damage due to endogenous factors. He es- timated that around 10,000 potentially mutagenic .... 3 –5 direction is defined as. 'upstream'. A single DNA strand is synthesized in a.

  9. HPV 9G DNA Chip: 100% Clinical Sensitivity and Specificity

    OpenAIRE

    An, Heejung; Song, Keum-Soo; Nimse, Satish Balasaheb; Kim, Junghoon; Nguyen, Van-Thuan; Ta, Van-Thao; Sayyed, Danishmalik Rafiq; Kim, Taisun

    2012-01-01

    We describe a novel HPV 9G DNA chip test for the accurate and reliable genotyping of human papillomavirus (HPV). The HPV 9G DNA chip test established its efficiency in terms of a signal-to-background ratio (SBR) of 200, which is 50 times superior to commercial HPV DNA chips, and 100% target-specific hybridization at 25°C. We compared the genotyping results for the 439 clinical samples by the HPV 9G DNA chip test with the sequencing results for the MY11/GP6+ (M2) primer set-mediated PCR produc...

  10. DNA damage response and Autophagy: a meaningful partnership

    Directory of Open Access Journals (Sweden)

    ARISTIDES G ELIOPOULOS

    2016-11-01

    Full Text Available Autophagy and the DNA damage response (DDR are biological processes essential for cellular and organismal homeostasis. Herein we summarize and discuss emerging evidence linking DDR to autophagy. We highlight published data suggesting that autophagy is activated by DNA damage and is required for several functional outcomes of DDR signaling, including repair of DNA lesions, senescence, cell death, and cytokine secretion. Uncovering the mechanisms by which autophagy and DDR are intertwined provides novel insight into the pathobiology of conditions associated with accumulation of DNA damage, including cancer and aging, and novel concepts for the development of improved therapeutic strategies against these pathologies.

  11. Animal Mitochondrial DNA Replication

    Science.gov (United States)

    Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

    2016-01-01

    Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

  12. 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.

  13. SMC1-mediated intra-S-phase arrest facilitates bocavirus DNA replication.

    Science.gov (United States)

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

    2013-04-01

    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.

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

    Science.gov (United States)

    2012-01-01

    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. PMID:22289628

  15. 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.

  16. 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.

  17. 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.

  18. DNA Microarray-Based Diagnostics.

    Science.gov (United States)

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  19. 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.

  20. 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

  1. Nanopore-CMOS Interfaces for DNA Sequencing.

    Science.gov (United States)

    Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

    2016-08-06

    DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces.

  2. 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%.

  3. Ultrasensitive chemiluminescence biosensors using nucleic acid-functionalized silver-cysteine nanowires as signal amplifying labels.

    Science.gov (United States)

    Chen, Wenjuan; Chen, Yuan; Wang, Meilan; Chi, Yuwu

    2018-03-26

    Ultrasensitive chemiluminescence (CL) sensors for biomolecules (DNA and proteins) have been developed by adopting DNA-functionalized silver-cysteine hybrid nanowires (p-SCNWs) as signal amplifying labels. The sensing is established from a sandwich-type DNA hybridization, where the target DNA strands are initially hybridized with the capture DNA located at paramagnetic microspheres (PMs) and subsequently hybridized with p-SCNWs functionalized with the signal DNA probe. After magnetic separation, p-SCNWs on the hybrids were completely decomposed with HNO3 to release numerous silver ions. The powerful catalysis of silver ions toward the redox reaction of K2S2O8-Mn2+-H3PO4 causes the generation of KMnO4 that is capable of oxidizing luminol at high pH, triggering an amplified chemiluminescent signal emission. The sensing combines the extraordinary sensitivity of the catalytic chemiluminescence technology and the amplifying strategy via releasing large quantities of silver ions as the catalyst from each hybrid, enabling the assay of target DNA strands at a concentration as low as 0.34 fM. The CL signals associated with single-base pair mismatched DNA strands and non-complementary DNA strands are able to be discriminated well from the CL signal related to the complementary DNA hybridization. Likewise, the combination of p-SCNWs functionalized with an aptamer and PMs/aptamer/thrombin complex allowed the chemiluminescence sensing of thrombin with a low limit of detection corresponding to 0.17 pM.

  4. 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...... for a long period of time before its information is accessed by the cell. Although DNA plays a critical role as an informational storage molecule, it is by no means as unexciting as a computer tape or disk drive. The structure of the DNA described by Watson and Crick in 1953 is a right handed helix of two...

  5. 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....

  6. DNA Sequencing apparatus

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  7. 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...

  8. 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.

  9. DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae).

    Science.gov (United States)

    Bugrov, Alexander G; Karamysheva, Tatiana V; Perepelov, Eugeny A; Elisaphenko, Eugeny A; Rubtsov, Denis N; Warchałowska-Sliwa, Elzbieta; Tatsuta, Haruki; Rubtsov, Nikolay B

    2007-01-01

    A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty of them were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the DDBJ/GenBank/EMBL database ( http://www.ddbj.nig.ac.jp/ ) was performed. Different patterns of signals were observed after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes. Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telemetric regions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely related Podisma species.

  10. 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...

  11. 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.

  12. 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.

  13. Cellular sensing of viral DNA and viral evasion mechanisms.

    Science.gov (United States)

    Orzalli, Megan H; Knipe, David M

    2014-01-01

    Mammalian cells detect foreign DNA introduced as free DNA or as a result of microbial infection, leading to the induction of innate immune responses that block microbial replication and the activation of mechanisms that epigenetically silence the genes encoded by the foreign DNA. A number of DNA sensors localized to a variety of sites within the cell have been identified, and this review focuses on the mechanisms that detect viral DNA and how the resulting responses affect viral infections. Viruses have evolved mechanisms that inhibit these host sensors and signaling pathways, and the study of these antagonistic viral strategies has provided insight into the mechanisms of these host responses. The field of cellular sensing of foreign DNA is in its infancy, but our currently limited knowledge has raised a number of important questions for study.

  14. 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.

  15. Signal Processing and Restoration

    NARCIS (Netherlands)

    Biemond, J.; Slump, C.H.; Lagendijk, R.L.; Tolhuizen, L.M.G.M.; de With, P.H.N.

    2004-01-01

    Digital Signal Processing (DSP) concerns the theoretical and practical aspects of representing information-bearing signals in digital form and the use of processors or special purpose hardware to extract that information or to transform the signals in useful ways. Areas where digital signal

  16. 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.

  17. Signal verification can promote reliable signalling.

    Science.gov (United States)

    Broom, Mark; Ruxton, Graeme D; Schaefer, H Martin

    2013-11-22

    The central question in communication theory is whether communication is reliable, and if so, which mechanisms select for reliability. The primary approach in the past has been to attribute reliability to strategic costs associated with signalling as predicted by the handicap principle. Yet, reliability can arise through other mechanisms, such as signal verification; but the theoretical understanding of such mechanisms has received relatively little attention. Here, we model whether verification can lead to reliability in repeated interactions that typically characterize mutualisms. Specifically, we model whether fruit consumers that discriminate among poor- and good-quality fruits within a population can select for reliable fruit signals. In our model, plants either signal or they do not; costs associated with signalling are fixed and independent of plant quality. We find parameter combinations where discriminating fruit consumers can select for signal reliability by abandoning unprofitable plants more quickly. This self-serving behaviour imposes costs upon plants as a by-product, rendering it unprofitable for unrewarding plants to signal. Thus, strategic costs to signalling are not a prerequisite for reliable communication. We expect verification to more generally explain signal reliability in repeated consumer-resource interactions that typify mutualisms but also in antagonistic interactions such as mimicry and aposematism.

  18. Engineering of DNA-mediated assemblies for biosensing applications

    Science.gov (United States)

    Xu, Phyllis F.

    Traditionally known as the genetic code, in recent years DNA has been engineered in new ways and applied toward novel applications in materials science and bio-nanotechnology. The nanometer features and highly specific base-pairing of DNA has enabled its use to build complex 2- and 3D nano-architectures and nano-structures. Specific DNA scaffolds include branched DNA junctions, 2D and 3D DNA "tiles" and "bricks", and DNA hydrogels that can be used for applications across various fields of materials science. Another unique feature of DNA is its ability to bind other materials through molecular recognition which has made the use of DNA aptamer or DNA-protein conjugates highly useful for applications in biosensing and identification. The main focus of this thesis is to demonstrate the use of DNA as an invaluable tool for engineering specific nanostructures for biosensing. The first chapter introduces the basics of DNA and current research in various DNA structures and assemblies and their applications. The second and third chapters describe the self-assembly of two structurally different nanomaterials using DNA, carbon nanotubes and gold nanoparticles. In the second chapter, the successful large-scale alignment of carbon nanotubes on a surface is shown. The study goes into the factors that affect the quality of alignment, including salt concentration, length of DNA, and annealing. In the third chapter, we show how DNA can be used to engineer discrete gold nanocrystal assemblies-"nanodumbbells"- that can adopt different structural conformations through DNA interactions. By incorporating DNA aptamers in the nanoparticle structure, these nanodumbbells can be used to sense particular analytes in solution. Chapter 4 continues the study of the nanodumbbell structures and their potential use as surface-enhanced Raman spectroscopy (SERS) biosensors. A large measurable difference in Raman signal was experimentally obtained from the two distinct nanodumbbell conformations. This

  19. Mitochondrial DNA in the regulation of innate immune responses

    Directory of Open Access Journals (Sweden)

    Chunju Fang

    2015-10-01

    Full Text Available Abstract Mitochondrion is known as the energy factory of the cell, which is also a unique mammalian organelle and considered to be evolved from aerobic prokaryotes more than a billion years ago. Mitochondrial DNA, similar to that of its bacterial ancestor’s, consists of a circular loop and contains significant number of unmethylated DNA as CpG islands. The innate immune system plays an important role in the mammalian immune response. Recent research has demonstrated that mitochondrial DNA (mtDNA activates several innate immune pathways involving TLR9, NLRP3 and STING signaling, which contributes to the signaling platforms and results in effector responses. In addition to facilitating antibacterial immunity and regulating antiviral signaling, mounting evidence suggests that mtDNA contributes to inflammatory diseases following cellular damage and stress. Therefore, in addition to its well-appreciated roles in cellular metabolism and energy production, mtDNA appears to function as a key member in the innate immune system. Here, we highlight the emerging roles of mtDNA in innate immunity.

  20. Statistical analysis of molecular signal recording.

    Directory of Open Access Journals (Sweden)

    Joshua I Glaser

    Full Text Available A molecular device that records time-varying signals would enable new approaches in neuroscience. We have recently proposed such a device, termed a "molecular ticker tape", in which an engineered DNA polymerase (DNAP writes time-varying signals into DNA in the form of nucleotide misincorporation patterns. Here, we define a theoretical framework quantifying the expected capabilities of molecular ticker tapes as a function of experimental parameters. We present a decoding algorithm for estimating time-dependent input signals, and DNAP kinetic parameters, directly from misincorporation rates as determined by sequencing. We explore the requirements for accurate signal decoding, particularly the constraints on (1 the polymerase biochemical parameters, and (2 the amplitude, temporal resolution, and duration of the time-varying input signals. Our results suggest that molecular recording devices with kinetic properties similar to natural polymerases could be used to perform experiments in which neural activity is compared across several experimental conditions, and that devices engineered by combining favorable biochemical properties from multiple known polymerases could potentially measure faster phenomena such as slow synchronization of neuronal oscillations. Sophisticated engineering of DNAPs is likely required to achieve molecular recording of neuronal activity with single-spike temporal resolution over experimentally relevant timescales.

  1. 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...

  2. 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...... to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby...... 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....

  3. Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

    Science.gov (United States)

    Yoshida, Hiroyuki; Nishikawa, Makiya; Yasuda, Sachiyo; Toyota, Hiroyasu; Kiyota, Tsuyoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2012-10-01

    Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. 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.

  5. 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)

  6. 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.

  7. MITOCHONDRIAL DNA- REVOLUTIONARY EVOLUTION

    Directory of Open Access Journals (Sweden)

    Vaidhehi Narayan Nayak

    2017-07-01

    Full Text Available BACKGROUND Mitochondrion, the sausage-shaped organelle residing in the cytoplasm of all eukaryotic cells, apart from being the power house, represents endosymbiotic evolution of a free living organism to intracellular structure. Anthropologically, mitochondrial DNA is the fossilised source to trace the human ancestry particularly of maternal lineage. This article attempts to highlight the various biological functions of mitochondrial DNA (mtDNA with a note on its forensic application.

  8. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

    2008-01-01

    Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...... independently of DNA damage signaling pathways during the very early stages of apoptosis....

  9. Influence of killing method on Lepidoptera DNA barcode recovery.

    Science.gov (United States)

    Willows-Munro, Sandi; Schoeman, M Corrie

    2015-05-01

    The global DNA barcoding initiative has revolutionized the field of biodiversity research. Such large-scale sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA-friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA-friendly protocol for killing. In this study, we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). Previous studies have suggested that chemicals, such as ethyl acetate and formaldehyde, degraded DNA and as such may not be appropriate for the collection of insects for DNA-based research. All Lepidoptera collected produced DNA barcodes of good quality, and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis. © 2014 John Wiley & Sons Ltd.

  10. 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...

  11. DNA ELECTROPHORESIS AT SURFACES

    Energy Technology Data Exchange (ETDEWEB)

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  12. Aptamer based voltammetric determination of ampicillin using a single-stranded DNA binding protein and DNA functionalized gold nanoparticles.

    Science.gov (United States)

    Wang, Jun; Ma, Kui; Yin, Huanshun; Zhou, Yunlei; Ai, Shiyun

    2017-12-20

    An aptamer based method is described for the electrochemical determination of ampicillin. It is based on the use of DNA aptamer, DNA functionalized gold nanoparticles (DNA-AuNPs), and single-stranded DNA binding protein (ssDNA-BP). When the aptamer hybridizes with the target DNA on the AuNPs, the ssDNA-BP is captured on the electrode surface via its specific interaction with ss-DNA. This results in a decreased electrochemical signal of the redox probe Fe(CN) 6 3- which is measured best at a voltage of 0.188 mV (vs. reference electrode). In the presence of ampicillin, the formation of aptamer-ampicillin conjugate blocks the further immobilization of DNA-AuNPs and ssDNA-BP, and this leads to an increased response. The method has a linear reposne that convers the 1 pM to 5 nM ampicillin concentration range, with a 0.38 pM detection limit (at an S/N ratio of 3). The assay is selective, stable and reproducible. It was applied to the determination of ampicillin in spiked milk samples where it gave recoveries ranging from 95.5 to 105.5%. Graphical abstract Schematic of a simple and sensitive electrochemical apta-biosensor for ampicillin detection. It is based on the use of gold nanoparticles (AuNPs), DNA aptamer, DNA functionalized AuNPs (DNA-AuNPs), and single-strand DNA binding protein (SSBP).

  13. Electrochemical DNA sensor by the assembly of graphene and DNA-conjugated gold nanoparticles with silver enhancement strategy.

    Science.gov (United States)

    Lin, Lei; Liu, Yang; Tang, Longhua; Li, Jinghong

    2011-11-21

    Sensitive and selective detection of DNA is in urgent need due to its important role in human bodies. Many disorders, such as Alzheimer's disease and various cancers, are closely related with DNA damage. In this work, a novel electrochemical DNA biosensor was constructed on a DNA-assembling graphene platform which provided a robust, simple and biocompatible platform with large surface area for DNA immobilization. The as-designed DNA sensor was fabricated by directly assembling captured ssDNA on a graphene-modified electrode through the π-π stacking interaction between graphene and ssDNA bases. Then, the target DNA sequence and oligonucleotide probes-labeled AuNPs were able to hybridize in a sandwich assay format, following the AuNPs-catalyzed silver deposition. The deposited silver was further detected by differential pulse voltammetry. Owing to the high DNA loading ability of graphene and the distinct signal amplification by AuNPs-catalyzed silver staining, the resulting biosensor exhibited a good analytical performance with a wide detection linear range from 200 pM to 500 nM, and a low detection limit of 72 pM. Additionally, the biosensor was proved to be able to discriminate the complementary sequence from the single-base mismatch sequence. The simple biosensor is promising in developing electronic, on-chip assays in clinical diagnosis, environmental control, and drug discovery.

  14. 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).

  15. 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.

  16. Nanoprobe-Initiated Enzymatic Polymerization for Highly Sensitive Electrochemical DNA Detection.

    Science.gov (United States)

    Wan, Ying; Wang, Pengjuan; Su, Yan; Wang, Lihua; Pan, Dun; Aldalbahi, Ali; Yang, Shulin; Zuo, Xiaolei

    2015-11-25

    Electrochemical DNA (E-DNA) sensors have been greatly developed and play an important role in early diagnosis of different diseases. To determine the extremely low abundance of DNA biomarkers in clinical samples, scientists are making unremitting efforts toward achieving highly sensitive and selective E-DNA sensors. Here, a novel E-DNA sensor was developed taking advantage of the signal amplification efficiency of nanoprobe-initiated enzymatic polymerization (NIEP). In the NIEP based E-DNA sensor, the capture probe DNA was thiolated at its 3'-terminal to be immobilized onto gold electrode, and the nanoprobe was fabricated by 5'-thiol-terminated signal probe DNA conjugated gold nanoparticles (AuNPs). Both of the probes could simultaneously hybridize with the target DNA to form a "sandwich" structure followed by the terminal deoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3'-terminal of DNA on the nanoprobe. During the DNA elongation, biotin labels were incorporated into the NIEP-generated long single-stranded DNA (ssDNA) tentacles, leading to specific binding of avidin modified horseradish peroxidase (Av-HRP). Since there are hundreds of DNA probes on the nanoprobe, one hybridization event would generate hundreds of long ssDNA tentacles, resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. By employing nanoprobe and TdT, it is demonstrated that the NIEP amplified E-DNA sensor has a detection limit of 10 fM and excellent differentiation ability for even single-base mismatch.

  17. A spatially localized architecture for fast and modular DNA computing

    Science.gov (United States)

    Chatterjee, Gourab; Dalchau, Neil; Muscat, Richard A.; Phillips, Andrew; Seelig, Georg

    2017-09-01

    Cells use spatial constraints to control and accelerate the flow of information in enzyme cascades and signalling networks. Synthetic silicon-based circuitry similarly relies on spatial constraints to process information. Here, we show that spatial organization can be a similarly powerful design principle for overcoming limitations of speed and modularity in engineered molecular circuits. We create logic gates and signal transmission lines by spatially arranging reactive DNA hairpins on a DNA origami. Signal propagation is demonstrated across transmission lines of different lengths and orientations and logic gates are modularly combined into circuits that establish the universality of our approach. Because reactions preferentially occur between neighbours, identical DNA hairpins can be reused across circuits. Co-localization of circuit elements decreases computation time from hours to minutes compared to circuits with diffusible components. Detailed computational models enable predictive circuit design. We anticipate our approach will motivate using spatial constraints for future molecular control circuit designs.

  18. 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 ...

  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. DNA Repair Systems

    Indian Academy of Sciences (India)

    D N Rao is a professor at the. Department of Biochemistry,. Indian Institute of Science,. Bengaluru. His research work primarily focuses on. DNA interacting proteins in prokaryotes. This includes restriction-modification systems, DNA repair proteins from pathogenic bacteria and and proteins involved in horizontal gene ...

  1. Human Mitochondrial DNA Replication

    Science.gov (United States)

    Holt, Ian J.; Reyes, Aurelio

    2012-01-01

    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  2. 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.

  3. 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

  4. 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.

  5. DNA Microarray Technology

    Science.gov (United States)

    ... this page. En Español: Tecnología de micromatriz de ADN DNA Microarray Technology What is a DNA microarray? ... this page. En Español: Tecnología de micromatriz de ADN Get Email Updates Privacy Copyright Contact Accessibility Plug- ...

  6. An electrochemical alkaline phosphatase biosensor fabricated with two DNA probes coupled with λ exonuclease.

    Science.gov (United States)

    Miao, Peng; Ning, Limin; Li, Xiaoxi; Shu, Yongqian; Li, Genxi

    2011-09-15

    In this work we have developed a novel electrochemical biosensor for the detection of alkaline phosphatase (AP) by the use of two complementary DNA probes (DNA 1 and DNA 2) coupled with λ exonuclease (λ exo). Firstly, the 5'-phosphoryl end of DNA 1 is dephosphorylated by AP. Then DNA 1 hybridizes with DNA 2, previously modified on a gold electrode surface. In this double-strand DNA, DNA 2 strand will be promptly cleaved by λ exo with its phosphoryl at the 5' end. After the DNA 2 strand is completely digested, DNA 1 will be released from the double strands and then hybridizes with another DNA 2 strand on the electrode surface, thus the cycle of the release of DNA 1 and the digestion of DNA 2 continues. Since the DNA probes may absorb hexaammineruthenium(III) chloride, the electrochemical species, and the removal of the DNA 2 strand from the electrode surface will result in the decrease of the detected electrochemical signal, which is initially activated by AP, an electrochemical biosensor to assay the activity of AP is proposed in this work. This method may have a linear detection range from 1 to 20 unit/mL with a detection limit of 0.1 unit/mL, and the detection of the enzymatic activity in complex biological fluids can also be realized. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. PIG3 Functions in DNA Damage Response through Regulating DNA-PKcs Homeostasis

    OpenAIRE

    Li, Bing; Shang, Zeng-Fu; Yin, Jiao-Jiao; Xu, Qin-Zhi; Liu, Xiao-Dan; Wang, Yu; Zhang, Shi-Meng; Guan, Hua; Zhou, Ping-Kun

    2013-01-01

    The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnor...

  8. 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

  9. Traffic signal timing manual

    Science.gov (United States)

    2008-06-01

    This report serves as a comprehensive guide to traffic signal timing and documents the tasks completed in association with its development. The focus of this document is on traffic signal control principles, practices, and procedures. It describes th...

  10. 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.

  11. 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...

  12. 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...

  13. 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.

  14. Biomedical signal processing

    CERN Document Server

    Akay, Metin

    1994-01-01

    Sophisticated techniques for signal processing are now available to the biomedical specialist! Written in an easy-to-read, straightforward style, Biomedical Signal Processing presents techniques to eliminate background noise, enhance signal detection, and analyze computer data, making results easy to comprehend and apply. In addition to examining techniques for electrical signal analysis, filtering, and transforms, the author supplies an extensive appendix with several computer programs that demonstrate techniques presented in the text.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Protoparvovirus Interactions with the Cellular DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Kinjal Majumder

    2017-10-01

    Full Text Available Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM infects murine and transformed human cells provoking a sustained DNA damage response (DDR. This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.

  20. Protoparvovirus Interactions with the Cellular DNA Damage Response

    Science.gov (United States)

    Majumder, Kinjal; Etingov, Igor

    2017-01-01

    Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection. PMID:29088070

  1. 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.

  2. DNA translocations through solid-state plasmonic nanopores.

    Science.gov (United States)

    Nicoli, Francesca; Verschueren, Daniel; Klein, Misha; Dekker, Cees; Jonsson, Magnus P

    2014-12-10

    Nanopores enable label-free detection and analysis of single biomolecules. Here, we investigate DNA translocations through a novel type of plasmonic nanopore based on a gold bowtie nanoantenna with a solid-state nanopore at the plasmonic hot spot. Plasmonic excitation of the nanopore is found to influence both the sensor signal (nanopore ionic conductance blockade during DNA translocation) and the process that captures DNA into the nanopore, without affecting the duration time of the translocations. Most striking is a strong plasmon-induced enhancement of the rate of DNA translocation events in lithium chloride (LiCl, already 10-fold enhancement at a few mW of laser power). This provides a means to utilize the excellent spatiotemporal resolution of DNA interrogations with nanopores in LiCl buffers, which is known to suffer from low event rates. We propose a mechanism based on plasmon-induced local heating and thermophoresis as explanation of our observations.

  3. 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

    2013-02-07

    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.

  4. 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

  5. The Signal Distribution System

    CERN Document Server

    Belohrad, D; CERN. Geneva. AB Department

    2005-01-01

    For the purpose of LHC signal observation and high frequency signal distribution, the Signal Distribution System (SDS) was built. The SDS can contain up to 5 switching elements, where each element allows the user to switch between one of the maximum 8 bi-directional signals. The coaxial relays are used to switch the signals. Depending of the coaxial relay type used, the transfer bandwidth can go up to 18GHz. The SDS is controllable via TCP/IP, parallel port, or locally by rotary switch.

  6. 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...

  7. 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

  8. Information Thermodynamics of Cytosine DNA Methylation.

    Science.gov (United States)

    Sanchez, Robersy; Mackenzie, Sally A

    2016-01-01

    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 rules as do current

  9. 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.

  10. 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.

  11. DNA Bending elasticity

    Science.gov (United States)

    Sivak, David Alexander

    DNA bending elasticity on length scales of tens of basepairs is of critical importance in numerous biological contexts. Even the simplest models of DNA bending admit of few simple analytic results, thus there is a need for numerical methods to calculate experimental observables, such as distance distributions, forces, FRET efficiencies, and timescales of particular large-scale motions. We have implemented and helped develop a coarse-grained representation of DNA and various other covalently-linked groups that allows simple calculation of such observables for varied experimental systems. The simple freely-jointed chain (FJC) model and extremely coarse resolution proved useful in understanding DNA threading through nanopores, identifying steric occlusion by other parts of the chain as a prime culprit for slower capture as distance to the pore decreased. Enhanced sampling techniques of a finer resolution discrete wormlike chain (WLC) model permitted calculation of cyclization rates for small chains and identified the ramifications of a thermodynamically-sound treatment of thermal melts. Adding treatment of double-stranded DNA's helical nature and single-stranded DNA provided a model system that helped demonstrate the importance of statistical fluctuations in even highly-stressed DNA mini-loops, and allowed us to verify that even these constructs show no evidence of excitation-induced softening. Additional incorporation of salt-sensitivity to the model allowed us to calculate forces and FRET efficiencies for such mini-loops and their uncircularized precursors, thereby furthering the understanding of the nature of IHF binding and bending of its recognition sequence. Adding large volume-excluding spheres linked to the ends of the dsDNA permits calculation of distance distributions and thus small-angle X-ray scattering, whereby we demonstrated the validity of the WLC in describing bending fluctuations in DNA chains as short as 42 bp. We also make important connections

  12. DNA-PK assay

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  13. DNA damage checkpoint recovery and cancer development

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Haiyong [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Zhang, Xiaoshan [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States); Teng, Lisong, E-mail: lsteng@zju.edu.cn [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Legerski, Randy J., E-mail: rlegersk@mdanderson.org [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States)

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  14. 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.

  15. DNA Mismatch Repair

    Science.gov (United States)

    MARINUS, M. G.

    2014-01-01

    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  16. "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....

  17. Apoptosis and DNA Methylation

    International Nuclear Information System (INIS)

    Meng, Huan X.; Hackett, James A.; Nestor, Colm; Dunican, Donncha S.; Madej, Monika; Reddington, James P.; Pennings, Sari; Harrison, David J.; Meehan, Richard R.

    2011-01-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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 these modifications, it is likely that the primary use of DNA vaccines may be as primers for viral-vectored vaccines, rather than as single agents. This review discusses the approaches used to enhance DNA vaccine immunogenicity, with a primary focus on fusion strategies that enhance antigen presentation....

  5. 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

  6. 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...

  7. 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...

  8. 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...

  9. 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

  10. Experimental DNA computing

    NARCIS (Netherlands)

    Henkel, Christiaan

    2005-01-01

    Because of their information storing and processing capabilities, nucleic acids are interesting building blocks for molecular scale computers. Potential applications of such DNA computers range from massively parallel computation to computational gene therapy. In this thesis, several implementations

  11. DNA Vaccines Against Anthrax

    National Research Council Canada - National Science Library

    Galloway, Darrell R; Baillie, Les

    2004-01-01

    DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development...

  12. 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...

  13. 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

  14. 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.)

  15. 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....

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. Effect of irradiation on the detection of bacterial DNA in contaminated food samples by DNA hybridization

    International Nuclear Information System (INIS)

    Rowe, T.F.; Towner, K.J.

    1994-01-01

    A membrane-based DNA hybridization technique was used in a model system to examine the effect of irradiation treatment on the detection of bacterial contamination in foodstuffs. Although hybridization signals were reduced compared with otherwise identical unirradiated food samples, artificial contamination levels in excess of 10 5 cfu per test could be distinguished in 12 of the 13 foods examined following the irradiation process. In no case were viable bacteria detected following irradiation treatment. (Author)

  1. 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.

  2. 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

  3. Toward larger DNA origami.

    Science.gov (United States)

    Marchi, Alexandria N; Saaem, Ishtiaq; Vogen, Briana N; Brown, Stanley; LaBean, Thomas H

    2014-10-08

    Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

  4. DNA Polymerase Gamma in Mitochondrial DNA Replication and Repair

    Directory of Open Access Journals (Sweden)

    William C. Copeland

    2003-01-01

    Full Text Available Mutations in mitochondrial DNA (mtDNA are associated with aging, and they can cause tissue degeneration and neuromuscular pathologies known as mitochondrial diseases. Because DNA polymerase γ (pol γ is the enzyme responsible for replication and repair of mitochondrial DNA, the burden of faithful duplication of mitochondrial DNA, both in preventing spontaneous errors and in DNA repair synthesis, falls on pol γ. Investigating the biological functions of pol γ and its inhibitors aids our understanding of the sources of mtDNA mutations. In animal cells, pol γ is composed of two subunits, a larger catalytic subunit of 125–140 kDa and second subunit of 35–55 kDa. The catalytic subunit contains DNA polymerase activity, 3’-5’ exonuclease activity, and a 5’-dRP lyase activity. The accessory subunit is required for highly processive DNA synthesis and increases the affinity of pol gamma to the DNA.

  5. Ribonucleotide triggered DNA damage and RNA-DNA damage responses.

    Science.gov (United States)

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.

  6. 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

  7. Identifying Fishes through DNA Barcodes and Microarrays.

    Science.gov (United States)

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N; Weber, Hannes; Blohm, Dietmar

    2010-09-07

    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.

  8. Identifying Fishes through DNA Barcodes and Microarrays.

    Directory of Open Access Journals (Sweden)

    Marc Kochzius

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  9. Gold-based optical biosensor for single-mismatched DNA detection using salt-induced hybridization

    DEFF Research Database (Denmark)

    Zhan, Zongrui; Ma, Xingyi; Cao, Cuong

    2011-01-01

    In this study, a gold nanoparticle (Au-NP)-based detection method for sensitive and specific DNA-based diagnostic applications is described. A sandwich format consisting of Au-NPs/DNA/PMP (Streptavidin-coated MagnetSphere Para-Magnetic Particles) was fabricated. PMPs captured and separated target...... DNA while Au-NPs modified with oligonucleotide detection sequences played a role in recognition and signal production. Due to the much lower stability of mismatched DNA strands caused by unstable duplex structures in solutions of relatively low salt concentration, hybridization efficiency...... in the presence of different buffers was well investigated, and thus, the optimized salt concentration allowed for discrimination of single-mismatched DNA (MMT) from perfectly matched DNA (PMT). Therefore, quantitative information concerning the target analyte was translated into a colorimetric signal, which...

  10. DNA-Damage-Induced Type I Interferon Promotes Senescence and Inhibits Stem Cell Function

    Directory of Open Access Journals (Sweden)

    Qiujing Yu

    2015-05-01

    Full Text Available Expression of type I interferons (IFNs can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

  11. Chiroptical properties, binding affinity, and photostability of a conjugated zinc porphyrin dimer complexed with left-handed Z-DNA and right-handed B-DNA.

    Science.gov (United States)

    Choi, Jung Kyu; Reed, Aisha; Balaz, Milan

    2014-01-14

    We have studied the UV-vis absorption and chiroptical properties, binding affinity and photostability of a conjugated positively charged butadiyne-linked Zn(ii) porphyrin dimer bound to DNA sequence poly(dG-dC)2. Right-handed B-DNA, spermine-induced Z-DNA and Co(iii)-induced Z-DNA have been explored. Resonance light scattering (RLS) spectra showed formation of porphyrin aggregates in the presence of all DNA forms with the largest aggregates formed with B-DNA. The porphyrin dimer gave rise to induced bisignate circular dichroism (CD) signals in the presence of the left-handed Z-DNA conformations. On the other hand, the dimer stayed nearly chiroptically silent when complexed with the B-form of poly(dG-dC)2. Our results indicated that the conjugated Zn(ii) porphyrin dimer can be used as a sensor for the chiroptical detection of Z-DNA in the visible (400-500 nm) and near-infrared region of the electromagnetic spectrum (700-800 nm). The helicity of DNA had little effect on the dimer binding affinities. The photostability of the porphyrin dimer complexed with any form of DNA was higher than that of the free molecule. The porphyrin dimer bound to Z-DNA exhibited slower photobleaching than the B-DNA dimer complex.

  12. 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

  13. 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

  14. Geolocation of RF signals

    CERN Document Server

    Progri, Ilir

    2011-01-01

    ""Geolocation of RF Signals - Principles and Simulations"" offers an overview of the best practices and innovative techniques in the art and science of geolocation over the last twenty years. It covers all research and development aspects including theoretical analysis, RF signals, geolocation techniques, key block diagrams, and practical principle simulation examples in the frequency band from 100 MHz to 18 GHz or even 60 GHz. Starting with RF signals, the book progressively examines various signal bands - such as VLF, LF, MF, HF, VHF, UHF, L, S, C, X, Ku, and, K and the corresponding geoloca

  15. Supramolecular Complexes of DNA

    Science.gov (United States)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  16. Metric representation of DNA sequences.

    Science.gov (United States)

    Wu, Z B

    2000-07-01

    A metric representation of DNA sequences is borrowed from symbolic dynamics. In view of this method, the pattern seen in the chaos game representation of DNA sequences is explained as the suppression of certain nucleotide strings in the DNA sequences. Frequencies of short nucleotide strings and suppression of the shortest ones in the DNA sequences can be determined by using the metric representation.

  17. Cytomegalovirus Infection Drives Adaptive Epigenetic Diversification of NK Cells with Altered Signaling and Effector Function

    OpenAIRE

    Schlums, Heinrich; Cichocki, Frank; Tesi, Bianca; Theorell, Jakob; Beziat, Vivien; Holmes, Tim D.; Han, Hongya; Chiang, Samuel C.C.; Foley, Bree; Mattsson, Kristin; Larsson, Stella; Schaffer, Marie; Malmberg, Karl-Johan; Ljunggren, Hans-Gustaf; Miller, Jeffrey S.

    2015-01-01

    The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins after human cytomegalovirus (HCMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hyperme-thylation. Genome-wide DNA methylation patterns were strikingly similar between HCMV-a...

  18. Cip29 is phosphorylated following activation of the DNA damage response in Xenopus egg extracts.

    Directory of Open Access Journals (Sweden)

    Janet Holden

    Full Text Available Acting through a complex signalling network, DNA lesions trigger a range of cellular responses including DNA repair, cell cycle arrest, altered gene expression and cell death, which help to limit the mutagenic effects of such DNA damage. RNA processing factors are increasingly being recognised as important targets of DNA damage signalling, with roles in the regulation of gene expression and also more directly in the promotion of DNA repair. In this study, we have used a Xenopus laevis egg extract system to analyse the DNA damage-dependent phosphorylation of a putative RNA export factor, Cip29. We have found that Cip29 is rapidly phosphorylated in response to DNA double-strand breaks in this experimental system. We show that the DNA damage-inducible modification of Cip29 is dependent on the activity of the key double-strand break response kinase, ATM, and we have identified a conserved serine residue as a damage-dependent phosphorylation site. Finally, we have determined that Cip29 is not required for efficient DNA end-joining in egg extracts. Taken together, these data identify Cip29 as a novel target of the DNA damage response and suggest that the damage-dependent modification of Cip29 may relate to a role in the regulation of gene expression after DNA damage.

  19. Target specificity of mammalian DNA methylation and demethylation machinery.

    Science.gov (United States)

    Ravichandran, M; Jurkowska, R Z; Jurkowski, T P

    2018-02-28

    DNA methylation is an essential epigenetic modification for mammalian embryonic development and biology. The DNA methylation pattern across the genome, together with other epigenetic signals, is responsible for the transcriptional profile of a cell and thus preservation of the cell's identity. Equally, the family of TET enzymes which triggers the initiation of the DNA demethylation cycle plays a vital role in the early embryonic development and a lack of these enzymes at later stages leads to a diseased state and dysregulation of the epigenome. DNA methylation has long been considered a very stable modification; however, it has become increasingly clear that for the establishment and maintenance of the methylation pattern, both generation of DNA methylation and its removal are important, and that a delicate balance of ongoing DNA methylation and demethylation shapes the final epigenetic methylation pattern of the cell. Although this epigenetic mark has been investigated in great detail, it still remains to be fully understood how specific DNA methylation imprints are precisely generated, maintained, read or erased in the genome. Here, we provide a biochemist's view on how both DNA methyltransferases and TET enzymes are recruited to specific genomic loci, and how their chromatin interactions, as well as their intrinsic sequence specificities and molecular mechanisms, contribute to the methylation pattern of the cell.

  20. 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

  1. DNA triplex structures in neurodegenerative disorder, Friedreich's ...

    Indian Academy of Sciences (India)

    canonical B-DNA structure or 'unusual' DNA structure. The unusual DNA structures like DNA-hairpin, cruciform, Z-DNA, triplex and tetraplex are represented as hotspots of chromosomal breaks, homologous recombination and gross ...

  2. 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.

  3. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb...

  4. Defects of mitochondrial DNA replication.

    Science.gov (United States)

    Copeland, William C

    2014-09-01

    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease. © The Author(s) 2014.

  5. Physical mapping of 5S and 45S rDNA in Chrysanthemum and ...

    Indian Academy of Sciences (India)

    2012-08-09

    Aug 9, 2012 ... The present study has shown that fluorescence in situ hybridization (FISH) physical mapping of rDNA sites is a valuable method to gain insight into the genome in Chrysan- themum and related genera of the Anthemideae. FISH signals revealed that 45S rDNA sites were localized on the terminal regions of ...

  6. DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing

    NARCIS (Netherlands)

    Stoddart, David; Franceschini, Lorenzo; Heron, Andrew; Bayley, Hagan; Maglia, Giovanni

    2015-01-01

    In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we

  7. 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...

  8. ROS signalling in the biology of cancer.

    Science.gov (United States)

    Moloney, Jennifer N; Cotter, Thomas G

    2017-06-03

    Increased reactive oxygen species (ROS) production has been detected in various cancers and has been shown to have several roles, for example, they can activate pro-tumourigenic signalling, enhance cell survival and proliferation, and drive DNA damage and genetic instability. Counterintuitively ROS can also promote anti-tumourigenic signalling, initiating oxidative stress-induced tumour cell death. Tumour cells express elevated levels of antioxidant proteins to detoxify elevated ROS levels, establish a redox balance, while maintaining pro-tumourigenic signalling and resistance to apoptosis. Tumour cells have an altered redox balance to that of their normal counterparts and this identifies ROS manipulation as a potential target for cancer therapies. This review discusses the generation and sources of ROS within tumour cells, the regulation of ROS by antioxidant defence systems, as well as the effect of elevated ROS production on their signalling targets in cancer. It also provides an insight into how pro- and anti-tumourigenic ROS signalling pathways could be manipulated in the treatment of cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Dissection of DNA damage responses using multiconditional genetic interaction maps

    NARCIS (Netherlands)

    Guénolé, Aude

    2013-01-01

    To protect the genome, cells have evolved a diverse set of pathways designed to sense, signal, and repair multiple types of DNA damage. To assess the degree of coordination and crosstalk among these pathways, we systematically mapped changes in the cell's genetic network across a panel of different

  10. 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....

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    acids allows only for the superimposition of punctual and loosely positioned signals to conserved amino acid sequences. The degree of flexibility of the genetic code is low or average with respect to several classes of alternative codes. This result is consistent with the view that DNA structure...

  12. The signal peptidase II (lsp) gene of Bacillus subtilis

    NARCIS (Netherlands)

    Pragai, Z; Tjalsma, H; Bolhuis, A; vanDijl, JM; Venema, G; Bron, S

    The gene encoding the type II signal peptidase (SPase III) of Bacillus subtilis was isolated by screening a genomic DNA library of this bacterium for the ability of increase the levels of globomycin resistance in Escherichia coli, and to complement the growth deficiency at the non-permissive

  13. 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)

  14. Targeting RAGE Signaling in Inflammatory Disease.

    Science.gov (United States)

    Hudson, Barry I; Lippman, Marc E

    2018-01-29

    The receptor for advanced glycation end-products (RAGE) is a multiligand pattern recognition receptor implicated in diverse chronic inflammatory states. RAGE binds and mediates the cellular response to a range of damage-associated molecular pattern molecules (DAMPs) including AGEs, HMGB1, S100s, and DNA. RAGE can also act as an innate immune sensor of microbial pathogen-associated molecular pattern molecules (PAMPs) including bacterial endotoxin, respiratory viruses, and microbial DNA. RAGE is expressed at low levels under normal physiology, but it is highly upregulated under chronic inflammation because of the accumulation of various RAGE ligands. Blocking RAGE signaling in cell and animal models has revealed that targeting RAGE impairs inflammation and progression of diabetic vascular complications, cardiovascular disease (CVD), and cancer progression and metastasis. The clinical relevance of RAGE in inflammatory disease is being demonstrated in emerging clinical trials of novel small-molecule RAGE inhibitors.

  15. 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

  16. Biomolecular Simulation of Base Excision Repair and Protein Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Straatsma, TP; McCammon, J A; Miller, John H; Smith, Paul E; Vorpagel, Erich R; Wong, Chung F; Zacharias, Martin W

    2006-03-03

    The goal of the Biomolecular Simulation of Base Excision Repair and Protein Signaling project is to enhance our understanding of the mechanism of human polymerase-β, one of the key enzymes in base excision repair (BER) and the cell-signaling enzymes cyclic-AMP-dependent protein kinase. This work used molecular modeling and simulation studies to specifically focus on the • dynamics of DNA and damaged DNA • dynamics and energetics of base flipping in DNA • mechanism and fidelity of nucleotide insertion by BER enzyme human polymerase-β • mechanism and inhibitor design for cyclic-AMP-dependent protein kinase. Molecular dynamics simulations and electronic structure calculations have been performed using the computer resources at the Molecular Science Computing Facility at the Environmental Molecular Sciences Laboratory.

  17. Signaling in symbiosis

    NARCIS (Netherlands)

    Limpens, E.H.M.; Bisseling, T.

    2003-01-01

    In recent years, the major focus in nodulation research has been on the genetic dissection of Nod-factor signaling. Components of this pathway appear to be shared with signaling processes that are induced during the formation of mycorrhiza. With the cloning of orthologs of the NIN and DMI2 genes

  18. 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.

  19. Digital Signal Processors

    Indian Academy of Sciences (India)

    A computer controlling the motion of a satellite should acquire signals from the satellite while it is in motion, compute corrections (if required) to the trajectory and send control signals back within a specified time for effective control. Delays may be fatal to ..... emulators and system evaluation tools have facilitated concurrent.

  20. 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

  1. Second-hand signals

    DEFF Research Database (Denmark)

    Bergenholtz, Carsten

    2014-01-01

    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...

  2. Quantum cloning without signaling

    OpenAIRE

    Gisin, Nicolas

    1998-01-01

    Perfect Quantum Cloning Machines (QCM) would allow to use quantum nonlocality for arbitrary fast signaling. However perfect QCM cannot exist. We derive a bound on the fidelity of QCM compatible with the no-signaling constraint. This bound equals the fidelity of the Bu\\v{z}ek-Hillery QCM.

  3. 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

  4. 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

  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. Capacitive DNA sensor for rapid and sensitive detection of whole genome human herpesvirus-1 dsDNA in serum.

    Science.gov (United States)

    Cheng, Cheng; Oueslati, Rania; Wu, Jayne; Chen, Jiangang; Eda, Shigetoshi

    2017-06-01

    This work presents a rapid, highly sensitive, low-cost, and specific capacitive DNA sensor for detection of whole genome human herpesvirus-1 DNA. This sensor is capable of direct DNA detection with a response time of 30 s, and it can be used to test standard buffer or serum samples. The sensing approach for DNA detection is based on alternating current (AC) electrokinetics. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate DNA hybridization. The same applied AC signal also directly measures the hybridization of target with the probe on the sensor surface. Experiments are conducted to optimize the AC signal, as well as the buffers for probe immobilization and target DNA hybridization. The assay is highly sensitive and specific, with no response to human herpesvirus-2 DNA at 5 ng/mL and a LOD of 1.0 pg/mL (6.5 copies/μL or 10.7 aM) in standard buffer. When testing the double stranded (ds) DNA spiked in human serum samples, the sensor yields a LOD of 20.0 pg/mL (129.5 copies/μL or 0.21 femtomolar (fM)) in neat serum. In this work, the target is whole genome dsDNA, consequently the test can be performed without the use of enzyme or amplification, which considerably simplifies the sensor operation and is highly suitable for point of care disease diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Glutathione: new roles in redox signalling for an old antioxidant

    Directory of Open Access Journals (Sweden)

    KATIA eAQUILANO

    2014-08-01

    Full Text Available The physiological roles played by the tripeptide glutathione have greatly advanced over the past decades superimposing the research on free radicals, oxidative stress and, more recently, redox signalling. In particular, GSH is involved in nutrient metabolism, antioxidant defence and regulation of cellular metabolic functions ranging from gene expression, DNA and protein synthesis to signal transduction, cell proliferation and apoptosis. This review will be focused on the role of GSH in cell signalling by analysing the more recent advancements about its capability to modulate nitroxidative stress, autophagy and viral infection.

  8. 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.

  9. Neutron signal transfer analysis

    CERN Document Server

    Pleinert, H; Lehmann, E

    1999-01-01

    A new method called neutron signal transfer analysis has been developed for quantitative determination of hydrogenous distributions from neutron radiographic measurements. The technique is based on a model which describes the detector signal obtained in the measurement as a result of the action of three different mechanisms expressed by signal transfer functions. The explicit forms of the signal transfer functions are determined by Monte Carlo computer simulations and contain only the distribution as a variable. Therefore an unknown distribution can be determined from the detector signal by recursive iteration. This technique provides a simple and efficient tool for analysis of this type while also taking into account complex effects due to the energy dependency of neutron interaction and single and multiple scattering. Therefore this method provides an efficient tool for precise quantitative analysis using neutron radiography, as for example quantitative determination of moisture distributions in porous buil...

  10. 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 ...

  11. 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.

  12. 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

  13. Bioelectronic Interface Connecting Reversible Logic Gates Based on Enzyme and DNA Reactions.

    Science.gov (United States)

    Guz, Nataliia; Fedotova, Tatiana A; Fratto, Brian E; Schlesinger, Orr; Alfonta, Lital; Kolpashchikov, Dmitry M; Katz, Evgeny

    2016-07-18

    It is believed that connecting biomolecular computation elements in complex networks of communicating molecules may eventually lead to a biocomputer that can be used for diagnostics and/or the cure of physiological and genetic disorders. Here, a bioelectronic interface based on biomolecule-modified electrodes has been designed to bridge reversible enzymatic logic gates with reversible DNA-based logic gates. The enzyme-based Fredkin gate with three input and three output signals was connected to the DNA-based Feynman gate with two input and two output signals-both representing logically reversible computing elements. In the reversible Fredkin gate, the routing of two data signals between two output channels was controlled by the control signal (third channel). The two data output signals generated by the Fredkin gate were directed toward two electrochemical flow cells, responding to the output signals by releasing DNA molecules that serve as the input signals for the next Feynman logic gate based on the DNA reacting cascade, producing, in turn, two final output signals. The Feynman gate operated as the controlled NOT gate (CNOT), where one of the input channels controlled a NOT operation on another channel. Both logic gates represented a highly sophisticated combination of input-controlled signal-routing logic operations, resulting in redirecting chemical signals in different channels and performing orchestrated computing processes. The biomolecular reaction cascade responsible for the signal processing was realized by moving the solution from one reacting cell to another, including the reacting flow cells and electrochemical flow cells, which were organized in a specific network mimicking electronic computing circuitries. The designed system represents the first example of high complexity biocomputing processes integrating enzyme and DNA reactions and performing logically reversible signal processing. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. 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

  15. 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.

  16. 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....

  17. 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.

  18. 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.

  19. DNA templated magnetic nanoparticles

    Science.gov (United States)

    Kinsella, Joseph M.

    Recent discoveries in nanoscience are predicted to potentially revolutionize future technologies in an extensive number of fields. These developments are contingent upon discovering new and often unconventional methods to synthesize and control nanoscale components. Nature provides several examples of working nanotechnology such as the use of programmed self assembly to build and deconstruct complex molecular systems. We have adopted a method to control the one dimensional assembly of magnetic nanoparticles using DNA as a scaffold molecule. With this method we have demonstrated the ability to organize 5 nm particles into chains that stretch up to ˜20 mum in length. One advantage of using DNA compared is the ability of the molecule to interact with other biomolecules. After assembling particles onto DNA we have been able to cleave the molecule into smaller fragments using restriction enzymes. Using ligase enzymes we have re-connected these fragments, coated with either gold or iron oxide, to form long one-dimensional arrangements of the two different types of nanoparticles on a single molecular guide. We have also created a sensitive magnetic field sensor by incorporating magnetic nanoparticle coated DNA strands with microfabricated electrodes. The IV characteristics of the aligned nanoparticles are dependant on the magnitude of an externally applied magnetic field. This transport phenomenon known as tunneling magnetoresistance (TMR) shows room temperature resistance of our devices over 80% for cobalt ferrite coated DNA when a field of 20 kOe is applied. In comparison, studies using two dimensional nanoparticle films of irox oxides xii only exhibit a 35% MR effect. Confinement into one dimension using the DNA guide produces a TMR mechanism which produces significant increases in magnetoresistance. This property can be utilized for applications in magnetic field sensing, data storage, and logic elements.

  20. Nanowire-nanopore transistor sensor for DNA detection during translocation

    Science.gov (United States)

    Xie, Ping; Xiong, Qihua; Fang, Ying; Qing, Quan; Lieber, Charles

    2011-03-01

    Nanopore sequencing, as a promising low cost, high throughput sequencing technique, has been proposed more than a decade ago. Due to the incompatibility between small ionic current signal and fast translocation speed and the technical difficulties on large scale integration of nanopore for direct ionic current sequencing, alternative methods rely on integrated DNA sensors have been proposed, such as using capacitive coupling or tunnelling current etc. But none of them have been experimentally demonstrated yet. Here we show that for the first time an amplified sensor signal has been experimentally recorded from a nanowire-nanopore field effect transistor sensor during DNA translocation. Independent multi-channel recording was also demonstrated for the first time. Our results suggest that the signal is from highly localized potential change caused by DNA translocation in none-balanced buffer condition. Given this method may produce larger signal for smaller nanopores, we hope our experiment can be a starting point for a new generation of nanopore sequencing devices with larger signal, higher bandwidth and large-scale multiplexing capability and finally realize the ultimate goal of low cost high throughput sequencing.

  1. Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA.

    Directory of Open Access Journals (Sweden)

    Ian C Nova

    Full Text Available Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (cis and trans by varying cis and trans [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl-] produces only small effects. Good signal-to-noise can be maintained with cis [Cl-] as small as 20 mM, if the concentration of KCl on the trans side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.

  2. 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.

  3. Highly sensitive surface plasmon resonance biosensor for the detection of HIV-related DNA based on dynamic and structural DNA nanodevices.

    Science.gov (United States)

    Diao, Wei; Tang, Min; Ding, Shijia; Li, Xinmin; Cheng, Wenbin; Mo, Fei; Yan, Xiaoyu; Ma, Hongmin; Yan, Yurong

    2018-02-15

    Early detection, diagnosis and treatment of human immune deficiency virus (HIV) infection is the key to reduce acquired immunodeficiency syndrome (AIDS) mortality. In our research, an innovative surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions (ESDRs) and double-layer DNA tetrahedrons (DDTs). ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA, leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA (dsDNA) products. Subsequently, the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures. Due to the high efficiency of ESDRs and large molecular weight of DDTs, the SPR response signal was enhanced dramatically. The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1pM to 150nM with a detection limit of 48fM. In addition, the whole detecting process can be accomplished in 60min with high accuracy and duplicability. In particular, the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample, indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Orexin/Hypocretin Signaling.

    Science.gov (United States)

    Kukkonen, Jyrki P

    Orexin/hypocretin peptide (orexin-A and orexin-B) signaling is believed to take place via the two G-protein-coupled receptors (GPCRs), named OX 1 and OX 2 orexin receptors, as described in the previous chapters. Signaling of orexin peptides has been investigated in diverse endogenously orexin receptor-expressing cells - mainly neurons but also other types of cells - and in recombinant cells expressing the receptors in a heterologous manner. Findings in the different systems are partially convergent but also indicate cellular background-specific signaling. The general picture suggests an inherently high degree of diversity in orexin receptor signaling.In the current chapter, I present orexin signaling on the cellular and molecular levels. Discussion of the connection to (potential) physiological orexin responses is only brief since these are in focus of other chapters in this book. The same goes for the post-synaptic signaling mechanisms, which are dealt with in Burdakov: Postsynaptic actions of orexin. The current chapter is organized according to the tissue type, starting from the central nervous system. Finally, receptor signaling pathways are discussed across tissues, cell types, and even species.

  5. NMR studies of artificial double-crossover DNA tiles.

    Science.gov (United States)

    Veerapandian, Murugan; Kim, Byeonghoon; Amin, Rashid; Lee, Junwye; Yun, Kyusik; Park, Sung Ha

    2012-03-01

    This report documents the design and characterization of DNA molecular nanoarchitectures consisting of artificial double crossover DNA tiles with different geometry and chemistry. The Structural characterization of the unit tiles, including normal, biotinylated and hairpin loop structures, are morphologically studied by atomic force microscopy. The specific proton resonance of the individual tiles and their intra/inter nucleotide relationships are verified by proton nuclear magnetic resonance spectroscopy and 2-dimensional correlation spectral studies, respectively. Significant up-field and down-field shifts in the resonance signals of the individual residues at various temperatures are discussed. The results suggest that with artificially designed DNA tiles it is feasible to obtain structural information of the relative base sequences. These tiles were later fabricated into 2D DNA lattice structures for specific applications such as protein arrangement by biotinylated bulged loops or pattern generation using a hairpin structure.

  6. 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.

  7. Electrochemical Detection of DNA Using Nanomaterials Based Sensors

    Science.gov (United States)

    Marín, Sergio; Merkoçi, Arben

    The detection of non-amplified DNA sequences plays a crucial role in the rapid diagnosis of genetic-related diseases especially for early stage treatment. Among the various biosensors that have been used for DNA detection, electrochemical sensors show great promise because they present efficient signal transduction and are capable of precise DNA recognition at a relatively low cost in addition to the broad range of designs with interest to be applied in different kinds of samples. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific electrochemical sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed electrochemical DNA detection essential for point of care clinical diagnostics.

  8. DNA nanomechanical devices for molecular biology and DNA nanotechnology

    Science.gov (United States)

    Gu, Hongzhou

    The aim of nanotechnology is to put specific atomic and molecular species where we want them, when we want them there. Achieving such a dynamic and functional control could lead to molecular programming. Structural DNA nanotechnology offers a powerful route to this goal by combining stable branched DNA motifs with cohesive ends to produce objects, programmed nanomechanical devices and fixed or modified patterned lattices. In Chapter II, a two-armed nanorobotic device is built based on a DNA origami substrate. The arms face each other, ready to capture different DNA nanostructures into distinguishable nanopatterns. Combining with a simple error-correction protocol, we are able to achieve this goal in a nearly flawless fashion. In Chapter III, a DNA-based programmable assembly line is developed by combining three PX/JX2 cassettes and a novel DNA walker on a DNA origami substrate. This programmable assembly line can generate eight products by switching the cassettes to PX (ON) or JX2 (OFF) state when the DNA walker passes by. DNA nanomechanical devices hold the promise of controlling structure and performing exquisitely fine measurements on the molecular scale. Several DNA nanomechanical devices based on different biochemistry phenomena have been reported before. In Chapter IV, a scissors-based DNA device is built to measure the work that can be done by a DNA-bending protein (MutS) when it binds to DNA.

  9. Signal flow analysis

    CERN Document Server

    Abrahams, J R; Hiller, N

    1965-01-01

    Signal Flow Analysis provides information pertinent to the fundamental aspects of signal flow analysis. This book discusses the basic theory of signal flow graphs and shows their relation to the usual algebraic equations.Organized into seven chapters, this book begins with an overview of properties of a flow graph. This text then demonstrates how flow graphs can be applied to a wide range of electrical circuits that do not involve amplification. Other chapters deal with the parameters as well as circuit applications of transistors. This book discusses as well the variety of circuits using ther

  10. 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.

  11. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    Directory of Open Access Journals (Sweden)

    Eun Ah Song

    2016-08-01

    Full Text Available The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA, shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.

  12. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells.

    Science.gov (United States)

    Song, Eun Ah; Kim, Hyeyoung

    2016-08-03

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells' molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.

  13. New tangles in the auxin signaling web

    Science.gov (United States)

    Wright, R. Clay

    2015-01-01

    Plants use auxin to relay critical information that shapes their growth and development. Auxin perception and transcriptional activation are mediated by the degradation of Aux/IAA repressor proteins. Degradation of Aux/IAAs relieves repression on Auxin Response Factors (ARFs), which bind DNA sequences called Auxin Response Elements (AuxREs). In most higher plant genomes, multiple paralogs exist for each part of the auxin nuclear signaling pathway. This potential combinatorial diversity in signaling pathways likely contributes to the myriad of context-specific responses to auxin. Recent structures of several domains from ARF proteins have exposed new modes of ARF dimerization, new models for ARF-AuxRE specificity, and the strong likelihood of larger order complexes formed by ARF and Aux/IAA homo- and heteromultimerization. Preliminary experiments support a role for these novel interactions in planta, further increasing the potential architectural complexity of this seemingly simple pathway. PMID:25750737

  14. Proteomics insights into DNA damage response and translating this knowledge to clinical strategies

    DEFF Research Database (Denmark)

    von Stechow, Louise; Olsen, Jesper V

    2017-01-01

    Genomic instability is a critical driver in the process of cancer formation. At the same time, inducing DNA damage by irradiation or genotoxic compounds constitutes a key therapeutic strategy to kill fast-dividing cancer cells. Sensing of DNA lesions initiates a complex set of signalling pathways......, collectively known as the DNA damage response (DDR). Deciphering DDR signalling pathways with high-throughput technologies could provide insights into oncogenic transformation, metastasis formation and therapy responses, and could build a basis for better therapeutic interventions in cancer treatment. Mass...

  15. 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.

  16. 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

  17. DNA polymerase having modified nucleotide binding site for DNA sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  18. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ...

    Indian Academy of Sciences (India)

    fluoroquinolone complexes. Mohan N ... DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as their HS DNA cleavage properties by means of agarose gel ...

  19. Viruses and the DNA Damage Response: Activation and Antagonism.

    Science.gov (United States)

    Luftig, Micah A

    2014-11-01

    Viruses must interact with their hosts in order to replicate; these interactions often provoke the evolutionarily conserved response to DNA damage, known as the DNA damage response (DDR). The DDR can be activated by incoming viral DNA, during the integration of retroviruses, or in response to the aberrant DNA structures generated upon replication of DNA viruses. Furthermore, DNA and RNA viral proteins can induce the DDR by promoting inappropriate S phase entry, by modifying cellular DDR factors directly, or by unintentionally targeting host DNA. The DDR may be antiviral, although viruses often require proximal DDR activation of repair and recombination factors to facilitate replication as well as downstream DDR signaling suppression to ensure cell survival. An unintended consequence of DDR attenuation during infection is the long-term survival and proliferation of precancerous cells. Therefore, the molecular basis for DDR activation and attenuation by viruses remains an important area of study that will likely provide key insights into how viruses have evolved with their hosts.

  20. Purinergic signalling and diabetes

    DEFF Research Database (Denmark)

    Burnstock, Geoffrey; Novak, Ivana

    2013-01-01

    The pancreas is an organ with a central role in nutrient breakdown, nutrient sensing and release of hormones regulating whole body nutrient homeostasis. In diabetes mellitus, the balance is broken-cells can be starving in the midst of plenty. There are indications that the incidence of diabetes...... molecules in purinergic signalling cascades. This signalling takes place at the level of the pancreas, where the close apposition of various cells-endocrine, exocrine, stromal and immune cells-contributes to the integrated function. Following an introduction to diabetes, the pancreas and purinergic...... signalling, we will focus on the role of purinergic signalling and its changes associated with diabetes in the pancreas and selected tissues/organ systems affected by hyperglycaemia and other stress molecules of diabetes. Since this is the first review of this kind, a comprehensive historical angle is taken...

  1. Topological signal processing

    CERN Document Server

    Robinson, Michael

    2014-01-01

    Signal processing is the discipline of extracting information from collections of measurements. To be effective, the measurements must be organized and then filtered, detected, or transformed to expose the desired information.  Distortions caused by uncertainty, noise, and clutter degrade the performance of practical signal processing systems. In aggressively uncertain situations, the full truth about an underlying signal cannot be known.  This book develops the theory and practice of signal processing systems for these situations that extract useful, qualitative information using the mathematics of topology -- the study of spaces under continuous transformations.  Since the collection of continuous transformations is large and varied, tools which are topologically-motivated are automatically insensitive to substantial distortion. The target audience comprises practitioners as well as researchers, but the book may also be beneficial for graduate students.

  2. Transmembrane Signalling: Membrane messengers

    Science.gov (United States)

    Cockroft, Scott L.

    2017-05-01

    Life has evolved elaborate means of communicating essential chemical information across cell membranes. Inspired by biology, two new artificial mechanisms have now been developed that use synthetic messenger molecules to relay chemical signals into or across lipid membranes.

  3. Ultrahigh bandwidth signal processing

    DEFF Research Database (Denmark)

    Oxenløwe, Leif Katsuo

    2016-01-01

    Optical time lenses have proven to be very versatile for advanced optical signal processing. Based on a controlled interplay between dispersion and phase-modulation by e.g. four-wave mixing, the processing is phase-preserving, an hence useful for all types of data signals including coherent multi......-level modulation founats. This has enabled processing of phase-modulated spectrally efficient data signals, such as orthogonal frequency division multiplexed (OFDM) signa In that case, a spectral telescope system was used, using two time lenses with different focal lengths (chirp rates), yielding a spectral...... regeneratio These operations require a broad bandwidth nonlinear platform, and novel photonic integrated nonlinear platform like aluminum gallium arsenide nano-waveguides used for 1.28 Tbaud optical signal processing will be described....

  4. Acoustic MIMO signal processing

    CERN Document Server

    Huang, Yiteng; Chen, Jingdong

    2006-01-01

    A timely and important book addressing a variety of acoustic signal processing problems under multiple-input multiple-output (MIMO) scenarios. It uniquely investigates these problems within a unified framework offering a novel and penetrating analysis.

  5. Signals and systems

    CERN Document Server

    Rao, K Deergha

    2018-01-01

    This textbook covers the fundamental theories of signals and systems analysis, while incorporating recent developments from integrated circuits technology into its examples. Starting with basic definitions in signal theory, the text explains the properties of continuous-time and discrete-time systems and their representation by differential equations and state space. From those tools, explanations for the processes of Fourier analysis, the Laplace transform, and the z-Transform provide new ways of experimenting with different kinds of time systems. The text also covers the separate classes of analog filters and their uses in signal processing applications. Intended for undergraduate electrical engineering students, chapter sections include exercise for review and practice for the systems concepts of each chapter. Along with exercises, the text includes MATLAB-based examples to allow readers to experiment with signals and systems code on their own. An online repository of the MATLAB code from this textbook can...

  6. Signal Station Inspection Reports

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Handwritten reports resulting from detailed inspections of US Army Signal Service Stations, 1871-1889. Features reported included instrument exposure and condition,...

  7. Foundations of signal processing

    CERN Document Server

    Vetterli, Martin; Goyal, Vivek K

    2014-01-01

    This comprehensive and engaging textbook introduces the basic principles and techniques of signal processing, from the fundamental ideas of signals and systems theory to real-world applications. Students are introduced to the powerful foundations of modern signal processing, including the basic geometry of Hilbert space, the mathematics of Fourier transforms, and essentials of sampling, interpolation, approximation and compression. The authors discuss real-world issues and hurdles to using these tools, and ways of adapting them to overcome problems of finiteness and localisation, the limitations of uncertainty and computational costs. Standard engineering notation is used throughout, making mathematical examples easy for students to follow, understand and apply. It includes over 150 homework problems and over 180 worked examples, specifically designed to test and expand students' understanding of the fundamentals of signal processing, and is accompanied by extensive online materials designed to aid learning, ...

  8. Traffic Signal Cycle Lengths

    Data.gov (United States)

    Town of Chapel Hill, North Carolina — Traffic signal location list for the town of Chapel Hill. This data set includes light cycle information as well as as intersection information.The Town of Chapel...

  9. Population distribution of 45S and 5S rDNA in golden mahseer, Tor ...

    Indian Academy of Sciences (India)

    But FISH data showed significant difference between the populations, four of the five populations showed six 18S (three pairs) and two 5S (one pair) signals with positional polymorphism, while one population showed eight 18S and four 5S signals, respectively. Southern blot data confirms that 5S rDNA clusters present on ...

  10. Fungal DNA barcoding.

    Science.gov (United States)

    Xu, Jianping

    2016-11-01

    Fungi are ubiquitous in both natural and human-made environments. They play important roles in the health of plants, animals, and humans, and in broad ecosystem functions. Thus, having an efficient species-level identification system could significantly enhance our ability to treat fungal diseases and to monitor the spatial and temporal patterns of fungal distributions and migrations. DNA barcoding is a potent approach for rapid identification of fungal specimens, generating novel species hypothesis, and guiding biodiversity and ecological studies. In this mini-review, I briefly summarize (i) the history of DNA sequence-based fungal identification; (ii) the emergence of the ITS region as the consensus primary fungal barcode; (iii) the use of the ITS barcodes to address a variety of issues on fungal diversity from local to global scales, including generating a large number of species hypothesis; and (iv) the problems with the ITS barcode region and the approaches to overcome these problems. Similar to DNA barcoding research on plants and animals, significant progress has been achieved over the last few years in terms of both the questions being addressed and the foundations being laid for future research endeavors. However, significant challenges remain. I suggest three broad areas of research to enhance the usefulness of fungal DNA barcoding to meet the current and future challenges: (i) develop a common set of primers and technologies that allow the amplification and sequencing of all fungi at both the primary and secondary barcode loci; (ii) compile a centralized reference database that includes all recognized fungal species as well as species hypothesis, and allows regular updates from the research community; and (iii) establish a consensus set of new species recognition criteria based on barcode DNA sequences that can be applied across the fungal kingdom.

  11. 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.

  12. Electrochemical DNA Sensors for Detection of DNA Damage

    Directory of Open Access Journals (Sweden)

    Ana Maria Oliveira Brett

    2005-11-01

    Full Text Available Electrochemical devices have received particular attention due to their rapiddetection and great sensitivity for the evaluation of DNA-hazard compounds interactionmechanisms. Several types of bioanalytical method use nucleic acids probes to detect DNAdamage. This article reviews current directions and strategies in the development andapplications of electrochemical DNA sensors for the detection of DNA damage.

  13. 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.

  14. 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).

  15. Redox signaling in plants.

    Science.gov (United States)

    Foyer, Christine H; Noctor, Graham

    2013-06-01

    Our aim is to deliver an authoritative and challenging perspective of current concepts in plant redox signaling, focusing particularly on the complex interface between the redox and hormone-signaling pathways that allow precise control of plant growth and defense in response to metabolic triggers and environmental constraints and cues. Plants produce significant amounts of singlet oxygen and other reactive oxygen species (ROS) as a result of photosynthetic electron transport and metabolism. Such pathways contribute to the compartment-specific redox-regulated signaling systems in plant cells that convey information to the nucleus to regulate gene expression. Like the chloroplasts and mitochondria, the apoplast-cell wall compartment makes a significant contribution to the redox signaling network, but unlike these organelles, the apoplast has a low antioxidant-buffering capacity. The respective roles of ROS, low-molecular antioxidants, redox-active proteins, and antioxidant enzymes are considered in relation to the functions of plant hormones such as salicylic acid, jasmonic acid, and auxin, in the composite control of plant growth and defense. Regulation of redox gradients between key compartments in plant cells such as those across the plasma membrane facilitates flexible and multiple faceted opportunities for redox signaling that spans the intracellular and extracellular environments. In conclusion, plants are recognized as masters of the art of redox regulation that use oxidants and antioxidants as flexible integrators of signals from metabolism and the environment.

  16. An ultrasensitive supersandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide.

    Science.gov (United States)

    Wang, Jiao; Shi, Anqi; Fang, Xian; Han, Xiaowei; Zhang, Yuzhong

    2015-01-15

    In this article, a supersandwich-type electrochemical biosensor for sequence-specific DNA detection is described. In design, single-strand DNA labeled with methylene blue (MB) was used as signal probe, and auxiliary probe was designed to hybridize with two different regions of signal probe. The biosensor construction contained three steps: (i) capture DNA labeled with thiol was immobilized on the surface of gold nanoparticles decorated reduced graphene oxide (Au NPs/rGO); (ii) the sandwich structure formation contained "capture-target-signal probe"; and (iii) auxiliary probe was introduced to produce long concatamers containing signal molecule MB. Differential pulse voltammetry (DPV) was used to monitor the DNA hybridization event using peak current changes of MB in phosphate-buffered saline (PBS) containing 1.0M NaClO4. Under optimal conditions, the peak currents of MB were linear with the logarithm of the concentration of target DNA in the range of 0.1μM to 0.1fM with a detection limit of 35aM (signal/noise=3). In addition, this biosensor exhibited good selectivity even for single-base mismatched target DNA detection. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Scallop-Inspired DNA Nanomachine: A Ratiometric Nanothermometer for Intracellular Temperature Sensing.

    Science.gov (United States)

    Xie, Nuli; Huang, Jin; Yang, Xiaohai; He, Xiaoxiao; Liu, Jianbo; Huang, Jiaqi; Fang, Hongmei; Wang, Kemin

    2017-11-21

    Accurate measurement of intracellular temperature is of great significance in biology and medicine. With use of DNA nanotechnology and inspiration by nature's examples of "protective and reversible responses" exoskeletons, a scallop-inspired DNA nanomachine (SDN) is desgined as a ratiometric nanothermometer for intracellular temperature sensing. The SDN is composed of a rigid DNA tetrahedron, where a thermal-sensitive molecular beacon (MB) is embedded in one edge of the DNA tetrahedron. Relying on the thermal-sensitive MB and fluorescence resonance energy transfer (FRET) signaling mechanism, the "On" to "Off" signal is reversibly responding to "below" and "over" the melting temperature. Mimicking the functional anatomy of a scallop, the SDN exhibits high cellular permeability and resistance to enzymatic degradation, good reversibility, and tunable response range. Furthermore, FRET ratiometric signal that allows the simultaneous recording of two emission intensities at different wavelengths can provide a feasible approach for precise detection, minimizing the effect of system fluctuations.

  18. Digital Signal Processing applied to Physical Signals

    CERN Document Server

    Alberto, Diego; Musa, L

    2011-01-01

    It is well known that many of the scientific and technological discoveries of the XXI century will depend on the capability of processing and understanding a huge quantity of data. With the advent of the digital era, a fully digital and automated treatment can be designed and performed. From data mining to data compression, from signal elaboration to noise reduction, a processing is essential to manage and enhance features of interest after every data acquisition (DAQ) session. In the near future, science will go towards interdisciplinary research. In this work there will be given an example of the application of signal processing to different fields of Physics from nuclear particle detectors to biomedical examinations. In Chapter 1 a brief description of the collaborations that allowed this thesis is given, together with a list of the publications co-produced by the author in these three years. The most important notations, definitions and acronyms used in the work are also provided. In Chapter 2, the last r...

  19. DNA Damage Induced Neuronal Death

    National Research Council Canada - National Science Library

    Kisby, Glen

    1999-01-01

    ... (nitrogen mustard or HN2) and the neurotoxic DNA-damaging agent methylazoxymethanol (MAM) using neuronal and astrocyte cell cultures from different brain regions of mice with perturbed DNA repair...

  20. The Dynamics of DNA Sequencing.

    Science.gov (United States)

    Morvillo, Nancy

    1997-01-01

    Describes a paper-and-pencil activity that helps students understand DNA sequencing and expands student understanding of DNA structure, replication, and gel electrophoresis. Appropriate for advanced biology students who are familiar with the Sanger method. (DDR)